- Email: [email protected]
[25] Quantitation of antigens by densitometric scanning of immunoelectrophoretic precipitates
370
A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y
[25]
[25] Q u a n t i t a t i o n o f A n t i g e n s b y D e n s i t o m e t r i c S c a n n i n g o f Imrnunoelectrophoretic Precipitates By JENS KROLL Densitometric scanning of precipitates formed by immunodiffusion techniques have been used for the quantitation of antigens 1-'~ as well as for the evaluation of reactions of partial identityY This approach has the advantage of being less dependent on monospecificity of antisera and purity of antigen standards compared with procedures based on precipitin reaction in suspension. 8 For the densitometric analysis of complex precipitating systems, it is essential that the line immunoelectrophoretic techniques enable identification and quantitation of the individual components of composite line patterns. 9-11 Materials and Methods Line Immunoelectrophoresis a'l° Principle. Antigens are forced to migrate into an antiserum-containing agarose gel from a uniform rectangular sample gel by electrophoresis at right angles to the origin. Precipitin lines are formed in parallel to the origin after a distance of migration of the antigen front determined by the antibody : antigen concentration ratio. Sets of different precipitin lines developed from samples run side by side can be directly compared as a consequence of the continuity of identical precipitin lines between adjoining patterns. Identification of individual precipitin lines can be achieved by addition of pure antigens or by local absorption with monospecific antisera (Figs. 1 and 2). A template indicating the location of sample A. J. Crowle, " I m m u n o d i f f u s i o n , " 2nd ed., p. 333. Academic Press, N e w York, 1961. z E. L. Becker, Arch. Biochem. Biophys. 93, 617 (196D. a W. G. Glenn, Aeromed. Rev. I, 1 (1968). 4 T. A. E1-Sharkawy and D. Huisingh, Infect. lmmun. 3, 711 (1971). C. Wadsworth, Scand. J. lmmunol. 6, 97 (1977). 6 j. E. Butler and C. A. Leone, Comp. Biochem. Physiol. 25, 417 (1968). 7 p. j. Gaffney, M. Mahmoud, and T. B. L. Kickwood, J. lmmunol. Methods 14, 25 (1977). C. A. Williams, in " M e t h o d s in Immunology and I m m u n o c h e m i s t r y " (C. A. Williams and M. W. Chase, eds.), Vol. 3, p. 94. Academic Press, N e w York, 1970. a j. Kroll, Scand. J. Imrnunol. Suppl. I, 61 (1973). 10 j. Kr011 and M. M. Andersen, J. Immunol. Methods 9, 141 (1975). H j. Kr011, J. lrnmunol. Methods 19, 41 (1978).
METHODS IN ENZYMOLOGY, VOL. 73
Copyright © 1981 by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181973-6
[25]
QUANTITATION OF ANTIGENS
anti-abe ,
@
371
II/ a n t i
IO2o xy
abe
FIG. 1. Schematic illustration of the line-immunoelectrophoretic technique used for the comparison of antigens and antibodies. Unknown antigens (x, y) and reference antigens (a, b, c) placed in adjoining sample gel sections (dashed line) are forced by electrophoresis to migrate into the antiserum gel. This gel is molded in two sections (dashed line) the left containing antibodies against the teference antigens (anti-abc) and the right containing antibodies against the unknown antigens (anti-xy). The circular wells contain reference samples of monospecific anti-c (1) and pure antigen b (2). Identification: In the resulting pattern the individual antigens are precipitated as separate lines designated x, y, a, b, and c. Encircled in the pattern are reactions of identity (3), partial identity (4), and nonidentity (5). It appears that antigen y is identical to c, and x is partially identical to b. The reference antigen sample forms two lines against anti-XY. The cathodic deflection of the upper line caused by local absorption with anti-c (1) shows that this line represents antigen c. Similarly, the anodic deflection of the lower line caused by addition of pure reference antigen (2) shows that this line represents antigen b. The same conclusion can be drawn from the continuity of lines between the patterns developed by the reference antigens against anti-abc and anti-xy. Quantitative estimation: The level of precipitation of the individual antigens is proportional to the antigen:antibody concentration ratio. Thus the unknown sample (xy) has a higher content of antigen c and a lower content of antigen b than the reference sample (abc). It also appears that anti-xy has a higher titer against antigen b and c than the reference antiserum (anti-abc).
antiserum and contact gel sections on a 100 x 200 mm glass plate is shown in Fig. 3.
Equipment Glass plates, 1.5 x 100 x 200 mm Leveling table with a surface area of 200 × 300 mm Erlenmeyer Pyrex glass bottles, 50 and 500 ml Electric heater with magnetic stirring Thermostatted water bath, 40-50 ° Glass tubes, 10 ml
372
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
AG
,5
S FIG. 2. Identification of precipitin lines in composite line patterns. S, rectangular sample gel (1 x 50 x 60 mm) containing 0.2% of human serum AG, antiserum gel containing 1% ofa polyspecific antiserum against human serum proteins, a-d: Circular wells (diameter 2 mm) containing l-/xl samples of 1 : 10 diluted monospecific antisera against a~-macroglobulin (a), cq-antitrypsin (b), orosomucoid (c), and haptoglobin (d). e: Circular well containing 0.6/zg of transferrin. Anode at top. Numbers indicate the precipitates of a~-macroglobulin (1), alantitrypsin (2), orosomucoid (3), haptoglobin (4), and transferrin (5). Precipitates I-4 are identified from the cathodic deflection of the respective lines caused by local specific absorption at the wells a-d. Similarly, identification of precipitate 5 is given by the anodic deflection of this line caused by addition of pure antigen to well e.
