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Chapter 7 Quantitative Immunoelectrophoresis
Chapter 7
Quantitative immunoelectrophoresis P. JUST SVENDSEN
CONTENTS Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apparatus and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practical application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed immunoelectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed immunoelectrophoresis with an intermediate gel . . . . . . . . . . . Procedure for fused rocket immunoelectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . Procedure for rocket immunoelectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed-line immunoelectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133 134 134 134 136 136 139 139 141 143 148 150 153
INTRODUCTION Classical immunoelectrophoresis, introduced by Grabar and Williams [ 11, was a great advance in the analysis of complex biological fluids, For the first time it was possible to resolve further components separated by electrophoresis in agar gel. However, the method did not give precise quantitative data and the resolution was adversely affected by a diffusion step. This problem was solved by Laurell, who introduced rocket immunoelectrophoresis [2] and crossed immunoelectrophoresis [3]. In the classical immunoelectrophoresis devised by Grabar and Williams [I], the antigens separated by electrophoresis are permitted to diffuse against a reservoir of precipitating antibodies. A number of arches will appear, one for each antigen in the sample, provided that the corresponding antibody is present in the antibody preparation. In rocket immunoelectrophoresis [2], specific antibodies are evenly distributed in an agarose gel, the samples are applied into sample wells punched out in this gel and a current is immediately passed through the gel. The antigens will migrate through the gel, and the antigen corresponding to the antibody in the gel will precipitate along the edges of the migrating sample, forming a peak when all antigen has been precipitated. The precipitate will appear with contours like that of a rocket, which led to the name of the method. The area enclosed by the precipitation line, or the peak height, is nearly proportional to the amount of antigen applied in the sample well. In crossed immunoelectrophoresis [3] the antigens are separated by agarose gel electrophoresis. The samples are applied in narrow application slits and, after electrophoresis, thin strips are cut out from the separation tracks and transferred to another plate.
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Antibody-containing gel is poured along the strips and a current is passed through these plates perpendicular to the direction of the current in the initial separation. A series of precipitates will occur in the antibodycontaining gel, each revealing the distribution of the corresponding antigen. This technique is primarily used for studying the heterogeneity of antigens and is only semi-quantitative, as not all of the antigen is transferred to the second-dimension plate. To obtain complete quantitative information, the crossed immunoelectrophoresis introduced by Laurell [3] was modified by Clarke and Freeman [4],who applied the sample in a circular application well and transferred the entire separation track to the seconddimension plate. They showed that the area enclosed by the precipitation line is proportional to the amount of antigen applied in the sample well. Kr6ll introduced line immunoelectrophoresis [5]. In this method, the antigens are evenly distributed in a rectangular gel slab in conjunction with the antibody-containing gel. A current is passed through the gels, and the precipitation lines will appear as straight lines parallel to the antigen-containing gel. The distance between the lines and the edge of the gel that contained the antigens at the start of the experiment is proportional to the amount of antigen introduced into the sample gel. Rocket immunoelectrophoresis according to Laurell [2], crossed immunoelectrophoresis according to Clarke and Freeman [4] and line immunoelectrophoresis according to KrQll [5] are the basic methods of quantitative immunoelectrophoresis. These three methods yield quantitative information that is proportional to the antigen concentration and inversely proportional to the antibody concentration. By combining and modifying these principles a great variety of methods have been developed for application in both routine clinical studies and research [6-91. This chapter describes the use of quantitative immunoelectrophoresis as a tool for following the fractionation of an antigen to purity as an example. An apparatus for conducting the experiments and the chemicals and solutions used are discussed. CHEMICALS AND SOLUTIONS Chemicals The following chemicals are used: 2-amino-2(hydroxymethyl)propane-l,3-diol(Tris), calcium lactate, 5,5-diethylbarbituric acid (Diemal), sodium azide, glacial acetic acid, sodium chloride, agarose Type H S A, Coomassie Brilliant Blue R 250, ethanol (96%), rabbit anti-human serum immunoglobulins, rabbit anti-human transferrin immunoglobulins. Solutions Tris-Diemal buffer, pH 8.6,ionic strength 0.1 (stock solution): 5,5-Diethylbarbituric acid (Diemal) 224 g 443 g Tris Calcium lactate 5.33 g Sodium azide 10 g Distilled water to 10 1
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(Dilute 1 part of stock solution plus 4 parts of distilled water to obtain an ionic strength of 0.02) The chemicals dissolve quickly in 5 1 of distilled water with magnetic stirring and no heating is necessary. This buffer is essentially the same as that recommended elsewhere (ref. 6, p. 25), except that the sodium ion has been replaced with Tris, thus increasing the buffering capacity several fold. There were some problems with the earlier buffer, as the buffering capacity often was exhausted, and as a consequence the pH in the cathodic vessel increased to over 11, resulting in dissolution of the immunoprecipitates from the cathodic side. This new buffer does not alter the immunoprecipitin pattern in quantitative immunoelectrophoresis, but stabilizes the reproducibility. 1 % Agarose in Tris-Diemal buffer, pH 8.6, ionic strength 0.02: Agarose ( 2 g) is added to 200 ml of the diluted buffer and dissolved by gentle heating with magnetic stirring. To ensure that the agarose is completely dissolved the solution should boil for 5-6 min. The solution is kept fluid at 56°C in a water-bath and is ready for use after temperature equilibration. Agarose (1%) can be stored at 4°C for several weeks and liquified repeatedly by heating. The agarose should exhibit an MR value of 0.1 3 to obtain good results with rabbit antibodies and Tris-Diemal (pH 8.6, ionic strength 0.02). Under these circumstances the average migration velocity of rabbit antibodies is close to zero, The MR value is a measure of the electroendosmosis and is obtained by subjecting a mixture of human albumin and polydextran to electrophoresis in the actual system. The shift of the polydextran is measured (the migration direction taken into consideration: negative for a cathodic shift, positive for an anodic shift), and this distance is related to the total distance between the polydextran and human albumin. The Litex agarose used in this laboratory is guaranteed to maintain the same electroendosmosis from batch to batch, which for Type H S A is 0.13%. 0.1 M sodium chloride for washing excess of protein out of the plates after immunoelectrophoresis. Staining solution: 4500 ml Ethanol (96%) Distilled water 4500 ml 1000ml Glacial acetic acid Coomassie Brilliant Blur R 250 50 g The dye is dissolved and the solution is heated to 6OoC, cooled to room temperature and finally filtered through filter-paper. Destaining solution: Ethanol (96%) 4500 ml Distilled water 4500 ml Glacial acetic acid 1000ml The destaining solution can be used repeatedly if filtered through activated charcoal after use.
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Antibodies Crude antisera can always be used directly in experiments. However, to obtain low background staining, a crude antibody preparation should be subjected to salting-out [250g of (N&)2S04 per 1000ml of rabbit anti-serum], dialysis and chromatography on DEAE-Sephadex A-50at pH 5 .O in sodium acetate-acetic acid buffer with an ionic strength of 0.05.The purified gamma-globulin fraction is then dialysed against 0.1 M NaC1-15 mM NaN3. This preparation will lose less than 2% of its activiiy per year when stored at 4OC. (For further details, see Chapter 23 of ref. 6.)
APPARATUS AND ACCESSORIES We use 1mm thick glass plates to support the agarose gels. These glass plates are made in the following standard sizes: 5 x 5 , 7 x 7 , 7 x 10,lO x 10 and 11 x 20.5 cm. The apparatus shown in Fig. 7.1A was therefore designed in such a fashion that it will accommodate several of the standard glass plates on the cooled surface (2 in Fig. 7.1B). The cooled surface measures 22 x 12 cm and is capable of holding one 1 1 x 20.5 cm plate, two 10 x 10 cm plates, three 7 x 10cm plates, three 7 x 7 cm plates or eight 5 x 5 cm plates. The design is similar to that described by Johansson [lo], and cooling is obtained by a serpentine-shaped cooling channel cut in a thick Perspex plate, leaving 1 mm of material between the cooling channel and the cooling surface. With a flow-rate of 1 l/min the cooling capacity of the apparatus is sufficient for quantitative immunoelectrophoresis. The temperature of the cooling water should be 10-1SoC. The experiments described below were conducted at a temperature of the cooling water of 15OC. The cooling water is circulated by a cooling thermostat. The cooling plate is attached to two supports (4in Fig. 7.1 B), with moulds attached for casting agarose gel connections to the electrode vessels (1 in Fig. 7.1B), each containing 1 1 of Tris-Diemal buffer (pH 8.6, ionic strength 0.02). The inner walls of the moulds are level with the cooling surface, and the outer walls of the moulds are 1 cm higher than the cooling surface and are level with the end pieces attached to the cooling plate. This central unit is placed in the electrode vessels, which are separated by a Perspex wall. When the lid (5 in Fig. 7.1B), is attached, the platinum-wire electrodes supported by PVC plates (3 in Fig. 7.1B), are automatically connected to the wire and jack-plugs (6 in Fig. 7.1B). The lid rests on the edge of the central unit and is approximately 1 mm from the upper edge of the electrode-vesselunit, thereby sealing the electrophoresis chamber completely from the surrounding air. Even when filter-paper wicks are used to connect the agarose gel plates to the electrode buffer, the gel moulds should not be removed from the central unit. As these moulds are immersed in the electrode buffer, they ensure complete sealing of the electrophoresis chamber. It is an advantage to keep the volume of the electrophoresis chamber small and well sealed from the surrounding air, including that above the electrode buffer, as water would otherwise condense on the cooled agarose gel on humid days or with very low cooling temperatures. If water condenses on the immunoelectrophoresis plate during the experiment, distorted immunoprecipitates will be formed. The lid (5 in Fig. 7.1B) is furnished with five pairs of holes 4cm apart, which provide
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Fig. 7.1. (A) Electrophoresis apparatus for quantitative immunoelectrophoresis, assembled. (B) Main parts of electrophoresis apparatus. 1 = Electrode vessels; 2 = cooled surface; 3 = electrodes on PVC supports; 4 = support with moulds for agarose gel connections; 5 = lid with electric connectors; 6 = jack-plugs for power supply.
access for a test probe (3 in Fig. 7.2) that is used to measure the potential gradient directly in the gel when the current has been switched on. The test probe is connected to the power supply (1 in Fig. 7.2), and a switch on the power supply sets the voltmeter so that the potential gradient can be read directly in volts per centimetre. At the same time, the correct polarity of the current is checked. The electrophoresis unit (2 in Fig. 7.2) is connected to one of the four power outlets on the power supply. Three channels in the power supply have the capacity of delivering 300V and lOOmAd.c., which is sufficient for quantitative immunoelectrophoresis. The fourth channel can deliver 300 V and 200 mA d.c., for use with buffers of higher ionic strength, e.g., electrophoresis in agarose gel using Tris-Diemal buffer (pH 8.6, ionic strength 0.075).
