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Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains
Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains John F. Kennedy,David Catty* and Patricia A. Keep Research Laboratory.]br the Chemistry of Bioactive Carbohydrates and Proteins, Department of Chemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TE, UK * Department of lmmunology, University of Birmingham, P.O. Box 363, Birmingham B15 2T-E UK
(Received 24 August 19791 The purification of rabbit immure{globulin molecules expressing kappa 0¢) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (~c)lgG. Cellulose trans-2,3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preserving immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit ala3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline cellulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (•) IgG, thus separating immunoglobulin, expressing kappa light chain,from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.
Introduction Structural studies on subpopulations of immunoglobulin (Ig) molecules require their separation from serum and from other immunoglobulin subpopulations by methods which preserve their activity as antigens and antibodies. Affinity chromatography in the form of immunoadsorption has been particularly useful in this respect. One example utilizes the presence of allotypic determinants on different Ig molecules and antisera raised to them. In this paper we demonstrate that such allotypic systems can be an effective means for purifying allotypically homogeneous Ig molecules which are of a single isotype. The original demonstration of allotypy in the rabbit was achieved by Oudin 1-3 by iso-immunization of certain rabbits with immunoglobulin from other rabbits. The nomenclature for allotypes was standardized by Dray et al. 4. (Different allotypic specificities are designated by Arabic numbers. The notation 'A' precedes the number indicating allotypic specificity. When the genetic locus is known the 'A' is omitted and the number indicating specificity is preceded by the locus designation.) The b locus allotypic specificities (b4, b5, b6 and b9) are present on kappa light chains only. Immunoglobulin carrying the b4 allotypic specificity was utilized in the work described here. The b locus allotypic markers correspond to multiple amino acid interchanges within the constant region of kappa light chain 5. There may, in addition, be some variable region differences correlating with these allotypes 6'7. Lambda light chain allotypic markers include the c7 and c21 specificities. Rabbit Ig allotypes have been reviewed extensively by Nisonoff et al. s and Kindt 9 and summarized by Mole 1°. In any process involving immunoadsorption, the matrix to which the antigen or antibody is coupled is 0141 8130/80/030137q)6502.00 © 1980 IPC Business Press
extremely important with respect to stability, ease of coupling reaction and flow properties. Cellulose trans-2,3carbonate derived from microcrystalline cellulose, first prepared by Barker et al.11 has been used previously 12 in a radioimmunoadsorbent method of estimating IgE. This method depended on the inhibition of uptake of labelled IgE antigen onto microcrystalline cellulose carbonate coupled to specific anti-IgE antibody. Catty et al.13 have applied microcrystalline cellulose carbonate to the preparation of water-insoluble immunoadsorbents used in the purification of antibodies to rabbit IgG and human IgM. These adsorbents possessed very high antibody binding capacities with respect to the amount of antigen coupled. Up to 86~o of the adsorbed antibody was recovered, and since antibody was found to bind to insolubilized antigen in ratios approaching those in free solution, the eluted antibodies differed little from those in whole antiserum with respect to heterogeneity in specificity and affinity. Macroporous cellulose has a very open structure and is porous to macromolecules, permitting free access of high molecular weight enzyme substrates. Cellulose carbonate derived from macroporous cellulose has been utilized previously ~4 in the insolubilization of chymotrypsin A such that it maintains much of its activity towards casein. Cellulose trans-2,3-carbonates, derived from both microcrystalline and macroporous celluloses, have been used and compared as matrices for the immobilization of immunoglobulin antigens and antibodies in the preparation of specific immunoadsorbents for the separation of rabbit immunoglobulin expressing kappa light chain from that expressing lambda light chain. Immunoglobulin expressing the antigenic determinants of the b4 allotypic system, present on kappa chains only, and a corresponding anti-b4 antiserum, were immobilized
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Production of immunoadsorbents: John F. Kennedy et al. on cellulose trans-2,3-carbonate and used in the immunoadsorption processes.
agar 1.5~o, w/v, in 0.1 M phosphate buffer, pH 7.2) against the corresponding antigen or antibody (Ouchterlony16).
Experimental
lmmunoelectrophoresis. This was performed in agar (Noble agar 1.5~o, w/v, in 0.1 M barbitone buffer, pH 8.6) by a method based on that of Grabar and Williams ~7. After electrophoresis with a constant current of 16 mA for 2 3 h, a trough was cut below the sample well and filled with sheep anti-rabbit whole serum antiserum (Z68) and immunodiffusion allowed to proceed overnight. The arcs of precipitate were then recorded.