Surgical knives and long razor blades, 20 x 120 mm Gel punchers for making circular sample wells, diameter 2 and 4 mm Constriction pipettes, 1, 3, 5, 10, 25, 50, 100, and 500/zl Electrophoresis table with a cooled area measuring 220 × 220 mm Buffer vessels, capacity 1-2 liters Power supply delivering a rectified current and stabilized voltage with an output of approximately 300 V and 100 mA Voltage probe fitted with a pair of platinum electrodes for measurement of the voltage gradient in the gel Staining and destaining vessels Plate holder for fixation of the electrophoresis plates during staining and destaining procedures This type of immunoelectrophoresis equipment is commercially available from several sources, e.g., Holm Nielsen Electrophoresis, ApS, Copenhagen, and Dansk Laboratorieudstyr A/S, Copenhagen. For further details see the survey given by Weeke. 12 12 B. Weeke, Scand. J. lmmunol. Suppl. I, 15 (1973).
[25]
QUANTITATION OF ANTIGENS
373
C
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. . . . . . . . . . . . . .
AG
c
o,~
s
C FIG. 3. Template showing the location and dimensions of the various gel sections for line immunoelectrophoretic comparison of different antigen and antibody samples on a 100 x 200 mm glass plate. C, Contact gel; S, antigen sample gel (1 × 20 x 90 mm); AG, antiserum containing gel (1 x 90 × 120 mm); a and b: circular wells (diameter 2-4 ram). In AG dashed lines demarcate different antiserum sections; in S, different samples.
Reagents Antigens: H u m a n s e r u m and rat ascites fluid proteins. A s t a n d a r d i z e d h u m a n reference s e r u m containing defined a m o u n t s o f 16 s e r u m proteins was o b t a i n e d f r o m B e h r i n g w e r k e A . G . , M a r b u r g , W. Germany. Antisera: Mono- and polyspecific rabbit antisera against h u m a n and rat s e r u m proteins w e r e o b t a i n e d f r o m D a k o - i m m u n o g l o b u l i n s A/S, C o p e n h a g e n , or f r o m B e h r i n g w e r k e Buffers: Barbital buffer A, 0.75 M, p H 8.6, containing sodium barbital 13.14 g/liter; barbital, 2.07 g/liter; and sodium azide, 1 g/liter. Barbital buffer B is a 1 : 4 dilution o f buffer A. Buffer A i~ used in the buffer vessels, and buffer B is used for p r e p a r a t i o n o f the a g a r o s e gel. A g a r o s e : T h e a g a r o s e used should have a m o d e r a t e e l e c t r o e n d o o s mosis flow (Mr 0.13) to p r o v i d e a net zero e l e c t r o p h o r e t i c mobility o f the rabbit i m m u n o g l o b u l i n s . This r e q u i r e m e n t is fulfilled f o r
374
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
LSA agarose, Litex A/S, Copenhagen, or for Indubiose A37, Findustrie Biologique Fran~aise, Paris. The agarose solution is stored for use in a thermostatted water bath adjusted to 3-5 ° above the gelation point of the agarose (37 ° for Indubiose A37 and 42° for LSA agarose). Staining and destaining solutions: The staining solution is Coomassie Brilliant Blue R-250, 5 g/liter dissolved in acid ethanol (equal volumes of 10% glacial acetic acid and 96% ethanol). The same solvent is used as destaining solution. Procedure
1. A 1.5 mm thick agarose gel is formed by pouring 30 ml of 1% agarose solution on a level 100 x 200 mm glass plate. 2. After congelation for 10-15 min, sections of the gel are cut out to form basins for the molding of sample and antiserum gel compartments as indicated on the template (Fig. 3). Gel sections cut free with the surgical knife are removed with the long razor blade. 3. Sample and antiserum gel sections, 1 mm thick, are formed on the level plate by pouring measured volumes of antigen or antibody solutions in 0.5% agarose into the respective basins, allowing one gel section to congeal for 5-10 rain before the next is applied. 4. Reference antigen or antibody samples are placed in circular wells punched out in the contact gel between the antiserum and sample gel sections (Fig. 3). 5. The wells are sealed with a drop of agarose after complete absorption of the samples in the gel. 6. Immunoelectrophoresis is carried out in a thermostatted electrophoresis cell adjusted to provide a gel temperature a few degrees below room temperature. Electrophoresis is carried out at 1.5-3 V/cm for 20-40 hr depending on the electrophoretic mobility of the antigen. Contact with the buffer is mediated by means of agarose bridges or filter paper wicks held in position by a glass rod. 7. After electrophoresis nonprecipitated proteins are removed from the gel by means of three shifts of blotting paper, and a 1 - c m layer of cellulose tissue is placed on the gel under a slight pressure. 8. The plates are dried, stained for 10 min in the solution of Coomassie Brilliant Blue, and finally washed in three changes of the destaining solution. C o m m e n t s on Procedure. The casting of antiserum and sample gel sections requires that the utensils and reactants be prewarmed to a temperature a little above the congelation point of the agarose solution. Antibodies are relatively heat stable, but for the preservation of some an-
[25]
QUANTITATION OF ANTIGENS
375
tigens it may be preferable to use an agarose with a low congelation point, such as Indubiose A377 A relatively weak (0.5%) and thin (1 mm) agarose gel is used in the antiserum and sample gel sections to facilitate the removal of nonprecipitated proteins from the gel after electrophoresis. Immunoelectrophoresis against an antiserum gradient as created by molding the antiserum gel in sections with increasing concentration of antiserum toward the anode (Fig. 3) may improve the conditions for the analysis of composite line spectra. 1° The same principle of electrophoresis against sequential antiserum gel sections can be used for the analysis of patterns developed against different antisera (see Fig. 6). With some experience, molding of sample and antiserum gel sections in basins formed in the contact gel can be achieved with a precision close to that attainable by molding the gel compartments between glass plates, a It is important to avoid gradients in pH or ionic strength across the gel as the resulting electroendoosmotic imbalance may give rise to distortion of the precipitin line patterns. Thus, using high concentrations of antiserum or sample material, dialysis of the reagents against the electrophoresis buffer (B) may be required. The circular sample wells are sealed with agarose to avoid retaining sample material in the bottom of the well and to prevent nonspecific deflections of the precipitin lines caused by heterogeneity of the electric field around the well. Electrophoresis is run at a gel temperature slightly below room temperature to avoid artifacts caused by excess evaporation or condensation of water on the gel.