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Pig. 7.2. Apparatus and accessories for quantitative immunoelectrophoresis. 1 = Power supply; 2 = electrophoresis apparatus; 3 = test probe for measuring the potential gradient; 4 = adjustable glass table for casting gels; 5 = device for levelling electrode buffers; 6 = gel punchers; 7 = adjustable template for punching samples wells; 8 = vessel for washing, staining and destaining; 9 = racks for holding glass plates; 10 = U-shaped frame for casting gels between glass plates; 1 1 = long razor blades for cutting and transferring gel slabs; 12 = slit formers.
The gels are usually cast on levelled glass plates, and for this purpose a levelling glass table (4 in Fig. 7.2) is used in combination with a precision spirit level. To ensure that no hydrodynamic flow occurs during immunoelectrophoresis due to a different level of the buffers in the electrode vessels, the buffer in the vessels is levelled by means of a U-tube (5 in Fig. 7.2). The U-tube is removed before immunoelectrophoresis. Through a template (7 in Fig. 7.2), the sample wells are punched out by means of a puncher [ 1 I ] (6 in Fig. 7.2), which in a single operation punches the well and removes the agarose gel plug. The puncher is connected to a suction device, and is made of two annular steel tubes. The outer tube has a sharp edge and cuts the gel on impact with it. The inner tube, which is spring-loaded, slides downwards when a mechanical pressure is applied, and sucks up the agarose gel plug. Slits in the outer tube serve as air inlets to release the vacuum introduced through the inner tube, thus avoiding damage to the walls of the well. The template is made of a base plate to which a sliding ruler is attached. The ruler has a pattern of holes with the same diameter as the upper part of the puncher, for punching all the patterns of sample wells needed in quantitative immunoelectrophoresis. The punchers are available for sample wells with diameters of 2.0,2.5,3.0 and 4.0 mm. Racks (9 in Fig. 7.2) for holding the various plate sizes fit into the vessels (8 in Fig. 7.2) for washing, staining and destaining purposes. For rocket immunoelectrophoresis on 1 1 x 20.5 cni glass plates, a matrix is made of a 1.5 mm thick U-shaped frame (10 in Fig. 7.2), set between two glass plates, held together by strong paper clamps. With a pipette the antibody-containing solution is poured into the matrix. If the rear glass plate is shifted a few millimetres relative to the front glass plate, the filling is greatly facilitated. In two-dimensional immunoelectrophoresis agarose gel slabs are transferred from one glass plate to another, by using long razor blades (1 1 in Fig. 7.2) measuring 2 x 15 cm. If narrow slits are desired, slit formers (1 2 in Fig. 7.2) are used. The slit formers rest on four legs, which are adjusted in such a way that the slit-forming blades are approximately
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I Fig. 7.3. Cross-sectional area of double constriction pipette.
0.2 mm from the glass plate. This prevents the sample from leaking between the gel and glass plate, as the slit is sealed in the bottom. When the gels have been poured on to the levelled glass plates, the slit formers are placed into the gels before they gel, and are removed just before sample application. Sample application is performed by using double constriction pipettes (Fig. 7.3). The double constriction pipette has the advantage over the single constriction pipette of not being fully emptied. This prevents air being blown into the filled wells, causing overflow and contamination of the surrounding gel. The double constriction pipette is filled to the upper constriction and emptied by blowing gently until the meniscus reaches the lower constriction. The tip of the pipette must touch the bottom of the sample well in order to deliver precise and reproducible volumes. These pipettes are available in various sizes from 1 p1 to several millilitres.
PRACTICAL APPLICATION Quantitative immunoelectrophoresis is an almost indispensable tool for following the fractionation of a protein t o purity, and for testing the isolated material for purity. Antibodies to the crude antigen mixture, in the following human serum, must be available; if not commercially available it may be raised in rabbits, following the procedure described in Chapter 23 of ref. 6 . The purification of human serum transferrin is described below. The first step is to investigate the crude antigen mixture by crossed immunoelectrophoresis according to Clarke and Freeman [4]. Procedure for crossed immunoelectrophoresis A 10 x 10 cm glass plate is washed with detergent, rinsed with ethanol and dried. To obtain a 1.5 mm thick agarose gel, 15 ml of agarose solution at 56°C is poured on to the glass plate, placed on the levelled glass table. After congelation of the agarose, five wells are punched out by means of a 2.5-mm gel puncher using the template shown in Fig. 7.4A. A 2 4 volume of human serum is applied into the sample well(s) S1 to S4 using a double constriction pipette. The fifth well, M, is used to apply a marker, e.g., albunin stained with bromophenol blue. The glass plate with the agarose gel is placed on the cooling surface of the electrophoresis apparatus. A conducting connection to the electrode vessels is obtained with buffer-saturated filter-paper wicks (Whatman No. 1, eight layers, 10 x I0 cm). It is important to ensure that the wicks have good contact with the gel. Electrophoresis in the first dimension is run with a field strength of 15 V/cni. This is tested in every run with a test probe in order to obtain the same potential gradient in the runs. The tinie for the run in the first dimension is approximately 55 min at I5'C. The electrophoresis is discontinued when the blue marker has migrated 5.5 cm. The agarose
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Fig. 7.4. Crossed emunoelectrophoresis. (A) Template for the first-dimensiongel. (B)Template for the seconddimension plate. The hatched area indicates the antibodycontaining gel. (C) Crossed immunoelectrophoresis of 2 pl of human serum into l000pl of anti-human serum (1 2.5 pl/cmagel).
gel is cut according to Fig. 7.4A following the dotted lines. The track with the marker is discarded. The first-dimension gel slabs are then transferred with a long razor blade to the seconddimension glass plates, which have been coated with agarose solution and dried (see Fig. 7.4B). The glass plates are then placed on the levelled glass table, and 12 ml of antibodycontaining solution (1 1ml of 1%agarose 1 ml of antihuman serum) are poured on to the upper part of the glass plate, corresponding to the shaded area in Fig. 7.4B. After 10 min the gel is ready, and the plate is placed on the electrophoresis apparatus and connected to the electrode buffer by means of filter-paper wicks (five layers of Whatman No. 1,lO x 10 cm). Immunoelectrophoresis in the second dimension is performed at 3 V/cm for 18-20 h. After electrophoresis, non-precipitated proteins must be removed, which can be ,effected efficiently by pressing the gel under six layers of filter-paper. Air trapped under
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the filter-paper will damage the gel, and therefore the application wells must be filled with distilled water prior to the pressing procedure. The first layer of filter-paper is applied wet, followed by five layers of dry filter-paper. A 6-8 mm thick glass plate should be placed on top of the filter-paper to sustain a smooth pressure all over the gel. After 5 min the upper five layers of filter-paper are renewed and the gel is pressed for another 10 min. A polyspecific antibody preparation of high titre usually contains a larger amount of proteins than a monospecific reagent. Therefore, to obtain low staining of the background, the plate should be rinsed for I 5 min with 0.1 M sodium chloride solution, pressed under filter-paper and rinsed for a further 15 min in distilled water. After a final pressing, the filter-paper is moistened with distilled water and removed. The plate is dried in a stream of hot air and then stained in Coomassie Brilliant Blue R 250 for 5 min, rinsed three times with ethanol-water-acetic acid and dried. Many enzymes remain active in immunoprecipitates, and if enzymes are to be stained with specific enzyme substrates the plate should be dried in cold air and developed before normal protein staining. Crossed immunoelectrophoresis performed as described here with 2 pl of human serum and 1 ml of anti-human serum is shown in Fig. 7.4C. Identification of the antigens causing the individual precipitates is performed immunochemically by using monospecific antibodies or pure antigens if available. Alternatively, the antigens can be identified by histochemical methods such as staining for lipid or staining for enzymatic activity, or by autoradiography for the detection of the sepcific binding of radioactively labelled substances. With Clarke and Freeman’s modification of Laurell’s crossed immunoelectrophoresis, individual proteins are quantitated by measuring the area below the corresponding peak. Quantitation by measuring the peak height may not always be possible as many of the peaks are asymmetric, and therefore the peak area is measured electronically. The stained plate is enlarged nine times in an enlarger, and with a light-spot planimeter the curves are integrated. The areas are expressed in arbitrary units on an electronic counter, and when compared with a standard serum the method can be used for quantitative determinations. The next step in the fractionation experiment is to establish which of the numerous peaks appearing on the plate in Fig. 7.4C corresponds to transferrin. This can be done immunochemically by employing the intermediate gel technique introduced by Svendsen and Axelsen [12], if a monospecific antibody preparation is available.
Procedure for crossed immunoelectrophoresis with an intermediate gel The firstdimension agarose gel electrophoresis and transfer of the agarose gel slabs t o the coated seconddimension glass plates is the same as for crossed immunoelectrophoresis. The casting of the antibody-containing gels is shown in Fig. 7.5A. A 1 mm thick glass plate is placed 2.2 cm from the first-dimension gel slab, along the dotted line. A 3.2-ml volume of agarose solution mixed with 75 p1 of anti-human transferrin is poured into the open space between the gel and the glass plate. A reference plate is made in parallel, replacing the anti-human transferrin with 0.1 M sodium chloride solution. When the gel has solidified, the glass plate is cut free, the gel is trimmed along the solid line below the dotted line, and the thin gel strip is discarded. An 8.2-ml volume of agarose solution is mixed with 800pl of anti-human serum and poured on to the remaining part
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Fig. 7.5. Crossed immunoelectrophoresk with an intermediate gel. (A) Template for the seconddimension plate. (B) Reference plate; 75 pl of 0.1 M NaCl were added to the intermediate gel. (C) 75 pl of anti-human serum transferrin were added to the intermediate gel. (B) and (C) 2 p1 of human serum were applied in the sample well. Antibody: 8 0 0 ~ of 1 anti-human serum in the upper gel.