Preparation of cellulose carbonates Cellulose carbonate derived from macroporous cellulose. Macroporous cellulose trans-2,3-carbonate was prepared essentially by the method of Kennedy et al.14, except that the addition of water was omitted. An aliquot (13 ml) of macroporous cellulose slurry (1 g cellulose; Pharmacia, particle size 4(~250 pm and hydroxypropylated at position 6) was filtered on sintered glass, then freed from water by solvent exchange. This was achieved by stirring the solid with fresh changes of dry dimethyl suiphoxide (DMSO; 6 x 25 ml) over a period of 18 h; the solvent being removed each time by filtration. The macroporous cellulose was suspended in a mixture of 10 mt dry DMSO, 1.5 ml dry 1,4-dioxan and 8 ml dry triethylamine. The mixture was stirred magnetically on an ice--salt bath while 16 ml ethyl chloroformate was added dropwise from a funnel over a period of 30 min. After stirring for a further 15 min, 20 ml dry 1,4-dioxan was added and the suspension filtered on sintered glass. The solid product was washed by stirring with dry 1,4-dioxan (9 x 20 ml), dry ethanol (10 x 20 ml) and dry diethyl ethel (3 x 20 ml), the solvent being removed eacl, time by filtration. The solid was freed from all solvent in a chloroform/P205 pistol drier and finally stored in a desiccator containing paraffin wax and P205. The cyclic 2,3-carbonate content was determined by titration with ammonia solution and by infrared spectroscopy. Cellulose carbonate derived J?om microcrystalline cellulose. The microcrystalline cellulose trans-2,3-carbonate was prepared using the optimum conditions determined by Barker et al. 1i. Microcrystalline cellulose (Sigmacell, particle size 38 pm) was dried for 24 h in a chloroform/P20 5 pistol drier. Dry cellulose (1.0 g) was suspended in 10 ml dry DMSO, 1.5 ml dry 1,4-dioxan and 8 ml dry triethylamine. The trans-2,3-carbonate was prepared as previously for macroporous cellulose. The degree of substitution (DS) of the product was determined by titration with ammonia and by infrared spectroscopy. Sera utilized in the purification of h4 IyG and in immunodiffusion The following rabbit sera, prepared in the Department of Immunology, University of Birmingham Medical School, were used: RB 9675 ala3/b4 serum; RB 8006/II a2/b6 anti-b4 antiserum; RB 913 a2/b4 serum; RB 9376 ala2/b5b6 anti-b4 antiserum; and in addition the following sheep sera: Z67 anti-rabbit IgG antiserum (sheep); Z68 anti-rabbit whole serum antiserum (sheep). Immunological techniques lmmunodij]usion. The quantitative determination of rabbit IgG was achieved by radial immunodiffusion of antigen in agar (Noble agar 1.5')0, w/v, in 0.1M phosphate buffer, pH 7.2) containing antiserum (Z67 sheep antirabbit IgG: 0.36 ml in 12 ml agar). This technique was developed by Mancini et al 15. Qualitative characterization of isolated antibody or antigen was achieved by double diffusion in agar (Noble
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Pur!fication of the 7-globulin j?action of rabbit ala3/b4 serum To rabbit ala3/b4 serum (RB 9675) was added dropwise an equal volume of saturated ammonium sulphate solution. After leaving at 4°C for 2 h to obtain maximum precipitation of the 7-globulin, the precipitate was centrifuged and washed once with 50~o saturated ammonium sulphate solution, then dissolved in phosphate buffer (0.01 M, pH 7.1) to the volume of the original serum. The 7globulin solution was dialysed against phosphate buffer (0.01 M, pH 7.1) at 4°C for 24 h with several changes of buffer, then purified further by stirring batchwise with DEAE-cellulose (Whatman DE52) equilibrated in phosphate buffer (0.01 M, pH 7.1). After leaving for 4 h at 4°C, then centrifuging, the supernatant was concentrated to a volume of 5 ml by ultrafiltration. The ~-globulin solution was centrifuged to remove denatured material and the IgG concentration determined by measuring absorbance at 280 nm, using a n El1'c'm for rabbit IgG of 14.6 (Ref 18). The purity of the rabbit ala3/b4 IgG was tested by immunoelectrophoresis against a polyvalent sheep antiserum to rabbit serum proteins (Z68), with rabbit whole serum for comparison. The presence of a single arc in the 7-region was indicative of a pure preparation. Chemical coupling o]" rabbit ala3/b4 IyG to cellulose carbonates Macroporous cellulose trans-2,3-carbonate. A solution of rabbit al a3/b4 IgG (2.95 mg ml- 1 in 0.01 M phosphate buffer, pH 7.1; 3.0 ml), derived from 10 ml of whole serum, was mixed with phosphate buffer (0.1 M, pH 6.8; 5.0 ml). Macroporous cellulose carbonate (DS 0.25; 200 mg) was added and the mixture stirred magnetically for 24 h at 2OC. After centrifuging, the supernatant was retained for measurement of IgG content and the product was washed with phosphate-buffered saline (PBS; 8 mM Na2HPO4.12H20; 3 mM NaHzPO4.2H20 , 0.15 M NaC1; 4 x 3 ml) to remove any non-specifically bound material. The washings were pooled and retained for IgG measurement. The macroporous cellulose carbonate,." (ala3/b4 IgG) product was suspended in 3.0 ml PBS and packed into a Pasteur pipette above a layer (0.3 ml settled volume) of Bio-Gel P-6. The immunoadsorbent column (0.75 ml settled volume) was washed with PBS. The amount of rabbit ala3/b4 IgG bound to the macroporous cellulose trans-2,3-carbonate was estimated by radial immunodiffusion assay [against sheep antirabbit IgG antiserum (Z67)] by comparison between the total IgG used in the coupling reaction and the residue in the supernatant and washings. This amount was calculated to be 3.95 mg (Table 1).
Production of immunoadsorbents: John F. Kennedy et al. Table 1 Use of macroporous cellulose trans-2,3-carbonate (MCC) in the purification of rabbit b4 (h-) IgG
Operation
Material
Ammonium sulphate IgG solution precipitation of rabbit ala3/b4 serum (10.0 ml) Preparation of MCC/ MCC (ala3/b4 IgG) column IRabbit ala3/b4 IgG (added to MCC] Rabbit ala3/b4 IgG (bound to MCC) Application of rabbit anti-b4 antiserum to MCC/(ala3/b4 IgG)
Rabbit a2/b6 anti-b4 serum applied Protein in glycine/HC1 eluate
Preparation of MCC/ MCC (anti-b4 IgG) column Rabbit anti-b4 IgG (added to MCC) Rabbit anti-b4 IgG (bound to MCC) Application of rabbit Rabbit a2/b4 IgG a2/b4 IgG to MCC/ applied (anti-b4 IgG) column
Total Volume yield (ml) (rag) 5.0
14.8
3.0
200.0 8.9