Densitometric Scanning Stained immunoplates are scanned in transmitted light at 570 nm using an integrating scanning densitometer with a resolution of at least 0.25 mm. Quantitation. Standard curves are made by densitometric scanning of precipitin lines formed by immunoelectrophoresis of serial dilutions of the standard serum against the corresponding mono- or polyspecific antisera as illustrated in Fig. 4A. For comparative analysis of different antigens the density of the respective immunoprecipitates correlates with the molar rather than the mass concentration of the antigens. This relationship holds true for antigens in the molecular weight range 4 to 15 × 104; a separate pattern is observed for immunoprecipitates of the macroglobulins 11 (Fig. 4B). The use of densitometric scanning of precipitin lines as a means for estimation of the relative concentration of individual components in composite antigen solutions is illustrated in Figs. 5 and 6. Sensitivity and Reproducibility. Reproducible line spectra are devel-
376
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
150
100
E u
e
L
50
I 0.5
I 1.0 Protein
I 1.5
I 2.5
I 2.0
iJglml
150
•EIO0 E
00
I
50
I
I
I
I
2
4
6
8
P r o t e i n pM FIG. 4. Optical density of immunoelectrophoretic precipitates of five serum proteins (peak area in square millimeters) plotted against the mass (A) or the molar (B) concentration of the antigens. In (A) individual standard curves are given for orosomucoid (IlL MW 44,000; albumin ([]), MW 69,000; transferrin (©), MW 90,000; immunoglobulin (A), MW 150,000; and a2-macroglobulin (&), MW 800,000.
[25]
QUANTITATION OF ANTIGENS
377
!
.a,
B I
. . . . . .
FIG. 5. (I) Line immunoelectrophoretic comparison of human serum and spinal fluid proteins. AB: Sample gel 1 x 20 z 60 mm molded in two sections (dashed line). Section A contains 30% of spinal fluid (total protein content 0.45 mg/ml) and section B contains 0.2% of serum from the same patient (total protein content 76 mg/ml). AG: Antiserum gel molded in two sections (dashed line), the lower section containing 0.25%, and the upper section 4%, of a polyspecific antiserum against human serum proteins. Immunoelectrophoresis was carried
378
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
II 8
9
7
10
FIG. 5. (Continued)
out at 1.5 V/cm for 20 hr. Anode at top. Numbers indicate precipitates of the following proteins: transferrin (1), IgG (2), haptoglobin (3), c~2-HS-globulin (4), cq-lipoprotein (5), Gc-globulin (6), a~-antitrypsin (7), orosomucoid (8), albumin (9), prealbumin (10), IgA (11), and c~-macroglobulin (12). It appears that the low molecular weight proteins (orosomucoid and prealbumin) constitute a relatively large proportion of spinal fluid proteins, and the high molecular weight proteins (IgA and c~2-macroglobulin) are absent. (II) Densitometric scanning of the spinal fluid precipitin line pattern. Numbers indicate the density peaks of the corresponding precipitin lines. o p e d by i m m u n o e l e c t r o p h o r e s i s o f a c o m p l e x mixture o f antigens (e.g., h u m a n serum) against the c o r r e s p o n d i n g polyspecific antiserum. T h e sensitivity defined as the l o w e r level o f quantitation is about 0.1 /xg/ml. T h e analytical error e x p r e s s e d as the plate-to-plate variation is 4 - 6 % p r o v i d e d a reference precipitate is included in e a c h run to c o m p e n s a t e for plate-toplate differences in staining intensity. Differences in the optical density o f
[9-5]
QUANTITATION OF ANTIGENS
379
/ 432
1
{~
FIG. 6. Densitometric evaluation of the proportion of tumor-associated antigens in Yoshida ascites tumor fluid. Immunoelectrophoresis: S, Sample gel (1.5 x 5 x 12 mm) containing 5% of Yoshida ascites fluid. AG, Antiserum gel (1 x 12 x 100 ram) molded in three sections (dashed demarcation lines); the right section contains 10% and the middle section 2.5% of a polyspecific antiserum against rat serum proteins; the left section contains 5% of a polyspecific antiserum against Yoshida tumor cell-associated antigens [J. Kr011, Protides of Biol. Fluids, Proc. Colloq. 27, 161 (1979)]. Immunoelectrophoresis was carried out at 1.5 V/cm for 40 hr. Anode to the right. Densitometric scanning: This method shows that the tumor-associated antigens (1-4) account for less than 0.5%0 of the total ascites fluid proteins. p r e c i p i t a t e s f o r m e d b y a g i v e n a m o u n t o f t h e s a m e a n t i g e n a g a i n s t different a n t i s e r a f r o m r a b b i t s i m m u n i z e d w i t h this a n t i g e n is w i t h i n t h e a n a l y t ical e r r o r o f t h e m e t h o d . Limitations. T h e r e s o l u t i o n o f c o n v e n t i o n a l d e n s i t o m e t e r s m a y b e insufficient for t h e a n a l y s i s o f c l o s e l y s p a c e d line p a t t e r n s . A l s o d e n s i t o m e t r y m a y b e i m p e d e d in c r o w d e d p a t t e r n s o w i n g to t h e fusion o f different lines. To o p t i m i z e line s e p a r a t i o n , i m m u n o e l e c t r o p h o r e s i s s h o u l d t h e n b e carried out against an antiserum concentration gradient or repeated after subfractionation of the antigens. R e s u l t s o b t a i n e d for e l e c t r o p h o r e t i c a l l y h e t e r o g e n e o u s a n t i g e n s m a y b e m i s l e a d i n g . T h u s for I g G t h e m e t h o d e s t i m a t e s t h e a n o d i c a l l y m i g r a t i n g component only. This problem can be overcome by carbamoylation of the a n t i g e n s . TM
380
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
A
j FIG. 7. The molecular antibody:antigen ratio of immunoelectrophoretic precipitates. The composition of immuneprecipitates of human albumin (A) and orosomucoid (B) is evaluated by SDS-polyacrylamide gel electrophoresis of the dissociated immune complex (representing approximately 5 ~xg of antigen). In the densitometric curves, peak 1 represents the antigen and peak 2 the antibody. Peak 3 is a macromolecular component (MW - 400,000) derived from the antiserum, probably a complement factor (C lq). The density ratio antibody (peak 2): antigen (peak 1) is 14.6 for the albumin complex (A), and 20.1 for the orosomucoid
[25]
QUANTITATION OF ANTIGENS
381
B
2
complex (B). Considering the molecular size of the components (orosmucoid, MW 44,000; albumin, MW 69,000; IgG, MW 150,000), the density values represent a molecular antibody : antigen ratio of 6.7 for the albumin complex and 5.9 for the orosomucoid complex. The latter value probably is an overestimation due to the weak stainability of orosomucoid.