o f t h e glass plate, and the same is done on the reference plate. After immunoelectrophoresis, rinsing, pressing, drying and staining the plates are ready (Fig. 7.5B and C). On the plate in Fig. 7.5B all antigens have passed freely through the intermediate gel. The skewed baseline of the immunoprecipitates is due to decreasing mobilities of the antigens and electroendosmotic transport of slowly migrating antibody molecules. On the plate in Fig. 7.4C all antigens except transferrin have passed through the intermediate gel. As transferrin has been trapped in the intermediate gel, it is missing from the upper gel. A simple comparison of the two plates easily indicates which of the peaks in the reference plate corresponds to transferrin. This peak is marked with a heavy vertical arrow, and the transferrin peak is marked in the same way in all subsequent immunoelectrophoresis plates. The horizontal line in Fig. 7.5C is caused by excess of antigen used to absorb the antibody preparation to make it monospecific. For further details, see Chapter 7 of ref. 6. We have now established which of the peaks in crossed immunoelectrophoresis
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Fig. 7.6. Crossed immunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well. (B) 5 PI of the supernatant from the salting-out experiment described in the text were applied to the sample well. (B) and (C): antibody: lOOOpl of anti-human serum.
corresponds to transferrin. The first step in the fractionation procedure is salting-out with (NH4)$04. To 218 ml of human serum (1000 donor pool) were added 54.3 g (NH4)*S04 and 10.9 mg of FeS04. This mixture was left at room temperature overnight with magnetic stirring, and then centrifuged at 20,000 r.p.m. for 20min. The supernatant was dialysed against water, and finally equilibrated against sodium acetate-acetic acid buffer (pH 5.6, ionic strength 0.05). The supernatant was subjected t o crossed imniunoelectrophoresis and compared with serum (see Fig. 7.61, and was then applied to a column containing 425 ml of DEAE-Sepharose CL-6B, which was equilibrated with the same buffer. The proteins were eluted by increasing the concentration of the buffer to an ionic strength of 0.7 (linear gradient), and fractions were collected in an LKB Ultrorac fraction collector at 2 O C , flow-rate 30 ml/h, one fraction per hour. After the experiment, 68 fractions were collected and analysed by fused rocket immunoelectrophoresis [ 13, 141, which is superior to W monitoring as an elution profile for each individual antigen is obtained in a single experiment. UV monitoring is employed for detecting only where the UV-absorbing substances have been collected. Procedure for fused rocket immunoelectrophoresis A 10 x 10 or 1 1 x 20.5 cm glass plate is used. Volumes for a 1 0 x 10 cm plate are given first, followed by volumes for an 11 x 20.5 cm plate in parentheses. A 10 x 10 cm coated glass plate is placed on the levelled glass table and a 1 mm thick glass plate is placed 2.7 cm from the lower edge of the coated glass plate along the dotted line in Fig. 7.7A.A 4.1 (8.3)ml volume of 1% agarose solution is poured on to the plate and allowed t o congeal. The glass plate is cut free and the gel is trimmed following the solid line below the dotted line in Fig. 7.7A. A 10 (23) ml volume of agarose solution is mixed with 1 (2)ml of anti-human serum, poured on to the remainder of the glass plate and allowed to congeal. Sample wells are then punched out through the template (7 in Fig. 7.2), according
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Fig. 7.7. Fused rocket immunoelectrophoresis. (A) Template for casting the gel and punching the sample wells. (B) Immunoelution profile from ion-exchange chromatography on DEAE-Sepharose C L 6 B ; 2-pl aliquots from the collected fractions were applied in the sample wells. Closed circles indicate the fractions pooled. Antibody: 2000 ~1 of anti-human serum.
to Fig. 7.7 in the antibody-free gel. Thirty-eight sample wells can be punched in a 10 x 10 cm plate and 79 in an 11 x 20.5 cm plate. Samples of constant volume from the collected fractions are then transferred to the sample wells, in the same order as they were collected, by means of a double constriction pipette. The pipette is not rinsed between the individual applications. The plate is left for diffusion for 60 min on the cooled surface in the electrophoresis apparatus. At room temperature in a humid chamber, 30 min will suffice. The diffusion step will cause a continuous precipitation line to be formed for each antigen in the sample, over the sample wells corresponding to the fractions in which they were eluted. The plate is then connected to the electrode buffer by means of five layers of fdter-paper, and immunoelectrophoresis is conducted at 3 V/cm overnight (1 8-20 h). After electrophoresis the plate is pressed, washed, stained and dried as described above. Fig. 7.7B shows fused rocket immunoelectrophoresis of the fractions collected from the DEAE-Sepharose CG6B experiment. A number of elution profiles appear, and
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Fig. 7.8. Crossed immunoelectrophoresis; 5 pl of the pool from the ion-exchange experiment on DEAE-Sepharose CLdB were applied in the sample well. Antibody: 1000 PIof anti-human serum. Fig. 7.9. Fused rocket immunoelectrophoresis; 1-pl aliquots of the fractions from the ion-exchange experiment on CM-Sepharose CL-6B were applied in the sample wells. Antibody: 2000pI of antihuman serum.
transferrin was detected by the intermediate gel immunoelectrophoresis technique (not shown) and is marked with a vertical arrow. The thinner horizontal arrow indicates haemopexin, w h c h is difficult to separate from transferrin. Haemopexin is marked in the same manner in subsequent plates. The fractions marked with closed circles (Nos. 13-35) were pooled and checked for purity by means of crossed immunoelectrophoresis (Fig. 7.8). The pool (640 ml) was then directly applied to a CM-Sepharose CL-6B ion-exchange column (bed volume 125 ml) equilibrated with the same starting buffer as in the preceding ionexchange experiment. (In Fig. 7.7B it can be seen that transferrin passed the matrix without being bound.) The proteins were eluted by increasing the concentration of the buffer to an ionic strength of 0.4 (linear gradient). Fractions were collected in an LKB Ultrorac fraction collector at 2"C, flow-rate 30 ml/h, one fraction per hour. Fortynine fractions were collected and subjected t o fused rocket immunoelectrophoresis and the result is shown in Fig. 7.9. The fractions marked with closed circles (Nos. 38-43) were pooled and tested for purity by crossed immunoelectrophoresis (Fig. 7.10). The amount of impurities had decreased, but a few can still be seen. Preparative isotachophoresis [ 14,15J was then applied to remove the last traces of impurities, especially haemopexin. The pool (1 64 ml) from the experiment on CM-Sepharose CLdB was concentrated to 25 ml on an Amicon filter and dialysed against the terminator, Tris6-aminocaproic acid (pH 8.9). The leading electrolyte was Tris-phosphate (pH 8.1). The supporting medium was 5% polyacrylamide (5% cross-linking) with N,N'-methylenebisacrylarnide as cross-linking agent, cast in the glass column, cross-sectional area 4.0 cm', with central cooling in the apparatus described in ref. 15. A 120O-pl volumeof Ampholine (pZ range 6-8) was added to the sample as spacers. The experiment was run overnight at 5°C with a constant current of 10mA d.c. The elution buffer was Tris-sulphate (pH 7.1, flow-rate 30ml/h). Fractions were collected every 20min. For more details, see ref. 15.
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Fig, 7.10. Crossed immunoelectrophoresh; 2 pl of the pool from the ion-exchange experiment on CMSepharose CLdB were applied in the sample well. Antibody: 1OOOwl of anti-human serum.
Fig. 7.11. (A) Fused rocket immunoelectrophoresis; 1-pl aliquots of the fractions from the isotachophoresis experiment were applied in the sample wells. Antibody: 1OOOwl of anti-human serum. (B) Crossed immunoelectrophoresis; 2 p1 of the pool from the isotachophoresis experiment were applied in the sample well. Antibody: 1000 pi of anti-human serum.
Fused rocket immunoelectrophoresis was employed to analyse the collected fractions, and the plate is shown in Fig. 7.1 1A. It appears that fractions 22-27 can be pooled with transferrin in very high purity. The pool was then checked in crossed immunoelectrophoresis (Fig. 7.1 1B) and only one peak can be seen, except for an extremely small trace of impurity, marked with a thin vertical arrow. To remove the Ampholine collected with the protein, as well as polymers and aggregates, the pool (56 ml) from the isotachophoretic experiment was concentrated to 15 ml and applied on an Ultrogel ACA 44 gel filtration matrix (gel bed 2000 ml) and eluted at 60 d / h . The buffer was sodium phosphate (PH 7.2, ionic strength 0.415). The collected fractions were analysed by fused rocket immunoelectrophoresis, and from the result in Fig. 7.12A it is evident that a small
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Fig. 7.12. (A) Fused rocket immunoelectrophoresis; 1-p1 aliquots of the fractions from the gel filtration experiment on Ultrogel ACA 44 were applied in the sample wells. Antibody: 100Opl of antihuman serum. (R) Crossed immunoelectrophoresis; 4 p1 of the pool from the gel filtration experiment were applied in the sample well. Antibody: l000pI of anti-human serum.
Fig. 7.13. Rocket immunoelectrophoresis. (A) Template. (B) Left: 5-111aliquots of the pool from the gel filtration experiment on Ultrogel ACA 44 diluted 1 20 were applied. Right: 5-111 aliquots of the starting material (human serum, lOOOdonor pool) for the fractionation experiment described in the text diluted 1 t 50, 1 + 20, 1 + 10 and 1 + 5. Antibody: loop1 of anti-human transferrin.
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impurity has been separated from the transferrin. Fractions 23-29 were pooled (note how precisely fractions can be selected) and finally crossed immunoelectrophoresis of this pool revealed that immunochemically pure human serum transferrin was isolated (Fig. 7.12B). The volume of this pool was 200ml. The recovery of transferrin compared to the starting material can now be measured by Laurel1 rocket immunoelectrophoresis.