bance of the unbound material (PBS eluate) and bound anti-b4 I g G (glycine/HC1 eluate) was measured at 280 nm and the total protein determined (Table 2). For the macroporous adsorbent the amount of anti-b4 I g G eluted was very small and the protein content was confirmed by Lowry assay 19 using rabbit IgG (D68; standards at concentrations of 0.25, 0.13 and 0.06 mg ml ~; Table 1). The PBS eluate and glycine/HCl eluate from each column was tested for the presence of anti-b4 lgG antibody activity by immunodiffusion against b4 IgG (Tables 3 and 4). For the macroporous cellulose trans-2,3-
4.0 Table 2 Use of microcrystalline cellulose trans-2,3-carbonate (CC) in the purification of rabbit b4 (h-) IgG
2.0 1.0
0.4
0.8
27.3 0.3 Trace
1.0
0.5
Microcrystalline cellulose trans-2,3-carbonate. Rabbit ala3/b4 IgG (27.9 mg/ml in 0.01 M phosphate buffer, pH 7.1; 3.2 ml) was mixed with phosphate buffer (0.1 M, pH 6.8; 20.0 ml). Microcrystalline cellulose trans-2,3carbonate (DS 0.22; 1.6 g) was added and the mixture stirred magnetically for 24 h at 20°C. The product was centrifuged, washed with PBS (4 × 10 ml), suspended in PBS (15.0 ml) then packed into a column (9.5 cm x 1.1 cm) above a layer of Bio-Gel P-6 (2 ml settled volume). From Mancini assay it was determined that 62.8 mg of rabbit ala3/b4 IgG was bound to the microcrystalline cellulose trans-2,3-carbonate (Table 2). Isolation of rabbit anti-b4 IgG antibody usin9 the macroporous cellulose carbonate/(ala3/b4 IgG) and microcrystalline cellulose carbonate/(ala3/b4 lgG) immunoadsorbent columns Each column was equilibrated by washing overnight with PBS, at a flow rate of 10 ml h-1, until a constant baseline absorbance was obtained at 280 nm. Rabbit antib4 serum (RB 8006/II allotypic specificity a2/b6) was alternately frozen and thawed to inactivate complement then applied to the respective columns in the amounts indicated in Tables 1 and 2. Each column was washed with PBS and fractions collected on a time basis (25 min) until the first peak (of unbound anti-b4 IgG and other serum proteins) was eluted as detected by absorbance at 280 nm. Bound rabbit anti-b4 antibody was eluted from each column with glycine/HCl buffer (0.1 M, pH 2.2). The fractions containing the PBS-eluted proteins were pooled and concentrated to the volumes shown in Tables 1 and 2. For each column the fractions containing the glycine/HCl-eluted protein (antibody) were pooled, concentrated by ultrafiltration then dialysed against Tris/HCl buffer (0.2 M, pH 8.0) for several days and centrifuged. For the microcrystalline derivative the absor-
Operation
Total Volume yield (ml) (mg)
Material
Ammonium sulphate IgG solution precipitation of rabbit ala3/b4 serum (30.0 ml) Preparation of CC/ CC (ala3/b4 IgG)column IRabbit ala3/b4 IgG (added to CC) Rabbit ala3/b4 IgG (bound to CC) Application of rabbit anti-b4 antiserum to CC/(ala3/b4 IgG] column
3.5
97.8
3.2
1600.0 89.4 62.8
Rabbit a2/b6 anti-b4 serum applied Protein in glycine/ HCI eluate
30.0
Preparation of CC/ CC (anti-b4 IgG) column Rabbit anti-b4 IgG (added to CC) Rabbit anti-b4 lgG (bound to CC)
19.2
8.8
18.7
200.0 8.6 2.0
1.0
Application of rabbit Rabbit a2/b4 |gG a2/b4 serum to CC/ applied (anti-b4 IgG) column b4 (•) IgG isolated b4-negative ()~) IgG isolated
9.0 3.0 0.6
Table 3 Use of macroporous cellulose trans-2,3-carbonate in the purification of b4 (K) IgG: immunodiffusion results
Operation
Sample
Application PBS eluate of rabbit anti-b4 Glycine/HCl antiserum to eluate MCC/(a 1a3/b4 lgG) column
Z67 sheep Rabbit b4 anti-rabbit RB9376 IgG IgG anti-b4 + +
Application PBS eluate of rabbit a2/b4 IgG to Glycine/HCl MCC/(anti-b4 eluate IgG] column
+
++
+
+, Weakprecipitin line; + +, strong precipitinline
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Production of immunoadsorbents: John F. Kennedy et al. Table 4 Use of microcrystalline cellulose trans-2,3-carbonate in the purification of b4 0c) IgG: immunodiffusion results
Operation
Sample
Application of rabbit anti-b4 antiserum to CC/(a 1a3/b4 IgG) column
PBS eluate Glycine/HCl eluate
Application of rabbit a2/b4 serum to CC/(anti-b4 lgG) column Initial PBS eluate application Glycine/HCl eluate
Z67 sheep Rabbit b4 RB 9376 anti-rabbit lgG anti-b4 lgG
+
Application of rabbit a2/h4 h lG to the macroporous cellulose trans-2,3-carbonate/(anti-h4 l qG) and microcrystalline cellulose trans-2,3-carbonate/(anti-h4 l qG) immunoadsorbeitt columns Macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG). Rabbit a2/b4 IgG was obtained by precipitation of
+ + + +
1st reapplication
PBS eluate Glycine/HC1 eluate
+ + + +
2nd reapplication
PBS eluate Glycine/HCl eluate
+ + + +
4th reapplication
PBS eluate Glycine/HCL eluate
200 mg) was added and the mixture stirred magnetically for 24 h at 2 0 C . The product was centrifuged, the supernatant removed and the adsorbent washed with PBS (4 x 3 ml). The washings were concentrated by ultrafiltration to 3.0 ml. The supernatant and washings from the coupling reaction were dialysed against PBS for 2 days with several changes of buffer and the protein concentration determined by measuring absorbance at 280 nm. The protein content of the original anti-b4 IgG solution used in the coupling reaction was also determined. The amount of anti-b4 antibody coupled to the cellulose carbonate was calculated 12 mg; Table 2). A column of the adsorbent was prepared in a Pasteur pipette above a layer of Bio-Gel P-6 (0.5 ml when packed).
+
+ +
+ , Weak precipitin line; + + , strong precipitin line; + + + . very strong precipitin line; , no precipitation
carbonate/(ala3/b4 IgG)adsorbent, the PBS eluate retained anti-b4 antibody activity indicating that not all of the applied antibody bound to the column. The acid eluate also contained anti-b4 antibody activity. The PBS eluate from the microcrystalline cellulose carbonate/(ala3/b4 IgG) column did not contain any anti-b4 antibody activity detectable by immunodiffusion therefore all anti-b4 IgG was bound to the adsorbent (Figure 1). The presence of anti-b4 antibody activity was demonstrated in the glycine/HC1 eluate.
a2/b4 serum (RB 913 a2/b4) with an equal volume of saturated a m m o n i u m sulphate solution as described previously. An aliquot of the IgG solution (1.0 ml: absorbance of 0.47 mg at 280 n m = 0.680) was applied to the macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG) adsorbent, which was washed with PBS to obtain unbound material [b4-negative (2 chain) IgG and unbound b4 IgG]. Bound b4 (h') lgG was eluted with glycine/HCl buffer (0.1 M, pH 2.2). The PBS eluate was concentrated by ultrafiltration to 1.3 ml. The glycine/HCl eluate was also concentrated to 1.3 ml, then dialysed against PBS overnight.