382
A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y
[25]
Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis was done according to Anderson et al. 13 using a gradient gel (7 to 20% polyacrylamide), 0.375 M Tris. HC1, pH 8.7. Samples in 0.08 M Tris • HCI, pH 6.8, containing I% SDS glycerol and 25% urea were heated on a boiling water bath for 5 min before loading. Electrophoresis was done at 20 V/cm and 10° for 4 hr. Figure 7 illustrates the use of this procedure for estimation of the antibody:antigen ratio in precipitin lines cut from the immunoelectrophoresis gel as described elsewhere in this volume. 14 Comments The present approach to a relative determination of antigens has the advantage compared with conventional quantitative immunoelectrophoretic techniques of being less dependent on the availability of defined standards. Compared with polyacrylamide gel electrophoretic techniques for the determination of relative protein concentrations, the present procedure provides a higher sensitivity and improves the conditions for the correlation of individual components in different patterns. The observation that the optical density of the precipitin lines correlates with the molar rather than with the mass concentration of the antigens considered is in accordance with standard quantitative precipitin tests showing that the molecular ratio of antibody to antigen is relatively invariant for antigens in the molecular weight range 4 to 16 x 104.1~ The high antibody : antigen molecular ratio found by polyacrylamide gel electrophoretic analysis of the dissociated immune complex is the ratio found in extreme antibody excess. 15 Probably the initial precipitate is formed at antigen-antibody equivalence, the remaining antigen combining sites being occupied by cathodically migrating antibodies during the electrophoresis. Supporting this notion is the observation that the immune precipitate, once formed, increases in density during continued electrophoresis? 2 Differences in stainability of proteins is a significant source of error in densitometry. The high antibody : antigen molecular ratio in the precipitin lines, however, tends to eliminate this type of error. In fact densitometric ~3 C. W. A n d e r s o n , P. R. B a u m , and R. F. Gesteland, J. Virol. 12, 241 (1973). 14 j. Kroll, this volume [3]. ~ E. A. Kabat and M. M. Mayer, " E x p e r i m e n t a l I m m u n o c h e m i s t r y , " p. 26. T h o m a s , Springfield, Illinois, 1961.
[26]
ULTRASENSITIVE ENZYMIC RADIOIMMUNOASSAY
383
scanning of precipitin lines might be used as a means for the quantitation of nonprotein antigens within the 4 to 16 × 104 molecular weight range provided the same high proportion of antibody protein is present in the precipitate. Acknowledgments The skillful technical assistance of Birthe Larsen, Anna Margrethe Poulsen, Lene Ahrenst, and John Post is gratefully acknowledged.
[26] U l t r a s e n s i t i v e E n z y m i c R a d i o i m m u n o a s y By IH-CHANG
Hsu, ROBERT H. YOLKEN, and CURTIS C. HARRIS
Ultrasensitive enzymic radioimmunoassay (USERIA) 1 combines the advantages of two useful techniques for the measurement of biologically important substances, radioimmunoassay (RIA)2"3 and enzyme-linked immunosorbent assay (ELISA). 4 However, USERIA is more sensitive than either of the parent assays. ELISA is similar in design to solid-phase RIA except that an enzyme is used as the immunoglobulin marker instead of a y-emitting isotope. This enzyme-antibody conjugate is bound to the solid phase by a series of antibody-antigen (Ab-Ag) reactions and converts the enzymic substrate to a visible colored product. ELISA is easier, safer, and less expensive than RIA, and no radioisotopes are required. However, ELISA and RIA have similar levels of sensitivity, which can be a limiting factor. ~Therefore USERIA was devised to provide a more sensitive system. USERIA has application in several areas of biomedical investigation. For example, it has been shown to be 100- to 1000-fold more sensitive than RIA in the detection of cholera toxin and rotavirus. 1 USERIA was also employed to measure covalent binding of chemical carcinogen to DNA. As few as 3 fmol of acetylaminofluorene-DNA (AAF-DNA) adducts 6 and C. C. Harris, R. H. Yolken, H. Krokan, and I.-C. Hsu, Proc. Natl. Acad. Sci. U.S.A. 76, 5336 (1979). 2 j. p. Feber, Adv. Clin. Chem. 20, 130 (1978). 3 j. Thorell and S. Larson, "Radioimmunoassay and Related Techniques: Methodology and Clinical Application." Mosby, St. Louis, Missouri, 1978. 4 E. Engvall and P. Perlmann, J. lmmunol. 109, 129 (1972). 5 R. H. Yolken, Hosp. trrac. 11, 123 (1978). I. C. Hsu, M. C. Poirier, S. H. Yuspa, R. H. Yolken, and C. C. Harris, Carcinogenesis 1, 455 (1980).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 73
,
Copyright (~ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181973-6
A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y
[25]