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Procedure for rocket immunoelectrophoresis
A 1 0 x 10 cm coated glass plate is placed on the levelled glass table, I 5 ml of agarose solution are mixed with loop1 of anti-human transferrin and the mixture is poured on to the glass plate. When the gel has solidified after approximately 10min the sample wells are punched out through a template according to Fig. 7.13A. When using an 11 x 20.5 cm glass plate, a matrix is made of a 1.5 mm thick U-shaped frame set between two glass plates (1 1 x 20.5 cm, one being coated), held together by strong paper clamps. With a pipette the antibodycontaining solution at 56°C (30 ml) is poured into the matrix. After lOmin at 4"C, the gel is ready, and the uncoated glass plate is removed by gently sliding it off. The U-shaped frame is kept on the glass plate until the pressing and staining procedure. An aliquot of the sample, in this instance the pool from the gel filtration experiment, is diluted as recommended by the antibody supplier, for human serum transferrin 1 20 with 0.1 M sodium chloride solution. To obtain a standard graph, aliquots from the starting material, in this instance the 1000-donor pool from which 1 ml was saved, are diluted 1 5 0 , l q.20, 1 10 and 1 + 5. The four standards are applied with a 5-pl double constriction pipette into four of the sample wells, and the sample dilution is applied in one or more sample wells, also in 5-4 aliquots. Immunoelectrophoresis is immediately started with a field strength of 3 V/cm, and the experiment is run for 18-20 h. After electrophoresis the plate is squeezed under filter-paper. No washing is necessary when using the immunoglobulin fraction of the antiserum rather than the total antiserum, if monospecific antibodies are employed. Therefore, the plate can be dried after pressing it once, stained for 5 min in Coomassie Brilliant Blue R 250, washed three times with destaining solution and dried. The plate is now ready for interpretation (see Fig. 7.13B). If the dilution 1 + 20 is assigned to represent loo%,then the other dilutions will represent the following: 1 5 = 350%, 1 1 0 = 191% (1 + 20 = 100%) and 1 50 = 41%. Laurel1 rocket immunoelectrophoresis offers an almost linear correlation between the antigen concentration and the area delineated by the precipitation line, and as this area is nearly proportional to the peak height the latter is used for the calculation, The peak heights are easily determined on graph paper, measured from the upper edge of the sample well, and a standard graph is drawn with the antigen concentration as the abscissa and the peak height as the ordinate. The peak height of the unknown is measured and interpolated on the diagram. The standard graph obtained from the plate in Fig. 7.13B is shown in Fig. 7.14. The interpolation for calculating the concentration in the transferrin pool is indicated by arrows, and the result is that the pool has a concentration of 45% relative to the starting material and the total yield is 41%. If corrections are made for samples taken during the fractionation and the known loss introduced by omitting to wash the precipitate in the salting-out experiment, then the recovery is 51%. If the precipitate is washed, the total yield would be 48%, so washing the precipitate defmitely has a significant effect on the recovery. As mentioned above, the peaks in crossed immunoelectrophoresis can be identified if a pure known antigen is available or, vice versa, an unknown antigen can be identified if the peaks in crossed immunoelectrophoresis can be recognized.
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45
Fig. 7.14. Standard graph for human serum transferrin determined by rocket immunoelectrophoresis. Data from the plate shown in Fig. 7.13B. The arrows indicate the interpolation to obtain the concentration of human transferrin in the pool from the gel fitration experiment on Ultrogel ACA 44. The dilution 1 + 20 is set as 100%.
This can be achieved simply by performing crossed immunoelectrophoresis on 2 p1 of human serum, and for the purpose of demonstration by adding 4 pl of the pool containing the purified human serum transferrin to the sample in the sample well. The result of such an experiment is shown in Fig. 7.1 5B. The plate in Fig. 7.15A is a reference plate, where 4 pl of 0.1 M sodium chloride solution were added to the serum in the sample well. By comparing the two plates, the increase in area of the transferrin peak is easily revealed. Another means of identifying an unknown antigen or a precipitation peak by means of a pure antigen is to absorb the antibodies with the antigen. A 2 - 4 volume of human serum was subjected to crossed immunoelectrophoresis, and 100 pl of the pool containing the pure human serum transferrin were added to the antibody-containing agarose solution before casting the gel. A reference plate was made in parallel, replacing 1OOpl of transferrin with lOOplO.1 Msodium chloride solution. Fig. 7.16B shows the resulting plate, and on comparison of this with the reference plate in Fig. 7.16A, the increase in the area of the transferrin peak is obvious. The background of the plate in Fig. 7.16B is dark, owing to the insoluble transferrinlanti-transferrincomplexes formed by the addition of transferrin to the antibodies. This effect can be prevented by centrifugation of the absorbed antibodies before addition to the agarose solution. However, larger volumes must be used in this instance in order to maintain the precision. Crossed-line immunoelectrophoresis [ 161 can be used for the same purpose.
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Fig. 15. Crossed immunoelectrophoresis. (A) 2 pl of human serum + 4 pl of 0.1 M NaCl were applied in the sample well. (B) 2 p1 of human serum t 4 pl of the pool from the gel filtration experiment on Ultrogel ACA 44 were applied in the sample well. Antibody: l O O O p l of anti-human serum.
Fig. 16. Crossed imrnunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well. Antibody: 1000 pl of anti-human serum. (B) 2 p1 of human serum were applied in the sample well. Antibody: 1OOOpl of anti-human serum absorbed with loop1 of the pool from the gel filtration experiment on Ultrogel ACA 44.
Procedure for crossed-line immunoelectrophoresis The first-dimension agarose gel electrophoresis and transfer of the agarose gel slabs t o the coated second-dimension glass plates is the same as for crossed immunoelectrophoresis. A 1 mm thick glass plate is placed 1.2 cm from the first-dimension gel slab (see Fig. 7.1 7) and 1.8 ml of agarose solution mixed with 20 pl of the pool containing the pure transferrin is cast into the open space between the first-dimension gel slab and the glass plate. A reference plate is made in parallel, replacing the transferrin with 20 jd of 0.1 M sodium chloride solution. The glass plate is cut free and the gel is trimmed along the solid
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Fig. 7.17. Template for crossed-line immunoelectrophoresis.
Fig, 7.18. Crossed-line immunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well; 20 p1 of 0.1 M NaCl were added to the gel slab between the first-dimension gel slab and the antibody-containing gel. (B) 2 pl of human serum were applied in the sample well; 20111 of the pool from the gel filtration experiment on Ultrogel ACA 44 were added to the gel between the first-dimension gel slab and the antibody-containing gel. Antibody: 900 pl of anti-human serum.
h e below the dotted line in Fig. 7.17. The thin gel strip is discarded. A 9.5-ml volume of agarose solution is mixed with 900 pl of anti-human serum and cast on to the remaining part of the plate; the same is done with the reference plate. After immunoelectrophoresis, the plate is pressed, washed, dried and stained as for crossed immunoelectrophoresis. The result is shown in Fig. 7.18B. The transferrin peak has been elevated by the transferrin line. If approximately 320jd of the pure transferrin had been included in the antigencontaining gel, the transferrin line would have absorbed all the anti-transferrin and no precipitate would have formed on the plate, i.e., the transferrin peak would have been missing. This method is therefore recommended for removing antibodies to cross-reacting antigens, e.g., when tissue-specific antigens are investigated [ 171.
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Fig. 7.19. Tandem-crossed immunoelectrophoresis. (A) Template. (B) 2 pl of human serum were applied in the sample well to the left; 4 pl of the pool from the gel fitration experiment on Ultrogel ACA 44 were applied in the sample well to the right. The plate was left for diffusion for 60min before electrophoresis in the fmst direction. Antibody: 1000 pl of anti-human serum.
The last method to be described is tandem-crossed immunoelectrophoresis [ 181, which is essentially the same as crossed immunoelectrophoresis, but two samples are applied in the first-dimension gel, the sample wells having a centre-to-centre distance of 10 mm (see Fig. 7.19A). Human serum (2 pl) is applied in the rear sample well and 4 pl of the pool containing the pure human transferrin is applied in the other well in front. Before electrophoresis in the first dimension is started, the plate is left for diffusion for 60 min on the cooled surface of the electrophoresis apparatus at 15'C. For more complex mixtures, a reference plate should be made in parallel, replacing in this instance transferrin with 4 p1 of 0.1 Msodium chloride solution. The diffusion causes the contents of the sample wells to mix partially in the gel. If identical antigens are present in both wells, the corresponding peaks will show reaction of identity, i.e., fuse into a double peak, as shown in Fig. 7.19B. The above methods can be used to analyse and characterize both antigen and antibody mixtures, including the study of partially identical antigens, e.g., comparing antigens from different species or tissues (see Chapter 11 of ref. 6). A prerequisite for employing the methods is that precipitating antibodies must be available or raised and that the antigens have mobilities different from that of the antibodies. If the mobilities of the antigens coincide with that of the antibodies, the former can be modified by carbamylation to change the pZ(see Chapter 20 of ref. 6 ) , and by carbamylation antibodies can be modified to exhibit zero net mobility in agarose at pH 5 , retaining 80%of their antibody titre. The sensitivity of the methods by normal staining with Coomassie Brilliant Blue R 250 is in the range 5-10 ng of antigen applied in the sample well. The sensitivity can be increased 15-30-fold by employing radioactively labelled reagents or enzyme-conjugated antibodies in a sandwich technique, but if the amount of antigen applied in the well is less than 0.3 ng the lower limit for forming a precipitate may be reached [ 191.
REFERENCES
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REFERENCES
7 8 9 10 11 12 13 14 15 16 17 18 19
P. Grabar and C. A. Williams, Biochirn. Biophvs. Acta, 10 (1953) 193. C. -B. Laurell, hotides Biol. Fluids, 14 (1967) 499. C. -B. Laurcll, Anal. Biochem., 10 (1965) 3 5 8 . t1. G. M. Clarke and T. Freeman, Protides Biol. I;luids, 14 (1967) 503. J . KrQli, Scand. J . Clin. Lab. Invest., 22 (1968) 11 2. N. H. Axelsen, J . KrQll and B. Weeke, A Manual of Quantitative ImmunoelectrophoresiscMethods and Applications, Universitetsforlaget , Oslo, 1973. N. H. Axelsen, Quantitative lmrnunoelectrophoresis New Developments and Applications, Universitetsforlaget. Oslo, 1975. R . Verbruggen, Clin. Chem., 21 (1975) 5 . C. -B. Laurell, Scand. J. Clin. Lab. Invest., Suppl., 29 (124) (1972). B. G. Johansson, Scand. J. Clin. Lab. Invest., Suppl., 29 (124) (1972) 7. B. Weeke and J . P. Thomsen, Scand. J. Clin. Lab. Invest., 22 (1968) 165. P. J . Svendsen and N. H . Axelsen,J. lmmunol. Methods, 1 (1972) 169. N. M. G . Harboe and P. J . Svendsen, unpublished work (1969). P. J. Svendsen and C. Rose, Sci. Tools, 17 (1970) 13. P. I . Svendsen, in 2. Deyl (Editor), Electrophoresis. A Survey of Techniques and Applications. Part A : Techniques, Elsevier, Amsterdam, Oxford, New York, 1979, Chapter 16. J . Krgll, Scand. J. Clin. Lab. Invest., 24 (1969) 5 5 . E. Bock,J. Neurochem., 19 (1972) 1731. J. KrQll, Scand. J. Clin. Lab. Invest., 22 (1968) 79. A. Ingild, personal communication.