Microcrystalline cellulose trans-2,3-carbonate/(anti-b4 l qG). Rabbit a2/b4 serum (RB 913 a2/b4; 1.0 ml containing 9.0 mg IgG) was applied to the microcrystalline cellulose trans-2,3-carbonate/(anti-b4 IgG) adsorbent. A large peak, due to unbound b4 IgG and other serum proteins, was obtained by washing the column with PBS. Bound b4 (h) IgG was obtained by elution with glycine/HC1 (0.1 M, pH 2.2). The PBS eluate was concentrated by ultrafiltration to 2.3 ml. The glycine/HCl eluate was concentrated, then dialysed against Tris/HCl (0.2 M, pH 8.0), giving a final volume of 2.6 ml.
Chemical couplin,q of rabbit anti-b4 IgG antibody to cellulose carbonates Macroporous cellulose trans-2,3-carbonate. Rabbit anti-b4 IgG antibody, purified on the macroporous cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent column (0.8 ml, 0.3 rag), was mixed with phosphate buffer (0.1 M, pH 6.8; 2.0 ml). Macroporous cellulose carbonate (DS 0.25; 27.3 mg) was added and the mixture shaken for 24 h at 2 0 C . The product was centrifuged, then washed with PBS (4 ×2 ml). The macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG) adsorbent was suspended in PBS then packed in a Pasteur pipette above a layer of Bio-Gel P-6 (0.75 ml when settled).
Microcrystalline cellulose trans-2,3-carbonate. Rabbit anti-b4 IgG antibody purified on the microcrystalline cellulose carbonate/(ala3/b4 IgG) immunoadsorbent column (18.7 ml, 8.6 mg) was taken and the volume made up to 19.0 ml with phosphate buffer (0.1 M, pH 6.8). Microcrystalline cellulose trans-2,3-carbonate (DS 0.22,
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Figure i
(at Isolation of rabbit anti-b4 antibody on the
CC/ala3/b4 IgG} immunoadsorbent. Note the precipitin line obtained with the glycine/HCl eluate (anti-b4 antibody; well 3) with rabbit b4 lgG (1 mg/ml; well I) forming a line of identity with rabbit anti-b4 serum control (well 2), and the absence of anti-b4 antibody activity in the PBS eluate (non-bound material; well 4). (b) Isolation of rabbit b4 tl,) lgG on the CC/(anti-b4 IgGl immunoadsorbent. Outer w e l l s doubling dilutions of glycine/HCl eluate (b4 (h) lgG). Centre well rabbit ala2/b5b6 anti-b4 antiserum
Production of immunoadsorbents.: John F. Kennedy et al. Since [he PBS eluate was subsequently found to contain b4 (~c) IgG by immunodiffusion, the unbound material was re-applied to the column four times, eluting first with PBS and secondly with glycine/HCl buffer (0.1 M, pH 2.2) each time. The PBS and acid eluates were concentrated after every application and the acid peak dialysed against Tris/HCl buffer (0.2 M, pH 8.0). The concentrated eluates were tested each time for the presence of b4 (K) IgG by immunodiffusion. The amount of protein in the pooled acid eluates was determined by absorbance measurement at 280 nm (Table 2). The protein content of the final PBS eluate was determined from absorbance at 280 nm after precipitation with an equal volume of saturated ammonium sulphate solution and dialysis against phosphate buffer (0.01 M, pH 7.1) to obtain the 7-globulin fraction as described previously. The amount of b4-negative (2) IgG isolated is indicated in Table 2 (0.6 mg).
Detection of b4 allotypic determinants in the PBS and glycine/HCl eluates by immunodiffusion Macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG). After 24 h the PBS eluate (unbound material) gave precipitin lines with sheep anti-rabbit IgG antiserum (Z67) and rabbit anti-b4 antiserum (RB 9376). A faint line due to interaction of the glycine/HCl eluate with sheep anti-rabbit IgG antiserum (Z67) was observed (Table 3).
M icrocrystalline cellulose trans-2,3-carbonate/(anti-b4 log ). The PBS and glycine/HCl eluates were tested after each application to the adsorbent for the presence of b4 IgG by precipitation with rabbit anti-b4 antiserum (RB 9376; Table 4, Figure 1). After the fifth application to the column the PBS eluate was also tested for the presence of rabbit IgG. A precipitin line was observed after 24 h against sheep anti-rabbit IgG antiserum (Z67). It was noted that the PBS peak showed no b4 activity after the fifth application to the column and all b4 activity was assumed to have been removed leaving only b4-negative ()0 IgG (Table 4). The acid eluate, after each application of the PBS eluate to the column, was shown to contain b4 (K) IgG by the presence ofa precipitin line against rabbit antib4 antiserum (RB 9376; Table 4).