[25] Q u a n t i t a t i o n o f A n t i g e n s b y D e n s i t o m e t r i c S c a n n i n g o f Imrnunoelectrophoretic Precipitates By JENS KROLL Densitometric scanning of precipitates formed by immunodiffusion techniques have been used for the quantitation of antigens 1-'~ as well as for the evaluation of reactions of partial identityY This approach has the advantage of being less dependent on monospecificity of antisera and purity of antigen standards compared with procedures based on precipitin reaction in suspension. 8 For the densitometric analysis of complex precipitating systems, it is essential that the line immunoelectrophoretic techniques enable identification and quantitation of the individual components of composite line patterns. 9-11 Materials and Methods Line Immunoelectrophoresis a'l° Principle. Antigens are forced to migrate into an antiserum-containing agarose gel from a uniform rectangular sample gel by electrophoresis at right angles to the origin. Precipitin lines are formed in parallel to the origin after a distance of migration of the antigen front determined by the antibody : antigen concentration ratio. Sets of different precipitin lines developed from samples run side by side can be directly compared as a consequence of the continuity of identical precipitin lines between adjoining patterns. Identification of individual precipitin lines can be achieved by addition of pure antigens or by local absorption with monospecific antisera (Figs. 1 and 2). A template indicating the location of sample A. J. Crowle, " I m m u n o d i f f u s i o n , " 2nd ed., p. 333. Academic Press, N e w York, 1961. z E. L. Becker, Arch. Biochem. Biophys. 93, 617 (196D. a W. G. Glenn, Aeromed. Rev. I, 1 (1968). 4 T. A. E1-Sharkawy and D. Huisingh, Infect. lmmun. 3, 711 (1971). C. Wadsworth, Scand. J. lmmunol. 6, 97 (1977). 6 j. E. Butler and C. A. Leone, Comp. Biochem. Physiol. 25, 417 (1968). 7 p. j. Gaffney, M. Mahmoud, and T. B. L. Kickwood, J. lmmunol. Methods 14, 25 (1977). C. A. Williams, in " M e t h o d s in Immunology and I m m u n o c h e m i s t r y " (C. A. Williams and M. W. Chase, eds.), Vol. 3, p. 94. Academic Press, N e w York, 1970. a j. Kroll, Scand. J. Imrnunol. Suppl. I, 61 (1973). 10 j. Kr011 and M. M. Andersen, J. Immunol. Methods 9, 141 (1975). H j. Kr011, J. lrnmunol. Methods 19, 41 (1978).
METHODS IN ENZYMOLOGY, VOL. 73
Copyright © 1981 by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181973-6
[25]
QUANTITATION OF ANTIGENS
anti-abe ,
@
371
II/ a n t i
IO2o xy
abe
FIG. 1. Schematic illustration of the line-immunoelectrophoretic technique used for the comparison of antigens and antibodies. Unknown antigens (x, y) and reference antigens (a, b, c) placed in adjoining sample gel sections (dashed line) are forced by electrophoresis to migrate into the antiserum gel. This gel is molded in two sections (dashed line) the left containing antibodies against the teference antigens (anti-abc) and the right containing antibodies against the unknown antigens (anti-xy). The circular wells contain reference samples of monospecific anti-c (1) and pure antigen b (2). Identification: In the resulting pattern the individual antigens are precipitated as separate lines designated x, y, a, b, and c. Encircled in the pattern are reactions of identity (3), partial identity (4), and nonidentity (5). It appears that antigen y is identical to c, and x is partially identical to b. The reference antigen sample forms two lines against anti-XY. The cathodic deflection of the upper line caused by local absorption with anti-c (1) shows that this line represents antigen c. Similarly, the anodic deflection of the lower line caused by addition of pure reference antigen (2) shows that this line represents antigen b. The same conclusion can be drawn from the continuity of lines between the patterns developed by the reference antigens against anti-abc and anti-xy. Quantitative estimation: The level of precipitation of the individual antigens is proportional to the antigen:antibody concentration ratio. Thus the unknown sample (xy) has a higher content of antigen c and a lower content of antigen b than the reference sample (abc). It also appears that anti-xy has a higher titer against antigen b and c than the reference antiserum (anti-abc).
antiserum and contact gel sections on a 100 x 200 mm glass plate is shown in Fig. 3.
Equipment Glass plates, 1.5 x 100 x 200 mm Leveling table with a surface area of 200 × 300 mm Erlenmeyer Pyrex glass bottles, 50 and 500 ml Electric heater with magnetic stirring Thermostatted water bath, 40-50 ° Glass tubes, 10 ml
372
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
AG
,5
S FIG. 2. Identification of precipitin lines in composite line patterns. S, rectangular sample gel (1 x 50 x 60 mm) containing 0.2% of human serum AG, antiserum gel containing 1% ofa polyspecific antiserum against human serum proteins, a-d: Circular wells (diameter 2 mm) containing l-/xl samples of 1 : 10 diluted monospecific antisera against a~-macroglobulin (a), cq-antitrypsin (b), orosomucoid (c), and haptoglobin (d). e: Circular well containing 0.6/zg of transferrin. Anode at top. Numbers indicate the precipitates of a~-macroglobulin (1), alantitrypsin (2), orosomucoid (3), haptoglobin (4), and transferrin (5). Precipitates I-4 are identified from the cathodic deflection of the respective lines caused by local specific absorption at the wells a-d. Similarly, identification of precipitate 5 is given by the anodic deflection of this line caused by addition of pure antigen to well e.