Quantitative immunoelectrophoresis P. JUST SVENDSEN
CONTENTS Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apparatus and accessories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practical application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed immunoelectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed immunoelectrophoresis with an intermediate gel . . . . . . . . . . . Procedure for fused rocket immunoelectrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . Procedure for rocket immunoelectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . Procedure for crossed-line immunoelectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133 134 134 134 136 136 139 139 141 143 148 150 153
INTRODUCTION Classical immunoelectrophoresis, introduced by Grabar and Williams [ 11, was a great advance in the analysis of complex biological fluids, For the first time it was possible to resolve further components separated by electrophoresis in agar gel. However, the method did not give precise quantitative data and the resolution was adversely affected by a diffusion step. This problem was solved by Laurell, who introduced rocket immunoelectrophoresis [2] and crossed immunoelectrophoresis [3]. In the classical immunoelectrophoresis devised by Grabar and Williams [I], the antigens separated by electrophoresis are permitted to diffuse against a reservoir of precipitating antibodies. A number of arches will appear, one for each antigen in the sample, provided that the corresponding antibody is present in the antibody preparation. In rocket immunoelectrophoresis [2], specific antibodies are evenly distributed in an agarose gel, the samples are applied into sample wells punched out in this gel and a current is immediately passed through the gel. The antigens will migrate through the gel, and the antigen corresponding to the antibody in the gel will precipitate along the edges of the migrating sample, forming a peak when all antigen has been precipitated. The precipitate will appear with contours like that of a rocket, which led to the name of the method. The area enclosed by the precipitation line, or the peak height, is nearly proportional to the amount of antigen applied in the sample well. In crossed immunoelectrophoresis [3] the antigens are separated by agarose gel electrophoresis. The samples are applied in narrow application slits and, after electrophoresis, thin strips are cut out from the separation tracks and transferred to another plate.
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Antibody-containing gel is poured along the strips and a current is passed through these plates perpendicular to the direction of the current in the initial separation. A series of precipitates will occur in the antibodycontaining gel, each revealing the distribution of the corresponding antigen. This technique is primarily used for studying the heterogeneity of antigens and is only semi-quantitative, as not all of the antigen is transferred to the second-dimension plate. To obtain complete quantitative information, the crossed immunoelectrophoresis introduced by Laurell [3] was modified by Clarke and Freeman [4],who applied the sample in a circular application well and transferred the entire separation track to the seconddimension plate. They showed that the area enclosed by the precipitation line is proportional to the amount of antigen applied in the sample well. Kr6ll introduced line immunoelectrophoresis [5]. In this method, the antigens are evenly distributed in a rectangular gel slab in conjunction with the antibody-containing gel. A current is passed through the gels, and the precipitation lines will appear as straight lines parallel to the antigen-containing gel. The distance between the lines and the edge of the gel that contained the antigens at the start of the experiment is proportional to the amount of antigen introduced into the sample gel. Rocket immunoelectrophoresis according to Laurell [2], crossed immunoelectrophoresis according to Clarke and Freeman [4] and line immunoelectrophoresis according to KrQll [5] are the basic methods of quantitative immunoelectrophoresis. These three methods yield quantitative information that is proportional to the antigen concentration and inversely proportional to the antibody concentration. By combining and modifying these principles a great variety of methods have been developed for application in both routine clinical studies and research [6-91. This chapter describes the use of quantitative immunoelectrophoresis as a tool for following the fractionation of an antigen to purity as an example. An apparatus for conducting the experiments and the chemicals and solutions used are discussed. CHEMICALS AND SOLUTIONS Chemicals The following chemicals are used: 2-amino-2(hydroxymethyl)propane-l,3-diol(Tris), calcium lactate, 5,5-diethylbarbituric acid (Diemal), sodium azide, glacial acetic acid, sodium chloride, agarose Type H S A, Coomassie Brilliant Blue R 250, ethanol (96%), rabbit anti-human serum immunoglobulins, rabbit anti-human transferrin immunoglobulins. Solutions Tris-Diemal buffer, pH 8.6,ionic strength 0.1 (stock solution): 5,5-Diethylbarbituric acid (Diemal) 224 g 443 g Tris Calcium lactate 5.33 g Sodium azide 10 g Distilled water to 10 1
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(Dilute 1 part of stock solution plus 4 parts of distilled water to obtain an ionic strength of 0.02) The chemicals dissolve quickly in 5 1 of distilled water with magnetic stirring and no heating is necessary. This buffer is essentially the same as that recommended elsewhere (ref. 6, p. 25), except that the sodium ion has been replaced with Tris, thus increasing the buffering capacity several fold. There were some problems with the earlier buffer, as the buffering capacity often was exhausted, and as a consequence the pH in the cathodic vessel increased to over 11, resulting in dissolution of the immunoprecipitates from the cathodic side. This new buffer does not alter the immunoprecipitin pattern in quantitative immunoelectrophoresis, but stabilizes the reproducibility. 1 % Agarose in Tris-Diemal buffer, pH 8.6, ionic strength 0.02: Agarose ( 2 g) is added to 200 ml of the diluted buffer and dissolved by gentle heating with magnetic stirring. To ensure that the agarose is completely dissolved the solution should boil for 5-6 min. The solution is kept fluid at 56°C in a water-bath and is ready for use after temperature equilibration. Agarose (1%) can be stored at 4°C for several weeks and liquified repeatedly by heating. The agarose should exhibit an MR value of 0.1 3 to obtain good results with rabbit antibodies and Tris-Diemal (pH 8.6, ionic strength 0.02). Under these circumstances the average migration velocity of rabbit antibodies is close to zero, The MR value is a measure of the electroendosmosis and is obtained by subjecting a mixture of human albumin and polydextran to electrophoresis in the actual system. The shift of the polydextran is measured (the migration direction taken into consideration: negative for a cathodic shift, positive for an anodic shift), and this distance is related to the total distance between the polydextran and human albumin. The Litex agarose used in this laboratory is guaranteed to maintain the same electroendosmosis from batch to batch, which for Type H S A is 0.13%. 0.1 M sodium chloride for washing excess of protein out of the plates after immunoelectrophoresis. Staining solution: 4500 ml Ethanol (96%) Distilled water 4500 ml 1000ml Glacial acetic acid Coomassie Brilliant Blur R 250 50 g The dye is dissolved and the solution is heated to 6OoC, cooled to room temperature and finally filtered through filter-paper. Destaining solution: Ethanol (96%) 4500 ml Distilled water 4500 ml Glacial acetic acid 1000ml The destaining solution can be used repeatedly if filtered through activated charcoal after use.
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Antibodies Crude antisera can always be used directly in experiments. However, to obtain low background staining, a crude antibody preparation should be subjected to salting-out [250g of (N&)2S04 per 1000ml of rabbit anti-serum], dialysis and chromatography on DEAE-Sephadex A-50at pH 5 .O in sodium acetate-acetic acid buffer with an ionic strength of 0.05.The purified gamma-globulin fraction is then dialysed against 0.1 M NaC1-15 mM NaN3. This preparation will lose less than 2% of its activiiy per year when stored at 4OC. (For further details, see Chapter 23 of ref. 6.)
APPARATUS AND ACCESSORIES We use 1mm thick glass plates to support the agarose gels. These glass plates are made in the following standard sizes: 5 x 5 , 7 x 7 , 7 x 10,lO x 10 and 11 x 20.5 cm. The apparatus shown in Fig. 7.1A was therefore designed in such a fashion that it will accommodate several of the standard glass plates on the cooled surface (2 in Fig. 7.1B). The cooled surface measures 22 x 12 cm and is capable of holding one 1 1 x 20.5 cm plate, two 10 x 10 cm plates, three 7 x 10cm plates, three 7 x 7 cm plates or eight 5 x 5 cm plates. The design is similar to that described by Johansson [lo], and cooling is obtained by a serpentine-shaped cooling channel cut in a thick Perspex plate, leaving 1 mm of material between the cooling channel and the cooling surface. With a flow-rate of 1 l/min the cooling capacity of the apparatus is sufficient for quantitative immunoelectrophoresis. The temperature of the cooling water should be 10-1SoC. The experiments described below were conducted at a temperature of the cooling water of 15OC. The cooling water is circulated by a cooling thermostat. The cooling plate is attached to two supports (4in Fig. 7.1 B), with moulds attached for casting agarose gel connections to the electrode vessels (1 in Fig. 7.1B), each containing 1 1 of Tris-Diemal buffer (pH 8.6, ionic strength 0.02). The inner walls of the moulds are level with the cooling surface, and the outer walls of the moulds are 1 cm higher than the cooling surface and are level with the end pieces attached to the cooling plate. This central unit is placed in the electrode vessels, which are separated by a Perspex wall. When the lid (5 in Fig. 7.1B), is attached, the platinum-wire electrodes supported by PVC plates (3 in Fig. 7.1B), are automatically connected to the wire and jack-plugs (6 in Fig. 7.1B). The lid rests on the edge of the central unit and is approximately 1 mm from the upper edge of the electrode-vesselunit, thereby sealing the electrophoresis chamber completely from the surrounding air. Even when filter-paper wicks are used to connect the agarose gel plates to the electrode buffer, the gel moulds should not be removed from the central unit. As these moulds are immersed in the electrode buffer, they ensure complete sealing of the electrophoresis chamber. It is an advantage to keep the volume of the electrophoresis chamber small and well sealed from the surrounding air, including that above the electrode buffer, as water would otherwise condense on the cooled agarose gel on humid days or with very low cooling temperatures. If water condenses on the immunoelectrophoresis plate during the experiment, distorted immunoprecipitates will be formed. The lid (5 in Fig. 7.1B) is furnished with five pairs of holes 4cm apart, which provide
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Fig. 7.1. (A) Electrophoresis apparatus for quantitative immunoelectrophoresis, assembled. (B) Main parts of electrophoresis apparatus. 1 = Electrode vessels; 2 = cooled surface; 3 = electrodes on PVC supports; 4 = support with moulds for agarose gel connections; 5 = lid with electric connectors; 6 = jack-plugs for power supply.
access for a test probe (3 in Fig. 7.2) that is used to measure the potential gradient directly in the gel when the current has been switched on. The test probe is connected to the power supply (1 in Fig. 7.2), and a switch on the power supply sets the voltmeter so that the potential gradient can be read directly in volts per centimetre. At the same time, the correct polarity of the current is checked. The electrophoresis unit (2 in Fig. 7.2) is connected to one of the four power outlets on the power supply. Three channels in the power supply have the capacity of delivering 300V and lOOmAd.c., which is sufficient for quantitative immunoelectrophoresis. The fourth channel can deliver 300 V and 200 mA d.c., for use with buffers of higher ionic strength, e.g., electrophoresis in agarose gel using Tris-Diemal buffer (pH 8.6, ionic strength 0.075).