carbonate was 0.25 and 0.22, respectively. However, the percentage of ala3/b4 IgG coupled was much higher for microcrystalline cellulose carbonate (Table 5), which may be accounted for by the greater external surface area per unit volume of the microcrystalline derivative. Rabbit anti-b4 antiserum was applied to the cellulose trans-2,3-carbonate/(ala3/b4 IgG)immunoadsorbents~ From Table 3 it may be seen that a relatively greater amount of anti-b4 IgG antibody was eluted with glycine/HCl buffer from the microcrystalline cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent. The ratio of anti-b4 IgG eluted (mg) to al a3/b4 IgG (rag) coupled to the cellulose trans-2,3-carbonate was calculated for both the macroporous and microcrystalline supports (1:10 and 1:7, respectively). However, it must be noted that the latter value is a minimum estimate since the binding capacity of the adsorbent was not exceeded. The eluant was the same for both adsorbents, therefore the microcrystalline cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent was the more efficient for the purification and isolation of rabbit anti-b4 antibody, and the antigenic sites were probably less sterically hindered in the microcrystalline derivative. Immunodiffusion results (Table 3) for the glycine/HC1 and PBS eluates from the macroporous immunoadsorbent indicated that not all of the rabbit anti-b4 IgG antibody applied to the column was adsorbed on first passag e, but the immunodiffusion results for the corresponding eluates from the microcrystalline immunoadsorbent (Table 4) indicated that with this matrix all of the anti-b4 IgG antibody applied to the column had been adsorbed as no residual anti-b4 antibody activity was detectable in the PBS eluate. The purified anti-b4 IgG antibody eluted from the macroporous and microcrystalline immunoadsorbents was coupled to macroporous cellulose trans-2,3-carbonate and microcrystalline cellulose trans-2,3-carbonate, respectively. The ratio of anti-b4 IgG (mg) to cellulose trans-2,3carbonate (mg) used in the coupling reaction was 1:91 for the macroporous derivative and 1:23 for the microcrystal-
Discussion
The purification of rabbit b4 (~-) IgG using cellulose carbonate derived from macroporous cellulose and microcrystalline cellulose was effected in the following stages: (a) preparation of a cellulose trans-2,3-carbonate/(ala3/b4 IgG)immunoadsorbent; (b) application of rabbit anti-b4 IgG antiserum to the above immunoadsorbent and its elution as a purified antibody; (c) preparation of a cellulose trans-2,3-carbonate/(antib4 IgG) immunoadsorbent; (d) application of rabbit a2/b4 IgG to the adsorbent prepared in (c) and elution of bound b4 (~) IgG with glycine/HCl buffer, pH 2.2. The results of each stage, including amount of antigen or antibody coupled or bound, are given in Tables 1 and 2. The weight ratio of ala3/b4 IgG to cellulose trans-2,3carbonate used in the coupling procedure was similar for the macroporous and microcrystalline derivatives, and the degree of cyclic carbonate substitution for the macroporous cellulose carbonate and microcrystalline cellulose
Comparison of immunoadsorbents prepared using macroporous cellulose trans-2,3-carbonate and microcrystalline cellulose trans-2,3-carbonate Table 5
MCC Cellulose trans-2,3-carbonate/(ala3/b4 IgG) Ratio ala3/b4 IgG added (mg)/cellulose trans2,3-carbonate (mg) 1:23 IgG bound (%) 44.6 Ratio ala3/b4 IgG coupled (mg)/cellulosetrans-2,3-carbonate (mg) 1:51 Anti-b4 IgG antibody eluted (rag) 0.4 Ratio anti-b4 IgG eluted (mg)/coupled ala3/b4 IgG (mg) 1:10 Cellulose-trans-2,3-carbonate/[antl-b4 lgG) Ratio anti-b4 IgG added (mg)/cellulose trans2,3-carbonate (mg) 1:91 IgG bound (%) trace
CC
1:18 70.2 1:39 8.8 1:7" 1:23 23.3
* minimum value since binding capacity of column was not exceeded
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Production of immunoadsorbents: John F. Kennedy et al. line derivative (Table 5). Only a trace of anti-b4 antibody was successfully coupled to the macroporous cellulose trans-2,3-carbonate (since bound a2/b4 IgG was eluted with glycine/HCl after application to the immunoadsorbent column) but the amount of coupled antibody could not be determined accurately. However, 2 mg (23!~,) of the available anti-b4 IgG antibody was coupled to microcrystalline cellulose trans-2,3-carbonate, giving a ratio of antib4 antibody (mg) to cellulose trans-2,3-carbonate (mg) of 1:100. On application of rabbit a2/b4 lgG to the macroporous cellulose-2,3-carbonate/(anti-b4 IgG) immunoadsorbent, one peak was obtained on washing with PBS (unbound material) and a second, smaller peak was eluted with .glycine/HC1 pH 2.2. The peaks obtained were then tested for b4 activity by immunodiffusion (Table 3). The PBS eluate still reacted strongly with anti-b4 antiserum showing the presence of residual unbound b4 (K) IgG. The acid peak, containing ---0.5 mg protein, did not give a precipitin line with anti-b4 antiserum but a line was obtained against sheep anti-rabbit IgG antiserum, indicating that b4-negative rabbit immunoglobulin was present. Rabbit a2/b4 serum was also applied to a column of microcrystalline cellulose trans-2,3-carbonate/(anti-b4 IgG). Five applications were necessary to remove all of the b4 (~c)IgG, and the decrease in the b4 IgG content of the PBS eluate on each application to the column can be seen from the immunodiffisuon results (Table 4). Glycine/HC1 buffer pH 2.2 was used to elute the bound b4 (K) IgG each time. Originally, 9.0 mg of a2/b4 IgG were applied to the column, and totals of ~ 3 mg of b4 (t,) IgG and 0.6 mg of b4-negative (2) IgG were isolated (Table 2), giving a final recovery of ~40°o. The presence of rabbit IgG was detected in the final PBS eluate (Table 4) in the absence of detectable b4 (K) determinants. This is consistent with the isolated material containing only b4-negative ().) IgG molecules. Losses would be expected to occur through dialysis, ultrafiltration and transference from one vessel to another. From Table 5 it can be seen that the trans-2,3carbonate derived from microcrystalline cellulose is superior to the macroporous derivative with respect to chemical coupling of rabbit ala3/b4 IgG antigen and binding of rabbit anti-b4 antibody. The microcrystalline cellulose carbonate is also more effective for the coupling of specific anti-b4 antibody and subsequent binding and elution of b4 (K) IgG. Use of the more efficient microcrystalline cellulose carbonate in the preparation of im-
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Int. J. Biol. Macromol., 1980, Vol 2, June
munoadsorbents for the purification of b4 (K) lgG provides a powerful technique for the separation of rabbit immunoglobulin expressing kappa light chain (90'};i of total immunoglobulin) from rabbit immunoglobulin expressing lambda light chain (10~0 of total immunoglobulin) thus enabling studies on immunological and chemical properties of the separate populations of molecules to be performed. The isolation of immunoglobulin expressing 2 light chain from normal rabbit immunoglobulin by immunoadsorption, as opposed to exclusive production of immunoglobulin with 2 light chain by K-suppression in rabbit, facilitates the study of )~ chain allotypic determinants in the normal situation and the production of specific antisera against these determinants. This work has demonstrated the use of allotypic specificities of rabbit immunoglobulin populations and may be extended to the use of different allotypic systems and antibody antigen populations.