Surgical knives and long razor blades, 20 x 120 mm Gel punchers for making circular sample wells, diameter 2 and 4 mm Constriction pipettes, 1, 3, 5, 10, 25, 50, 100, and 500/zl Electrophoresis table with a cooled area measuring 220 × 220 mm Buffer vessels, capacity 1-2 liters Power supply delivering a rectified current and stabilized voltage with an output of approximately 300 V and 100 mA Voltage probe fitted with a pair of platinum electrodes for measurement of the voltage gradient in the gel Staining and destaining vessels Plate holder for fixation of the electrophoresis plates during staining and destaining procedures This type of immunoelectrophoresis equipment is commercially available from several sources, e.g., Holm Nielsen Electrophoresis, ApS, Copenhagen, and Dansk Laboratorieudstyr A/S, Copenhagen. For further details see the survey given by Weeke. 12 12 B. Weeke, Scand. J. lmmunol. Suppl. I, 15 (1973).
[25]
QUANTITATION OF ANTIGENS
373
C
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. . . . . . . . . . . . . .
AG
c
o,~
s
C FIG. 3. Template showing the location and dimensions of the various gel sections for line immunoelectrophoretic comparison of different antigen and antibody samples on a 100 x 200 mm glass plate. C, Contact gel; S, antigen sample gel (1 × 20 x 90 mm); AG, antiserum containing gel (1 x 90 × 120 mm); a and b: circular wells (diameter 2-4 ram). In AG dashed lines demarcate different antiserum sections; in S, different samples.
Reagents Antigens: H u m a n s e r u m and rat ascites fluid proteins. A s t a n d a r d i z e d h u m a n reference s e r u m containing defined a m o u n t s o f 16 s e r u m proteins was o b t a i n e d f r o m B e h r i n g w e r k e A . G . , M a r b u r g , W. Germany. Antisera: Mono- and polyspecific rabbit antisera against h u m a n and rat s e r u m proteins w e r e o b t a i n e d f r o m D a k o - i m m u n o g l o b u l i n s A/S, C o p e n h a g e n , or f r o m B e h r i n g w e r k e Buffers: Barbital buffer A, 0.75 M, p H 8.6, containing sodium barbital 13.14 g/liter; barbital, 2.07 g/liter; and sodium azide, 1 g/liter. Barbital buffer B is a 1 : 4 dilution o f buffer A. Buffer A i~ used in the buffer vessels, and buffer B is used for p r e p a r a t i o n o f the a g a r o s e gel. A g a r o s e : T h e a g a r o s e used should have a m o d e r a t e e l e c t r o e n d o o s mosis flow (Mr 0.13) to p r o v i d e a net zero e l e c t r o p h o r e t i c mobility o f the rabbit i m m u n o g l o b u l i n s . This r e q u i r e m e n t is fulfilled f o r
374
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
LSA agarose, Litex A/S, Copenhagen, or for Indubiose A37, Findustrie Biologique Fran~aise, Paris. The agarose solution is stored for use in a thermostatted water bath adjusted to 3-5 ° above the gelation point of the agarose (37 ° for Indubiose A37 and 42° for LSA agarose). Staining and destaining solutions: The staining solution is Coomassie Brilliant Blue R-250, 5 g/liter dissolved in acid ethanol (equal volumes of 10% glacial acetic acid and 96% ethanol). The same solvent is used as destaining solution. Procedure
1. A 1.5 mm thick agarose gel is formed by pouring 30 ml of 1% agarose solution on a level 100 x 200 mm glass plate. 2. After congelation for 10-15 min, sections of the gel are cut out to form basins for the molding of sample and antiserum gel compartments as indicated on the template (Fig. 3). Gel sections cut free with the surgical knife are removed with the long razor blade. 3. Sample and antiserum gel sections, 1 mm thick, are formed on the level plate by pouring measured volumes of antigen or antibody solutions in 0.5% agarose into the respective basins, allowing one gel section to congeal for 5-10 rain before the next is applied. 4. Reference antigen or antibody samples are placed in circular wells punched out in the contact gel between the antiserum and sample gel sections (Fig. 3). 5. The wells are sealed with a drop of agarose after complete absorption of the samples in the gel. 6. Immunoelectrophoresis is carried out in a thermostatted electrophoresis cell adjusted to provide a gel temperature a few degrees below room temperature. Electrophoresis is carried out at 1.5-3 V/cm for 20-40 hr depending on the electrophoretic mobility of the antigen. Contact with the buffer is mediated by means of agarose bridges or filter paper wicks held in position by a glass rod. 7. After electrophoresis nonprecipitated proteins are removed from the gel by means of three shifts of blotting paper, and a 1 - c m layer of cellulose tissue is placed on the gel under a slight pressure. 8. The plates are dried, stained for 10 min in the solution of Coomassie Brilliant Blue, and finally washed in three changes of the destaining solution. C o m m e n t s on Procedure. The casting of antiserum and sample gel sections requires that the utensils and reactants be prewarmed to a temperature a little above the congelation point of the agarose solution. Antibodies are relatively heat stable, but for the preservation of some an-
[25]
QUANTITATION OF ANTIGENS
375
tigens it may be preferable to use an agarose with a low congelation point, such as Indubiose A377 A relatively weak (0.5%) and thin (1 mm) agarose gel is used in the antiserum and sample gel sections to facilitate the removal of nonprecipitated proteins from the gel after electrophoresis. Immunoelectrophoresis against an antiserum gradient as created by molding the antiserum gel in sections with increasing concentration of antiserum toward the anode (Fig. 3) may improve the conditions for the analysis of composite line spectra. 1° The same principle of electrophoresis against sequential antiserum gel sections can be used for the analysis of patterns developed against different antisera (see Fig. 6). With some experience, molding of sample and antiserum gel sections in basins formed in the contact gel can be achieved with a precision close to that attainable by molding the gel compartments between glass plates, a It is important to avoid gradients in pH or ionic strength across the gel as the resulting electroendoosmotic imbalance may give rise to distortion of the precipitin line patterns. Thus, using high concentrations of antiserum or sample material, dialysis of the reagents against the electrophoresis buffer (B) may be required. The circular sample wells are sealed with agarose to avoid retaining sample material in the bottom of the well and to prevent nonspecific deflections of the precipitin lines caused by heterogeneity of the electric field around the well. Electrophoresis is run at a gel temperature slightly below room temperature to avoid artifacts caused by excess evaporation or condensation of water on the gel.