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Pig. 7.2. Apparatus and accessories for quantitative immunoelectrophoresis. 1 = Power supply; 2 = electrophoresis apparatus; 3 = test probe for measuring the potential gradient; 4 = adjustable glass table for casting gels; 5 = device for levelling electrode buffers; 6 = gel punchers; 7 = adjustable template for punching samples wells; 8 = vessel for washing, staining and destaining; 9 = racks for holding glass plates; 10 = U-shaped frame for casting gels between glass plates; 1 1 = long razor blades for cutting and transferring gel slabs; 12 = slit formers.
The gels are usually cast on levelled glass plates, and for this purpose a levelling glass table (4 in Fig. 7.2) is used in combination with a precision spirit level. To ensure that no hydrodynamic flow occurs during immunoelectrophoresis due to a different level of the buffers in the electrode vessels, the buffer in the vessels is levelled by means of a U-tube (5 in Fig. 7.2). The U-tube is removed before immunoelectrophoresis. Through a template (7 in Fig. 7.2), the sample wells are punched out by means of a puncher [ 1 I ] (6 in Fig. 7.2), which in a single operation punches the well and removes the agarose gel plug. The puncher is connected to a suction device, and is made of two annular steel tubes. The outer tube has a sharp edge and cuts the gel on impact with it. The inner tube, which is spring-loaded, slides downwards when a mechanical pressure is applied, and sucks up the agarose gel plug. Slits in the outer tube serve as air inlets to release the vacuum introduced through the inner tube, thus avoiding damage to the walls of the well. The template is made of a base plate to which a sliding ruler is attached. The ruler has a pattern of holes with the same diameter as the upper part of the puncher, for punching all the patterns of sample wells needed in quantitative immunoelectrophoresis. The punchers are available for sample wells with diameters of 2.0,2.5,3.0 and 4.0 mm. Racks (9 in Fig. 7.2) for holding the various plate sizes fit into the vessels (8 in Fig. 7.2) for washing, staining and destaining purposes. For rocket immunoelectrophoresis on 1 1 x 20.5 cni glass plates, a matrix is made of a 1.5 mm thick U-shaped frame (10 in Fig. 7.2), set between two glass plates, held together by strong paper clamps. With a pipette the antibody-containing solution is poured into the matrix. If the rear glass plate is shifted a few millimetres relative to the front glass plate, the filling is greatly facilitated. In two-dimensional immunoelectrophoresis agarose gel slabs are transferred from one glass plate to another, by using long razor blades (1 1 in Fig. 7.2) measuring 2 x 15 cm. If narrow slits are desired, slit formers (1 2 in Fig. 7.2) are used. The slit formers rest on four legs, which are adjusted in such a way that the slit-forming blades are approximately
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I Fig. 7.3. Cross-sectional area of double constriction pipette.
0.2 mm from the glass plate. This prevents the sample from leaking between the gel and glass plate, as the slit is sealed in the bottom. When the gels have been poured on to the levelled glass plates, the slit formers are placed into the gels before they gel, and are removed just before sample application. Sample application is performed by using double constriction pipettes (Fig. 7.3). The double constriction pipette has the advantage over the single constriction pipette of not being fully emptied. This prevents air being blown into the filled wells, causing overflow and contamination of the surrounding gel. The double constriction pipette is filled to the upper constriction and emptied by blowing gently until the meniscus reaches the lower constriction. The tip of the pipette must touch the bottom of the sample well in order to deliver precise and reproducible volumes. These pipettes are available in various sizes from 1 p1 to several millilitres.
PRACTICAL APPLICATION Quantitative immunoelectrophoresis is an almost indispensable tool for following the fractionation of a protein t o purity, and for testing the isolated material for purity. Antibodies to the crude antigen mixture, in the following human serum, must be available; if not commercially available it may be raised in rabbits, following the procedure described in Chapter 23 of ref. 6 . The purification of human serum transferrin is described below. The first step is to investigate the crude antigen mixture by crossed immunoelectrophoresis according to Clarke and Freeman [4]. Procedure for crossed immunoelectrophoresis A 10 x 10 cm glass plate is washed with detergent, rinsed with ethanol and dried. To obtain a 1.5 mm thick agarose gel, 15 ml of agarose solution at 56°C is poured on to the glass plate, placed on the levelled glass table. After congelation of the agarose, five wells are punched out by means of a 2.5-mm gel puncher using the template shown in Fig. 7.4A. A 2 4 volume of human serum is applied into the sample well(s) S1 to S4 using a double constriction pipette. The fifth well, M, is used to apply a marker, e.g., albunin stained with bromophenol blue. The glass plate with the agarose gel is placed on the cooling surface of the electrophoresis apparatus. A conducting connection to the electrode vessels is obtained with buffer-saturated filter-paper wicks (Whatman No. 1, eight layers, 10 x I0 cm). It is important to ensure that the wicks have good contact with the gel. Electrophoresis in the first dimension is run with a field strength of 15 V/cni. This is tested in every run with a test probe in order to obtain the same potential gradient in the runs. The tinie for the run in the first dimension is approximately 55 min at I5'C. The electrophoresis is discontinued when the blue marker has migrated 5.5 cm. The agarose
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Fig. 7.4. Crossed emunoelectrophoresis. (A) Template for the first-dimensiongel. (B)Template for the seconddimension plate. The hatched area indicates the antibodycontaining gel. (C) Crossed immunoelectrophoresis of 2 pl of human serum into l000pl of anti-human serum (1 2.5 pl/cmagel).
gel is cut according to Fig. 7.4A following the dotted lines. The track with the marker is discarded. The first-dimension gel slabs are then transferred with a long razor blade to the seconddimension glass plates, which have been coated with agarose solution and dried (see Fig. 7.4B). The glass plates are then placed on the levelled glass table, and 12 ml of antibodycontaining solution (1 1ml of 1%agarose 1 ml of antihuman serum) are poured on to the upper part of the glass plate, corresponding to the shaded area in Fig. 7.4B. After 10 min the gel is ready, and the plate is placed on the electrophoresis apparatus and connected to the electrode buffer by means of filter-paper wicks (five layers of Whatman No. 1,lO x 10 cm). Immunoelectrophoresis in the second dimension is performed at 3 V/cm for 18-20 h. After electrophoresis, non-precipitated proteins must be removed, which can be ,effected efficiently by pressing the gel under six layers of filter-paper. Air trapped under
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the filter-paper will damage the gel, and therefore the application wells must be filled with distilled water prior to the pressing procedure. The first layer of filter-paper is applied wet, followed by five layers of dry filter-paper. A 6-8 mm thick glass plate should be placed on top of the filter-paper to sustain a smooth pressure all over the gel. After 5 min the upper five layers of filter-paper are renewed and the gel is pressed for another 10 min. A polyspecific antibody preparation of high titre usually contains a larger amount of proteins than a monospecific reagent. Therefore, to obtain low staining of the background, the plate should be rinsed for I 5 min with 0.1 M sodium chloride solution, pressed under filter-paper and rinsed for a further 15 min in distilled water. After a final pressing, the filter-paper is moistened with distilled water and removed. The plate is dried in a stream of hot air and then stained in Coomassie Brilliant Blue R 250 for 5 min, rinsed three times with ethanol-water-acetic acid and dried. Many enzymes remain active in immunoprecipitates, and if enzymes are to be stained with specific enzyme substrates the plate should be dried in cold air and developed before normal protein staining. Crossed immunoelectrophoresis performed as described here with 2 pl of human serum and 1 ml of anti-human serum is shown in Fig. 7.4C. Identification of the antigens causing the individual precipitates is performed immunochemically by using monospecific antibodies or pure antigens if available. Alternatively, the antigens can be identified by histochemical methods such as staining for lipid or staining for enzymatic activity, or by autoradiography for the detection of the sepcific binding of radioactively labelled substances. With Clarke and Freeman’s modification of Laurell’s crossed immunoelectrophoresis, individual proteins are quantitated by measuring the area below the corresponding peak. Quantitation by measuring the peak height may not always be possible as many of the peaks are asymmetric, and therefore the peak area is measured electronically. The stained plate is enlarged nine times in an enlarger, and with a light-spot planimeter the curves are integrated. The areas are expressed in arbitrary units on an electronic counter, and when compared with a standard serum the method can be used for quantitative determinations. The next step in the fractionation experiment is to establish which of the numerous peaks appearing on the plate in Fig. 7.4C corresponds to transferrin. This can be done immunochemically by employing the intermediate gel technique introduced by Svendsen and Axelsen [12], if a monospecific antibody preparation is available.
Procedure for crossed immunoelectrophoresis with an intermediate gel The firstdimension agarose gel electrophoresis and transfer of the agarose gel slabs t o the coated seconddimension glass plates is the same as for crossed immunoelectrophoresis. The casting of the antibody-containing gels is shown in Fig. 7.5A. A 1 mm thick glass plate is placed 2.2 cm from the first-dimension gel slab, along the dotted line. A 3.2-ml volume of agarose solution mixed with 75 p1 of anti-human transferrin is poured into the open space between the gel and the glass plate. A reference plate is made in parallel, replacing the anti-human transferrin with 0.1 M sodium chloride solution. When the gel has solidified, the glass plate is cut free, the gel is trimmed along the solid line below the dotted line, and the thin gel strip is discarded. An 8.2-ml volume of agarose solution is mixed with 800pl of anti-human serum and poured on to the remaining part
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Fig. 7.5. Crossed immunoelectrophoresk with an intermediate gel. (A) Template for the seconddimension plate. (B) Reference plate; 75 pl of 0.1 M NaCl were added to the intermediate gel. (C) 75 pl of anti-human serum transferrin were added to the intermediate gel. (B) and (C) 2 p1 of human serum were applied in the sample well. Antibody: 8 0 0 ~ of 1 anti-human serum in the upper gel.