References 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19
Oudin, J. C.R. Acad. Sci. Paris 1956, 242, 2489 Oudin, J. C.R. Acad. Sci. Paris 1956, 242, 2606 Oudin, J. J. Exp. Med. 1960, 112, 125 Dray, S., Dubiski, S., Kelus, A., Lennox, E. S. and Oudin, J. Nature (London) 1962, 195, 785 Appella, E., Chersi, A, Rejnek, J. and Reisfeld, R. A. in 'Developmental Aspects of Antibody Formation and Structure' (Eds J. Sterzl and J. Riha) Academic Press, New York, 1970, Vol. 1, p. 327 Hood, L., Waterfield, M. D., Morris, J. and Todd, C. W. Ann. N Y Acad. Sci. 1971, 190, 26 Chersi, A., Mage, R. G. and Appella, E. Fed. Proc. 1971, 30, 1307 Nisonoff, A., Hopper, J. E. and Spring, S. B, 'The Antibody Molecule' Academic Press, New York, 1975 Kindt, T. J. Adv. lmmunol. 1975, 21, 37 Mole, L. E. Biochem. Soc. Trans. 1976, 4, 33 Barker, S. A., Cho, Tun H., Doss, S. H., Gray, C. J. and Kennedy, J. F. Carbohydr. Res. 1971, 17, 471 McLaughlan, P., Stanworth, D. R., Kennedy, J. F. and Cho Tun, H. Nature New Biol. 1971, 232, 345 Catty, D., Kennedy, J. F., Drew, R. L. and Cho Tun H. J. tmmunol. Methods 1973, 2, 353 Kennedy, J. F., Barker, S. A. and Rosevear, A. J. Chem. Soc. Perkin Trans. 1. 1973, 2293 Mancini, G., Carbonara, A. O. and Heremans, J. F. lmmunochemistry 1965, 2, 235 Ouchterlony, O. Progr. Allergy 1958, 5, 1 Graber, P. and Williams, C. A. Biochim. Biophys. Acta 1953, 10, 193 Grossberg, A. L., Radzimski, G. and Pressman, D. Biochemistry 1962, 1,391 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. d. Biol. Chem. 1951, 193, 265
(Received 24 August 19791 The purification of rabbit immure{globulin molecules expressing kappa 0¢) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (~c)lgG. Cellulose trans-2,3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preserving immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit ala3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline cellulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (•) IgG, thus separating immunoglobulin, expressing kappa light chain,from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.
Introduction Structural studies on subpopulations of immunoglobulin (Ig) molecules require their separation from serum and from other immunoglobulin subpopulations by methods which preserve their activity as antigens and antibodies. Affinity chromatography in the form of immunoadsorption has been particularly useful in this respect. One example utilizes the presence of allotypic determinants on different Ig molecules and antisera raised to them. In this paper we demonstrate that such allotypic systems can be an effective means for purifying allotypically homogeneous Ig molecules which are of a single isotype. The original demonstration of allotypy in the rabbit was achieved by Oudin 1-3 by iso-immunization of certain rabbits with immunoglobulin from other rabbits. The nomenclature for allotypes was standardized by Dray et al. 4. (Different allotypic specificities are designated by Arabic numbers. The notation 'A' precedes the number indicating allotypic specificity. When the genetic locus is known the 'A' is omitted and the number indicating specificity is preceded by the locus designation.) The b locus allotypic specificities (b4, b5, b6 and b9) are present on kappa light chains only. Immunoglobulin carrying the b4 allotypic specificity was utilized in the work described here. The b locus allotypic markers correspond to multiple amino acid interchanges within the constant region of kappa light chain 5. There may, in addition, be some variable region differences correlating with these allotypes 6'7. Lambda light chain allotypic markers include the c7 and c21 specificities. Rabbit Ig allotypes have been reviewed extensively by Nisonoff et al. s and Kindt 9 and summarized by Mole 1°. In any process involving immunoadsorption, the matrix to which the antigen or antibody is coupled is 0141 8130/80/030137q)6502.00 © 1980 IPC Business Press
extremely important with respect to stability, ease of coupling reaction and flow properties. Cellulose trans-2,3carbonate derived from microcrystalline cellulose, first prepared by Barker et al.11 has been used previously 12 in a radioimmunoadsorbent method of estimating IgE. This method depended on the inhibition of uptake of labelled IgE antigen onto microcrystalline cellulose carbonate coupled to specific anti-IgE antibody. Catty et al.13 have applied microcrystalline cellulose carbonate to the preparation of water-insoluble immunoadsorbents used in the purification of antibodies to rabbit IgG and human IgM. These adsorbents possessed very high antibody binding capacities with respect to the amount of antigen coupled. Up to 86~o of the adsorbed antibody was recovered, and since antibody was found to bind to insolubilized antigen in ratios approaching those in free solution, the eluted antibodies differed little from those in whole antiserum with respect to heterogeneity in specificity and affinity. Macroporous cellulose has a very open structure and is porous to macromolecules, permitting free access of high molecular weight enzyme substrates. Cellulose carbonate derived from macroporous cellulose has been utilized previously ~4 in the insolubilization of chymotrypsin A such that it maintains much of its activity towards casein. Cellulose trans-2,3-carbonates, derived from both microcrystalline and macroporous celluloses, have been used and compared as matrices for the immobilization of immunoglobulin antigens and antibodies in the preparation of specific immunoadsorbents for the separation of rabbit immunoglobulin expressing kappa light chain from that expressing lambda light chain. Immunoglobulin expressing the antigenic determinants of the b4 allotypic system, present on kappa chains only, and a corresponding anti-b4 antiserum, were immobilized
Int. J. Biol. Macromol., 1980, Vol 2, June
137
Production of immunoadsorbents: John F. Kennedy et al. on cellulose trans-2,3-carbonate and used in the immunoadsorption processes.
agar 1.5~o, w/v, in 0.1 M phosphate buffer, pH 7.2) against the corresponding antigen or antibody (Ouchterlony16).
Experimental
lmmunoelectrophoresis. This was performed in agar (Noble agar 1.5~o, w/v, in 0.1 M barbitone buffer, pH 8.6) by a method based on that of Grabar and Williams ~7. After electrophoresis with a constant current of 16 mA for 2 3 h, a trough was cut below the sample well and filled with sheep anti-rabbit whole serum antiserum (Z68) and immunodiffusion allowed to proceed overnight. The arcs of precipitate were then recorded.