Densitometric Scanning Stained immunoplates are scanned in transmitted light at 570 nm using an integrating scanning densitometer with a resolution of at least 0.25 mm. Quantitation. Standard curves are made by densitometric scanning of precipitin lines formed by immunoelectrophoresis of serial dilutions of the standard serum against the corresponding mono- or polyspecific antisera as illustrated in Fig. 4A. For comparative analysis of different antigens the density of the respective immunoprecipitates correlates with the molar rather than the mass concentration of the antigens. This relationship holds true for antigens in the molecular weight range 4 to 15 × 104; a separate pattern is observed for immunoprecipitates of the macroglobulins 11 (Fig. 4B). The use of densitometric scanning of precipitin lines as a means for estimation of the relative concentration of individual components in composite antigen solutions is illustrated in Figs. 5 and 6. Sensitivity and Reproducibility. Reproducible line spectra are devel-
376
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
150
100
E u
e
L
50
I 0.5
I 1.0 Protein
I 1.5
I 2.5
I 2.0
iJglml
150
•EIO0 E
00
I
50
I
I
I
I
2
4
6
8
P r o t e i n pM FIG. 4. Optical density of immunoelectrophoretic precipitates of five serum proteins (peak area in square millimeters) plotted against the mass (A) or the molar (B) concentration of the antigens. In (A) individual standard curves are given for orosomucoid (IlL MW 44,000; albumin ([]), MW 69,000; transferrin (©), MW 90,000; immunoglobulin (A), MW 150,000; and a2-macroglobulin (&), MW 800,000.
[25]
QUANTITATION OF ANTIGENS
377
!
.a,
B I
. . . . . .
FIG. 5. (I) Line immunoelectrophoretic comparison of human serum and spinal fluid proteins. AB: Sample gel 1 x 20 z 60 mm molded in two sections (dashed line). Section A contains 30% of spinal fluid (total protein content 0.45 mg/ml) and section B contains 0.2% of serum from the same patient (total protein content 76 mg/ml). AG: Antiserum gel molded in two sections (dashed line), the lower section containing 0.25%, and the upper section 4%, of a polyspecific antiserum against human serum proteins. Immunoelectrophoresis was carried
378
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
II 8
9
7
10
FIG. 5. (Continued)
out at 1.5 V/cm for 20 hr. Anode at top. Numbers indicate precipitates of the following proteins: transferrin (1), IgG (2), haptoglobin (3), c~2-HS-globulin (4), cq-lipoprotein (5), Gc-globulin (6), a~-antitrypsin (7), orosomucoid (8), albumin (9), prealbumin (10), IgA (11), and c~-macroglobulin (12). It appears that the low molecular weight proteins (orosomucoid and prealbumin) constitute a relatively large proportion of spinal fluid proteins, and the high molecular weight proteins (IgA and c~2-macroglobulin) are absent. (II) Densitometric scanning of the spinal fluid precipitin line pattern. Numbers indicate the density peaks of the corresponding precipitin lines. o p e d by i m m u n o e l e c t r o p h o r e s i s o f a c o m p l e x mixture o f antigens (e.g., h u m a n serum) against the c o r r e s p o n d i n g polyspecific antiserum. T h e sensitivity defined as the l o w e r level o f quantitation is about 0.1 /xg/ml. T h e analytical error e x p r e s s e d as the plate-to-plate variation is 4 - 6 % p r o v i d e d a reference precipitate is included in e a c h run to c o m p e n s a t e for plate-toplate differences in staining intensity. Differences in the optical density o f
[9-5]
QUANTITATION OF ANTIGENS
379
/ 432
1
{~
FIG. 6. Densitometric evaluation of the proportion of tumor-associated antigens in Yoshida ascites tumor fluid. Immunoelectrophoresis: S, Sample gel (1.5 x 5 x 12 mm) containing 5% of Yoshida ascites fluid. AG, Antiserum gel (1 x 12 x 100 ram) molded in three sections (dashed demarcation lines); the right section contains 10% and the middle section 2.5% of a polyspecific antiserum against rat serum proteins; the left section contains 5% of a polyspecific antiserum against Yoshida tumor cell-associated antigens [J. Kr011, Protides of Biol. Fluids, Proc. Colloq. 27, 161 (1979)]. Immunoelectrophoresis was carried out at 1.5 V/cm for 40 hr. Anode to the right. Densitometric scanning: This method shows that the tumor-associated antigens (1-4) account for less than 0.5%0 of the total ascites fluid proteins. p r e c i p i t a t e s f o r m e d b y a g i v e n a m o u n t o f t h e s a m e a n t i g e n a g a i n s t different a n t i s e r a f r o m r a b b i t s i m m u n i z e d w i t h this a n t i g e n is w i t h i n t h e a n a l y t ical e r r o r o f t h e m e t h o d . Limitations. T h e r e s o l u t i o n o f c o n v e n t i o n a l d e n s i t o m e t e r s m a y b e insufficient for t h e a n a l y s i s o f c l o s e l y s p a c e d line p a t t e r n s . A l s o d e n s i t o m e t r y m a y b e i m p e d e d in c r o w d e d p a t t e r n s o w i n g to t h e fusion o f different lines. To o p t i m i z e line s e p a r a t i o n , i m m u n o e l e c t r o p h o r e s i s s h o u l d t h e n b e carried out against an antiserum concentration gradient or repeated after subfractionation of the antigens. R e s u l t s o b t a i n e d for e l e c t r o p h o r e t i c a l l y h e t e r o g e n e o u s a n t i g e n s m a y b e m i s l e a d i n g . T h u s for I g G t h e m e t h o d e s t i m a t e s t h e a n o d i c a l l y m i g r a t i n g component only. This problem can be overcome by carbamoylation of the a n t i g e n s . TM
380
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[25]
A
j FIG. 7. The molecular antibody:antigen ratio of immunoelectrophoretic precipitates. The composition of immuneprecipitates of human albumin (A) and orosomucoid (B) is evaluated by SDS-polyacrylamide gel electrophoresis of the dissociated immune complex (representing approximately 5 ~xg of antigen). In the densitometric curves, peak 1 represents the antigen and peak 2 the antibody. Peak 3 is a macromolecular component (MW - 400,000) derived from the antiserum, probably a complement factor (C lq). The density ratio antibody (peak 2): antigen (peak 1) is 14.6 for the albumin complex (A), and 20.1 for the orosomucoid
[25]
QUANTITATION OF ANTIGENS
381
B
2
complex (B). Considering the molecular size of the components (orosmucoid, MW 44,000; albumin, MW 69,000; IgG, MW 150,000), the density values represent a molecular antibody : antigen ratio of 6.7 for the albumin complex and 5.9 for the orosomucoid complex. The latter value probably is an overestimation due to the weak stainability of orosomucoid.