o f t h e glass plate, and the same is done on the reference plate. After immunoelectrophoresis, rinsing, pressing, drying and staining the plates are ready (Fig. 7.5B and C). On the plate in Fig. 7.5B all antigens have passed freely through the intermediate gel. The skewed baseline of the immunoprecipitates is due to decreasing mobilities of the antigens and electroendosmotic transport of slowly migrating antibody molecules. On the plate in Fig. 7.4C all antigens except transferrin have passed through the intermediate gel. As transferrin has been trapped in the intermediate gel, it is missing from the upper gel. A simple comparison of the two plates easily indicates which of the peaks in the reference plate corresponds to transferrin. This peak is marked with a heavy vertical arrow, and the transferrin peak is marked in the same way in all subsequent immunoelectrophoresis plates. The horizontal line in Fig. 7.5C is caused by excess of antigen used to absorb the antibody preparation to make it monospecific. For further details, see Chapter 7 of ref. 6. We have now established which of the peaks in crossed immunoelectrophoresis
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Fig. 7.6. Crossed immunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well. (B) 5 PI of the supernatant from the salting-out experiment described in the text were applied to the sample well. (B) and (C): antibody: lOOOpl of anti-human serum.
corresponds to transferrin. The first step in the fractionation procedure is salting-out with (NH4)$04. To 218 ml of human serum (1000 donor pool) were added 54.3 g (NH4)*S04 and 10.9 mg of FeS04. This mixture was left at room temperature overnight with magnetic stirring, and then centrifuged at 20,000 r.p.m. for 20min. The supernatant was dialysed against water, and finally equilibrated against sodium acetate-acetic acid buffer (pH 5.6, ionic strength 0.05). The supernatant was subjected t o crossed imniunoelectrophoresis and compared with serum (see Fig. 7.61, and was then applied to a column containing 425 ml of DEAE-Sepharose CL-6B, which was equilibrated with the same buffer. The proteins were eluted by increasing the concentration of the buffer to an ionic strength of 0.7 (linear gradient), and fractions were collected in an LKB Ultrorac fraction collector at 2 O C , flow-rate 30 ml/h, one fraction per hour. After the experiment, 68 fractions were collected and analysed by fused rocket immunoelectrophoresis [ 13, 141, which is superior to W monitoring as an elution profile for each individual antigen is obtained in a single experiment. UV monitoring is employed for detecting only where the UV-absorbing substances have been collected. Procedure for fused rocket immunoelectrophoresis A 10 x 10 or 1 1 x 20.5 cm glass plate is used. Volumes for a 1 0 x 10 cm plate are given first, followed by volumes for an 11 x 20.5 cm plate in parentheses. A 10 x 10 cm coated glass plate is placed on the levelled glass table and a 1 mm thick glass plate is placed 2.7 cm from the lower edge of the coated glass plate along the dotted line in Fig. 7.7A.A 4.1 (8.3)ml volume of 1% agarose solution is poured on to the plate and allowed t o congeal. The glass plate is cut free and the gel is trimmed following the solid line below the dotted line in Fig. 7.7A. A 10 (23) ml volume of agarose solution is mixed with 1 (2)ml of anti-human serum, poured on to the remainder of the glass plate and allowed to congeal. Sample wells are then punched out through the template (7 in Fig. 7.2), according
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Fig. 7.7. Fused rocket immunoelectrophoresis. (A) Template for casting the gel and punching the sample wells. (B) Immunoelution profile from ion-exchange chromatography on DEAE-Sepharose C L 6 B ; 2-pl aliquots from the collected fractions were applied in the sample wells. Closed circles indicate the fractions pooled. Antibody: 2000 ~1 of anti-human serum.
to Fig. 7.7 in the antibody-free gel. Thirty-eight sample wells can be punched in a 10 x 10 cm plate and 79 in an 11 x 20.5 cm plate. Samples of constant volume from the collected fractions are then transferred to the sample wells, in the same order as they were collected, by means of a double constriction pipette. The pipette is not rinsed between the individual applications. The plate is left for diffusion for 60 min on the cooled surface in the electrophoresis apparatus. At room temperature in a humid chamber, 30 min will suffice. The diffusion step will cause a continuous precipitation line to be formed for each antigen in the sample, over the sample wells corresponding to the fractions in which they were eluted. The plate is then connected to the electrode buffer by means of five layers of fdter-paper, and immunoelectrophoresis is conducted at 3 V/cm overnight (1 8-20 h). After electrophoresis the plate is pressed, washed, stained and dried as described above. Fig. 7.7B shows fused rocket immunoelectrophoresis of the fractions collected from the DEAE-Sepharose CG6B experiment. A number of elution profiles appear, and
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Fig. 7.8. Crossed immunoelectrophoresis; 5 pl of the pool from the ion-exchange experiment on DEAE-Sepharose CLdB were applied in the sample well. Antibody: 1000 PIof anti-human serum. Fig. 7.9. Fused rocket immunoelectrophoresis; 1-pl aliquots of the fractions from the ion-exchange experiment on CM-Sepharose CL-6B were applied in the sample wells. Antibody: 2000pI of antihuman serum.
transferrin was detected by the intermediate gel immunoelectrophoresis technique (not shown) and is marked with a vertical arrow. The thinner horizontal arrow indicates haemopexin, w h c h is difficult to separate from transferrin. Haemopexin is marked in the same manner in subsequent plates. The fractions marked with closed circles (Nos. 13-35) were pooled and checked for purity by means of crossed immunoelectrophoresis (Fig. 7.8). The pool (640 ml) was then directly applied to a CM-Sepharose CL-6B ion-exchange column (bed volume 125 ml) equilibrated with the same starting buffer as in the preceding ionexchange experiment. (In Fig. 7.7B it can be seen that transferrin passed the matrix without being bound.) The proteins were eluted by increasing the concentration of the buffer to an ionic strength of 0.4 (linear gradient). Fractions were collected in an LKB Ultrorac fraction collector at 2"C, flow-rate 30 ml/h, one fraction per hour. Fortynine fractions were collected and subjected t o fused rocket immunoelectrophoresis and the result is shown in Fig. 7.9. The fractions marked with closed circles (Nos. 38-43) were pooled and tested for purity by crossed immunoelectrophoresis (Fig. 7.10). The amount of impurities had decreased, but a few can still be seen. Preparative isotachophoresis [ 14,15J was then applied to remove the last traces of impurities, especially haemopexin. The pool (1 64 ml) from the experiment on CM-Sepharose CLdB was concentrated to 25 ml on an Amicon filter and dialysed against the terminator, Tris6-aminocaproic acid (pH 8.9). The leading electrolyte was Tris-phosphate (pH 8.1). The supporting medium was 5% polyacrylamide (5% cross-linking) with N,N'-methylenebisacrylarnide as cross-linking agent, cast in the glass column, cross-sectional area 4.0 cm', with central cooling in the apparatus described in ref. 15. A 120O-pl volumeof Ampholine (pZ range 6-8) was added to the sample as spacers. The experiment was run overnight at 5°C with a constant current of 10mA d.c. The elution buffer was Tris-sulphate (pH 7.1, flow-rate 30ml/h). Fractions were collected every 20min. For more details, see ref. 15.
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Fig, 7.10. Crossed immunoelectrophoresh; 2 pl of the pool from the ion-exchange experiment on CMSepharose CLdB were applied in the sample well. Antibody: 1OOOwl of anti-human serum.
Fig. 7.11. (A) Fused rocket immunoelectrophoresis; 1-pl aliquots of the fractions from the isotachophoresis experiment were applied in the sample wells. Antibody: 1OOOwl of anti-human serum. (B) Crossed immunoelectrophoresis; 2 p1 of the pool from the isotachophoresis experiment were applied in the sample well. Antibody: 1000 pi of anti-human serum.
Fused rocket immunoelectrophoresis was employed to analyse the collected fractions, and the plate is shown in Fig. 7.1 1A. It appears that fractions 22-27 can be pooled with transferrin in very high purity. The pool was then checked in crossed immunoelectrophoresis (Fig. 7.1 1B) and only one peak can be seen, except for an extremely small trace of impurity, marked with a thin vertical arrow. To remove the Ampholine collected with the protein, as well as polymers and aggregates, the pool (56 ml) from the isotachophoretic experiment was concentrated to 15 ml and applied on an Ultrogel ACA 44 gel filtration matrix (gel bed 2000 ml) and eluted at 60 d / h . The buffer was sodium phosphate (PH 7.2, ionic strength 0.415). The collected fractions were analysed by fused rocket immunoelectrophoresis, and from the result in Fig. 7.12A it is evident that a small
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Fig. 7.12. (A) Fused rocket immunoelectrophoresis; 1-p1 aliquots of the fractions from the gel filtration experiment on Ultrogel ACA 44 were applied in the sample wells. Antibody: 100Opl of antihuman serum. (R) Crossed immunoelectrophoresis; 4 p1 of the pool from the gel filtration experiment were applied in the sample well. Antibody: l000pI of anti-human serum.
Fig. 7.13. Rocket immunoelectrophoresis. (A) Template. (B) Left: 5-111aliquots of the pool from the gel filtration experiment on Ultrogel ACA 44 diluted 1 20 were applied. Right: 5-111 aliquots of the starting material (human serum, lOOOdonor pool) for the fractionation experiment described in the text diluted 1 t 50, 1 + 20, 1 + 10 and 1 + 5. Antibody: loop1 of anti-human transferrin.
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impurity has been separated from the transferrin. Fractions 23-29 were pooled (note how precisely fractions can be selected) and finally crossed immunoelectrophoresis of this pool revealed that immunochemically pure human serum transferrin was isolated (Fig. 7.12B). The volume of this pool was 200ml. The recovery of transferrin compared to the starting material can now be measured by Laurel1 rocket immunoelectrophoresis.