Preparation of cellulose carbonates Cellulose carbonate derived from macroporous cellulose. Macroporous cellulose trans-2,3-carbonate was prepared essentially by the method of Kennedy et al.14, except that the addition of water was omitted. An aliquot (13 ml) of macroporous cellulose slurry (1 g cellulose; Pharmacia, particle size 4(~250 pm and hydroxypropylated at position 6) was filtered on sintered glass, then freed from water by solvent exchange. This was achieved by stirring the solid with fresh changes of dry dimethyl suiphoxide (DMSO; 6 x 25 ml) over a period of 18 h; the solvent being removed each time by filtration. The macroporous cellulose was suspended in a mixture of 10 mt dry DMSO, 1.5 ml dry 1,4-dioxan and 8 ml dry triethylamine. The mixture was stirred magnetically on an ice--salt bath while 16 ml ethyl chloroformate was added dropwise from a funnel over a period of 30 min. After stirring for a further 15 min, 20 ml dry 1,4-dioxan was added and the suspension filtered on sintered glass. The solid product was washed by stirring with dry 1,4-dioxan (9 x 20 ml), dry ethanol (10 x 20 ml) and dry diethyl ethel (3 x 20 ml), the solvent being removed eacl, time by filtration. The solid was freed from all solvent in a chloroform/P205 pistol drier and finally stored in a desiccator containing paraffin wax and P205. The cyclic 2,3-carbonate content was determined by titration with ammonia solution and by infrared spectroscopy. Cellulose carbonate derived J?om microcrystalline cellulose. The microcrystalline cellulose trans-2,3-carbonate was prepared using the optimum conditions determined by Barker et al. 1i. Microcrystalline cellulose (Sigmacell, particle size 38 pm) was dried for 24 h in a chloroform/P20 5 pistol drier. Dry cellulose (1.0 g) was suspended in 10 ml dry DMSO, 1.5 ml dry 1,4-dioxan and 8 ml dry triethylamine. The trans-2,3-carbonate was prepared as previously for macroporous cellulose. The degree of substitution (DS) of the product was determined by titration with ammonia and by infrared spectroscopy. Sera utilized in the purification of h4 IyG and in immunodiffusion The following rabbit sera, prepared in the Department of Immunology, University of Birmingham Medical School, were used: RB 9675 ala3/b4 serum; RB 8006/II a2/b6 anti-b4 antiserum; RB 913 a2/b4 serum; RB 9376 ala2/b5b6 anti-b4 antiserum; and in addition the following sheep sera: Z67 anti-rabbit IgG antiserum (sheep); Z68 anti-rabbit whole serum antiserum (sheep). Immunological techniques lmmunodij]usion. The quantitative determination of rabbit IgG was achieved by radial immunodiffusion of antigen in agar (Noble agar 1.5')0, w/v, in 0.1M phosphate buffer, pH 7.2) containing antiserum (Z67 sheep antirabbit IgG: 0.36 ml in 12 ml agar). This technique was developed by Mancini et al 15. Qualitative characterization of isolated antibody or antigen was achieved by double diffusion in agar (Noble
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Pur!fication of the 7-globulin j?action of rabbit ala3/b4 serum To rabbit ala3/b4 serum (RB 9675) was added dropwise an equal volume of saturated ammonium sulphate solution. After leaving at 4°C for 2 h to obtain maximum precipitation of the 7-globulin, the precipitate was centrifuged and washed once with 50~o saturated ammonium sulphate solution, then dissolved in phosphate buffer (0.01 M, pH 7.1) to the volume of the original serum. The 7globulin solution was dialysed against phosphate buffer (0.01 M, pH 7.1) at 4°C for 24 h with several changes of buffer, then purified further by stirring batchwise with DEAE-cellulose (Whatman DE52) equilibrated in phosphate buffer (0.01 M, pH 7.1). After leaving for 4 h at 4°C, then centrifuging, the supernatant was concentrated to a volume of 5 ml by ultrafiltration. The ~-globulin solution was centrifuged to remove denatured material and the IgG concentration determined by measuring absorbance at 280 nm, using a n El1'c'm for rabbit IgG of 14.6 (Ref 18). The purity of the rabbit ala3/b4 IgG was tested by immunoelectrophoresis against a polyvalent sheep antiserum to rabbit serum proteins (Z68), with rabbit whole serum for comparison. The presence of a single arc in the 7-region was indicative of a pure preparation. Chemical coupling o]" rabbit ala3/b4 IyG to cellulose carbonates Macroporous cellulose trans-2,3-carbonate. A solution of rabbit al a3/b4 IgG (2.95 mg ml- 1 in 0.01 M phosphate buffer, pH 7.1; 3.0 ml), derived from 10 ml of whole serum, was mixed with phosphate buffer (0.1 M, pH 6.8; 5.0 ml). Macroporous cellulose carbonate (DS 0.25; 200 mg) was added and the mixture stirred magnetically for 24 h at 2OC. After centrifuging, the supernatant was retained for measurement of IgG content and the product was washed with phosphate-buffered saline (PBS; 8 mM Na2HPO4.12H20; 3 mM NaHzPO4.2H20 , 0.15 M NaC1; 4 x 3 ml) to remove any non-specifically bound material. The washings were pooled and retained for IgG measurement. The macroporous cellulose carbonate,." (ala3/b4 IgG) product was suspended in 3.0 ml PBS and packed into a Pasteur pipette above a layer (0.3 ml settled volume) of Bio-Gel P-6. The immunoadsorbent column (0.75 ml settled volume) was washed with PBS. The amount of rabbit ala3/b4 IgG bound to the macroporous cellulose trans-2,3-carbonate was estimated by radial immunodiffusion assay [against sheep antirabbit IgG antiserum (Z67)] by comparison between the total IgG used in the coupling reaction and the residue in the supernatant and washings. This amount was calculated to be 3.95 mg (Table 1).