382
A N T I G E N A N D A N T I B O D Y D E T E C T I O N BY I M M U N O A S S A Y
[25]
Polyacrylamide Gel Electrophoresis
Sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis was done according to Anderson et al. 13 using a gradient gel (7 to 20% polyacrylamide), 0.375 M Tris. HC1, pH 8.7. Samples in 0.08 M Tris • HCI, pH 6.8, containing I% SDS glycerol and 25% urea were heated on a boiling water bath for 5 min before loading. Electrophoresis was done at 20 V/cm and 10° for 4 hr. Figure 7 illustrates the use of this procedure for estimation of the antibody:antigen ratio in precipitin lines cut from the immunoelectrophoresis gel as described elsewhere in this volume. 14 Comments The present approach to a relative determination of antigens has the advantage compared with conventional quantitative immunoelectrophoretic techniques of being less dependent on the availability of defined standards. Compared with polyacrylamide gel electrophoretic techniques for the determination of relative protein concentrations, the present procedure provides a higher sensitivity and improves the conditions for the correlation of individual components in different patterns. The observation that the optical density of the precipitin lines correlates with the molar rather than with the mass concentration of the antigens considered is in accordance with standard quantitative precipitin tests showing that the molecular ratio of antibody to antigen is relatively invariant for antigens in the molecular weight range 4 to 16 x 104.1~ The high antibody : antigen molecular ratio found by polyacrylamide gel electrophoretic analysis of the dissociated immune complex is the ratio found in extreme antibody excess. 15 Probably the initial precipitate is formed at antigen-antibody equivalence, the remaining antigen combining sites being occupied by cathodically migrating antibodies during the electrophoresis. Supporting this notion is the observation that the immune precipitate, once formed, increases in density during continued electrophoresis? 2 Differences in stainability of proteins is a significant source of error in densitometry. The high antibody : antigen molecular ratio in the precipitin lines, however, tends to eliminate this type of error. In fact densitometric ~3 C. W. A n d e r s o n , P. R. B a u m , and R. F. Gesteland, J. Virol. 12, 241 (1973). 14 j. Kroll, this volume [3]. ~ E. A. Kabat and M. M. Mayer, " E x p e r i m e n t a l I m m u n o c h e m i s t r y , " p. 26. T h o m a s , Springfield, Illinois, 1961.
[26]
ULTRASENSITIVE ENZYMIC RADIOIMMUNOASSAY
383
scanning of precipitin lines might be used as a means for the quantitation of nonprotein antigens within the 4 to 16 × 104 molecular weight range provided the same high proportion of antibody protein is present in the precipitate. Acknowledgments The skillful technical assistance of Birthe Larsen, Anna Margrethe Poulsen, Lene Ahrenst, and John Post is gratefully acknowledged.
[26] U l t r a s e n s i t i v e E n z y m i c R a d i o i m m u n o a s y By IH-CHANG
Hsu, ROBERT H. YOLKEN, and CURTIS C. HARRIS
Ultrasensitive enzymic radioimmunoassay (USERIA) 1 combines the advantages of two useful techniques for the measurement of biologically important substances, radioimmunoassay (RIA)2"3 and enzyme-linked immunosorbent assay (ELISA). 4 However, USERIA is more sensitive than either of the parent assays. ELISA is similar in design to solid-phase RIA except that an enzyme is used as the immunoglobulin marker instead of a y-emitting isotope. This enzyme-antibody conjugate is bound to the solid phase by a series of antibody-antigen (Ab-Ag) reactions and converts the enzymic substrate to a visible colored product. ELISA is easier, safer, and less expensive than RIA, and no radioisotopes are required. However, ELISA and RIA have similar levels of sensitivity, which can be a limiting factor. ~Therefore USERIA was devised to provide a more sensitive system. USERIA has application in several areas of biomedical investigation. For example, it has been shown to be 100- to 1000-fold more sensitive than RIA in the detection of cholera toxin and rotavirus. 1 USERIA was also employed to measure covalent binding of chemical carcinogen to DNA. As few as 3 fmol of acetylaminofluorene-DNA (AAF-DNA) adducts 6 and C. C. Harris, R. H. Yolken, H. Krokan, and I.-C. Hsu, Proc. Natl. Acad. Sci. U.S.A. 76, 5336 (1979). 2 j. p. Feber, Adv. Clin. Chem. 20, 130 (1978). 3 j. Thorell and S. Larson, "Radioimmunoassay and Related Techniques: Methodology and Clinical Application." Mosby, St. Louis, Missouri, 1978. 4 E. Engvall and P. Perlmann, J. lmmunol. 109, 129 (1972). 5 R. H. Yolken, Hosp. trrac. 11, 123 (1978). I. C. Hsu, M. C. Poirier, S. H. Yuspa, R. H. Yolken, and C. C. Harris, Carcinogenesis 1, 455 (1980).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 73
,
Copyright (~ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181973-6