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Procedure for rocket immunoelectrophoresis
A 1 0 x 10 cm coated glass plate is placed on the levelled glass table, I 5 ml of agarose solution are mixed with loop1 of anti-human transferrin and the mixture is poured on to the glass plate. When the gel has solidified after approximately 10min the sample wells are punched out through a template according to Fig. 7.13A. When using an 11 x 20.5 cm glass plate, a matrix is made of a 1.5 mm thick U-shaped frame set between two glass plates (1 1 x 20.5 cm, one being coated), held together by strong paper clamps. With a pipette the antibodycontaining solution at 56°C (30 ml) is poured into the matrix. After lOmin at 4"C, the gel is ready, and the uncoated glass plate is removed by gently sliding it off. The U-shaped frame is kept on the glass plate until the pressing and staining procedure. An aliquot of the sample, in this instance the pool from the gel filtration experiment, is diluted as recommended by the antibody supplier, for human serum transferrin 1 20 with 0.1 M sodium chloride solution. To obtain a standard graph, aliquots from the starting material, in this instance the 1000-donor pool from which 1 ml was saved, are diluted 1 5 0 , l q.20, 1 10 and 1 + 5. The four standards are applied with a 5-pl double constriction pipette into four of the sample wells, and the sample dilution is applied in one or more sample wells, also in 5-4 aliquots. Immunoelectrophoresis is immediately started with a field strength of 3 V/cm, and the experiment is run for 18-20 h. After electrophoresis the plate is squeezed under filter-paper. No washing is necessary when using the immunoglobulin fraction of the antiserum rather than the total antiserum, if monospecific antibodies are employed. Therefore, the plate can be dried after pressing it once, stained for 5 min in Coomassie Brilliant Blue R 250, washed three times with destaining solution and dried. The plate is now ready for interpretation (see Fig. 7.13B). If the dilution 1 + 20 is assigned to represent loo%,then the other dilutions will represent the following: 1 5 = 350%, 1 1 0 = 191% (1 + 20 = 100%) and 1 50 = 41%. Laurel1 rocket immunoelectrophoresis offers an almost linear correlation between the antigen concentration and the area delineated by the precipitation line, and as this area is nearly proportional to the peak height the latter is used for the calculation, The peak heights are easily determined on graph paper, measured from the upper edge of the sample well, and a standard graph is drawn with the antigen concentration as the abscissa and the peak height as the ordinate. The peak height of the unknown is measured and interpolated on the diagram. The standard graph obtained from the plate in Fig. 7.13B is shown in Fig. 7.14. The interpolation for calculating the concentration in the transferrin pool is indicated by arrows, and the result is that the pool has a concentration of 45% relative to the starting material and the total yield is 41%. If corrections are made for samples taken during the fractionation and the known loss introduced by omitting to wash the precipitate in the salting-out experiment, then the recovery is 51%. If the precipitate is washed, the total yield would be 48%, so washing the precipitate defmitely has a significant effect on the recovery. As mentioned above, the peaks in crossed immunoelectrophoresis can be identified if a pure known antigen is available or, vice versa, an unknown antigen can be identified if the peaks in crossed immunoelectrophoresis can be recognized.
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Fig. 7.14. Standard graph for human serum transferrin determined by rocket immunoelectrophoresis. Data from the plate shown in Fig. 7.13B. The arrows indicate the interpolation to obtain the concentration of human transferrin in the pool from the gel fitration experiment on Ultrogel ACA 44. The dilution 1 + 20 is set as 100%.
This can be achieved simply by performing crossed immunoelectrophoresis on 2 p1 of human serum, and for the purpose of demonstration by adding 4 pl of the pool containing the purified human serum transferrin to the sample in the sample well. The result of such an experiment is shown in Fig. 7.1 5B. The plate in Fig. 7.15A is a reference plate, where 4 pl of 0.1 M sodium chloride solution were added to the serum in the sample well. By comparing the two plates, the increase in area of the transferrin peak is easily revealed. Another means of identifying an unknown antigen or a precipitation peak by means of a pure antigen is to absorb the antibodies with the antigen. A 2 - 4 volume of human serum was subjected to crossed immunoelectrophoresis, and 100 pl of the pool containing the pure human serum transferrin were added to the antibody-containing agarose solution before casting the gel. A reference plate was made in parallel, replacing 1OOpl of transferrin with lOOplO.1 Msodium chloride solution. Fig. 7.16B shows the resulting plate, and on comparison of this with the reference plate in Fig. 7.16A, the increase in the area of the transferrin peak is obvious. The background of the plate in Fig. 7.16B is dark, owing to the insoluble transferrinlanti-transferrincomplexes formed by the addition of transferrin to the antibodies. This effect can be prevented by centrifugation of the absorbed antibodies before addition to the agarose solution. However, larger volumes must be used in this instance in order to maintain the precision. Crossed-line immunoelectrophoresis [ 161 can be used for the same purpose.
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Fig. 15. Crossed immunoelectrophoresis. (A) 2 pl of human serum + 4 pl of 0.1 M NaCl were applied in the sample well. (B) 2 p1 of human serum t 4 pl of the pool from the gel filtration experiment on Ultrogel ACA 44 were applied in the sample well. Antibody: l O O O p l of anti-human serum.
Fig. 16. Crossed imrnunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well. Antibody: 1000 pl of anti-human serum. (B) 2 p1 of human serum were applied in the sample well. Antibody: 1OOOpl of anti-human serum absorbed with loop1 of the pool from the gel filtration experiment on Ultrogel ACA 44.
Procedure for crossed-line immunoelectrophoresis The first-dimension agarose gel electrophoresis and transfer of the agarose gel slabs t o the coated second-dimension glass plates is the same as for crossed immunoelectrophoresis. A 1 mm thick glass plate is placed 1.2 cm from the first-dimension gel slab (see Fig. 7.1 7) and 1.8 ml of agarose solution mixed with 20 pl of the pool containing the pure transferrin is cast into the open space between the first-dimension gel slab and the glass plate. A reference plate is made in parallel, replacing the transferrin with 20 jd of 0.1 M sodium chloride solution. The glass plate is cut free and the gel is trimmed along the solid
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Fig. 7.17. Template for crossed-line immunoelectrophoresis.
Fig, 7.18. Crossed-line immunoelectrophoresis. (A) 2 pl of human serum were applied in the sample well; 20 p1 of 0.1 M NaCl were added to the gel slab between the first-dimension gel slab and the antibody-containing gel. (B) 2 pl of human serum were applied in the sample well; 20111 of the pool from the gel filtration experiment on Ultrogel ACA 44 were added to the gel between the first-dimension gel slab and the antibody-containing gel. Antibody: 900 pl of anti-human serum.
h e below the dotted line in Fig. 7.17. The thin gel strip is discarded. A 9.5-ml volume of agarose solution is mixed with 900 pl of anti-human serum and cast on to the remaining part of the plate; the same is done with the reference plate. After immunoelectrophoresis, the plate is pressed, washed, dried and stained as for crossed immunoelectrophoresis. The result is shown in Fig. 7.18B. The transferrin peak has been elevated by the transferrin line. If approximately 320jd of the pure transferrin had been included in the antigencontaining gel, the transferrin line would have absorbed all the anti-transferrin and no precipitate would have formed on the plate, i.e., the transferrin peak would have been missing. This method is therefore recommended for removing antibodies to cross-reacting antigens, e.g., when tissue-specific antigens are investigated [ 171.
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Fig. 7.19. Tandem-crossed immunoelectrophoresis. (A) Template. (B) 2 pl of human serum were applied in the sample well to the left; 4 pl of the pool from the gel fitration experiment on Ultrogel ACA 44 were applied in the sample well to the right. The plate was left for diffusion for 60min before electrophoresis in the fmst direction. Antibody: 1000 pl of anti-human serum.
The last method to be described is tandem-crossed immunoelectrophoresis [ 181, which is essentially the same as crossed immunoelectrophoresis, but two samples are applied in the first-dimension gel, the sample wells having a centre-to-centre distance of 10 mm (see Fig. 7.19A). Human serum (2 pl) is applied in the rear sample well and 4 pl of the pool containing the pure human transferrin is applied in the other well in front. Before electrophoresis in the first dimension is started, the plate is left for diffusion for 60 min on the cooled surface of the electrophoresis apparatus at 15'C. For more complex mixtures, a reference plate should be made in parallel, replacing in this instance transferrin with 4 p1 of 0.1 Msodium chloride solution. The diffusion causes the contents of the sample wells to mix partially in the gel. If identical antigens are present in both wells, the corresponding peaks will show reaction of identity, i.e., fuse into a double peak, as shown in Fig. 7.19B. The above methods can be used to analyse and characterize both antigen and antibody mixtures, including the study of partially identical antigens, e.g., comparing antigens from different species or tissues (see Chapter 11 of ref. 6). A prerequisite for employing the methods is that precipitating antibodies must be available or raised and that the antigens have mobilities different from that of the antibodies. If the mobilities of the antigens coincide with that of the antibodies, the former can be modified by carbamylation to change the pZ(see Chapter 20 of ref. 6 ) , and by carbamylation antibodies can be modified to exhibit zero net mobility in agarose at pH 5 , retaining 80%of their antibody titre. The sensitivity of the methods by normal staining with Coomassie Brilliant Blue R 250 is in the range 5-10 ng of antigen applied in the sample well. The sensitivity can be increased 15-30-fold by employing radioactively labelled reagents or enzyme-conjugated antibodies in a sandwich technique, but if the amount of antigen applied in the well is less than 0.3 ng the lower limit for forming a precipitate may be reached [ 191.
REFERENCES
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REFERENCES
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P. Grabar and C. A. Williams, Biochirn. Biophvs. Acta, 10 (1953) 193. C. -B. Laurell, hotides Biol. Fluids, 14 (1967) 499. C. -B. Laurcll, Anal. Biochem., 10 (1965) 3 5 8 . t1. G. M. Clarke and T. Freeman, Protides Biol. I;luids, 14 (1967) 503. J . KrQli, Scand. J . Clin. Lab. Invest., 22 (1968) 11 2. N. H. Axelsen, J . KrQll and B. Weeke, A Manual of Quantitative ImmunoelectrophoresiscMethods and Applications, Universitetsforlaget , Oslo, 1973. N. H. Axelsen, Quantitative lmrnunoelectrophoresis New Developments and Applications, Universitetsforlaget. Oslo, 1975. R . Verbruggen, Clin. Chem., 21 (1975) 5 . C. -B. Laurell, Scand. J. Clin. Lab. Invest., Suppl., 29 (124) (1972). B. G. Johansson, Scand. J. Clin. Lab. Invest., Suppl., 29 (124) (1972) 7. B. Weeke and J . P. Thomsen, Scand. J. Clin. Lab. Invest., 22 (1968) 165. P. J . Svendsen and N. H . Axelsen,J. lmmunol. Methods, 1 (1972) 169. N. M. G . Harboe and P. J . Svendsen, unpublished work (1969). P. J. Svendsen and C. Rose, Sci. Tools, 17 (1970) 13. P. I . Svendsen, in 2. Deyl (Editor), Electrophoresis. A Survey of Techniques and Applications. Part A : Techniques, Elsevier, Amsterdam, Oxford, New York, 1979, Chapter 16. J . Krgll, Scand. J. Clin. Lab. Invest., 24 (1969) 5 5 . E. Bock,J. Neurochem., 19 (1972) 1731. J. KrQll, Scand. J. Clin. Lab. Invest., 22 (1968) 79. A. Ingild, personal communication.