Production of immunoadsorbents: John F. Kennedy et al. Table 1 Use of macroporous cellulose trans-2,3-carbonate (MCC) in the purification of rabbit b4 (h-) IgG
Operation
Material
Ammonium sulphate IgG solution precipitation of rabbit ala3/b4 serum (10.0 ml) Preparation of MCC/ MCC (ala3/b4 IgG) column IRabbit ala3/b4 IgG (added to MCC] Rabbit ala3/b4 IgG (bound to MCC) Application of rabbit anti-b4 antiserum to MCC/(ala3/b4 IgG)
Rabbit a2/b6 anti-b4 serum applied Protein in glycine/HC1 eluate
Preparation of MCC/ MCC (anti-b4 IgG) column Rabbit anti-b4 IgG (added to MCC) Rabbit anti-b4 IgG (bound to MCC) Application of rabbit Rabbit a2/b4 IgG a2/b4 IgG to MCC/ applied (anti-b4 IgG) column
Total Volume yield (ml) (rag) 5.0
14.8
3.0
200.0 8.9
bance of the unbound material (PBS eluate) and bound anti-b4 I g G (glycine/HC1 eluate) was measured at 280 nm and the total protein determined (Table 2). For the macroporous adsorbent the amount of anti-b4 I g G eluted was very small and the protein content was confirmed by Lowry assay 19 using rabbit IgG (D68; standards at concentrations of 0.25, 0.13 and 0.06 mg ml ~; Table 1). The PBS eluate and glycine/HCl eluate from each column was tested for the presence of anti-b4 lgG antibody activity by immunodiffusion against b4 IgG (Tables 3 and 4). For the macroporous cellulose trans-2,3-
4.0 Table 2 Use of microcrystalline cellulose trans-2,3-carbonate (CC) in the purification of rabbit b4 (h-) IgG
2.0 1.0
0.4
0.8
27.3 0.3 Trace
1.0
0.5
Microcrystalline cellulose trans-2,3-carbonate. Rabbit ala3/b4 IgG (27.9 mg/ml in 0.01 M phosphate buffer, pH 7.1; 3.2 ml) was mixed with phosphate buffer (0.1 M, pH 6.8; 20.0 ml). Microcrystalline cellulose trans-2,3carbonate (DS 0.22; 1.6 g) was added and the mixture stirred magnetically for 24 h at 20°C. The product was centrifuged, washed with PBS (4 × 10 ml), suspended in PBS (15.0 ml) then packed into a column (9.5 cm x 1.1 cm) above a layer of Bio-Gel P-6 (2 ml settled volume). From Mancini assay it was determined that 62.8 mg of rabbit ala3/b4 IgG was bound to the microcrystalline cellulose trans-2,3-carbonate (Table 2). Isolation of rabbit anti-b4 IgG antibody usin9 the macroporous cellulose carbonate/(ala3/b4 IgG) and microcrystalline cellulose carbonate/(ala3/b4 lgG) immunoadsorbent columns Each column was equilibrated by washing overnight with PBS, at a flow rate of 10 ml h-1, until a constant baseline absorbance was obtained at 280 nm. Rabbit antib4 serum (RB 8006/II allotypic specificity a2/b6) was alternately frozen and thawed to inactivate complement then applied to the respective columns in the amounts indicated in Tables 1 and 2. Each column was washed with PBS and fractions collected on a time basis (25 min) until the first peak (of unbound anti-b4 IgG and other serum proteins) was eluted as detected by absorbance at 280 nm. Bound rabbit anti-b4 antibody was eluted from each column with glycine/HCl buffer (0.1 M, pH 2.2). The fractions containing the PBS-eluted proteins were pooled and concentrated to the volumes shown in Tables 1 and 2. For each column the fractions containing the glycine/HCl-eluted protein (antibody) were pooled, concentrated by ultrafiltration then dialysed against Tris/HCl buffer (0.2 M, pH 8.0) for several days and centrifuged. For the microcrystalline derivative the absor-
Operation
Total Volume yield (ml) (mg)
Material
Ammonium sulphate IgG solution precipitation of rabbit ala3/b4 serum (30.0 ml) Preparation of CC/ CC (ala3/b4 IgG)column IRabbit ala3/b4 IgG (added to CC) Rabbit ala3/b4 IgG (bound to CC) Application of rabbit anti-b4 antiserum to CC/(ala3/b4 IgG] column
3.5
97.8
3.2
1600.0 89.4 62.8
Rabbit a2/b6 anti-b4 serum applied Protein in glycine/ HCI eluate
30.0
Preparation of CC/ CC (anti-b4 IgG) column Rabbit anti-b4 IgG (added to CC) Rabbit anti-b4 lgG (bound to CC)
19.2
8.8
18.7
200.0 8.6 2.0
1.0
Application of rabbit Rabbit a2/b4 |gG a2/b4 serum to CC/ applied (anti-b4 IgG) column b4 (•) IgG isolated b4-negative ()~) IgG isolated
9.0 3.0 0.6
Table 3 Use of macroporous cellulose trans-2,3-carbonate in the purification of b4 (K) IgG: immunodiffusion results
Operation
Sample
Application PBS eluate of rabbit anti-b4 Glycine/HCl antiserum to eluate MCC/(a 1a3/b4 lgG) column
Z67 sheep Rabbit b4 anti-rabbit RB9376 IgG IgG anti-b4 + +
Application PBS eluate of rabbit a2/b4 IgG to Glycine/HCl MCC/(anti-b4 eluate IgG] column
+
++
+
+, Weakprecipitin line; + +, strong precipitinline
Int. J. Biol. Macromol., 1980, Vol 2, June
139
Production of immunoadsorbents: John F. Kennedy et al. Table 4 Use of microcrystalline cellulose trans-2,3-carbonate in the purification of b4 0c) IgG: immunodiffusion results
Operation
Sample
Application of rabbit anti-b4 antiserum to CC/(a 1a3/b4 IgG) column
PBS eluate Glycine/HCl eluate
Application of rabbit a2/b4 serum to CC/(anti-b4 lgG) column Initial PBS eluate application Glycine/HCl eluate
Z67 sheep Rabbit b4 RB 9376 anti-rabbit lgG anti-b4 lgG
+
Application of rabbit a2/h4 h lG to the macroporous cellulose trans-2,3-carbonate/(anti-h4 l qG) and microcrystalline cellulose trans-2,3-carbonate/(anti-h4 l qG) immunoadsorbeitt columns Macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG). Rabbit a2/b4 IgG was obtained by precipitation of
+ + + +
1st reapplication
PBS eluate Glycine/HC1 eluate
+ + + +
2nd reapplication
PBS eluate Glycine/HCl eluate
+ + + +
4th reapplication
PBS eluate Glycine/HCL eluate
200 mg) was added and the mixture stirred magnetically for 24 h at 2 0 C . The product was centrifuged, the supernatant removed and the adsorbent washed with PBS (4 x 3 ml). The washings were concentrated by ultrafiltration to 3.0 ml. The supernatant and washings from the coupling reaction were dialysed against PBS for 2 days with several changes of buffer and the protein concentration determined by measuring absorbance at 280 nm. The protein content of the original anti-b4 IgG solution used in the coupling reaction was also determined. The amount of anti-b4 antibody coupled to the cellulose carbonate was calculated 12 mg; Table 2). A column of the adsorbent was prepared in a Pasteur pipette above a layer of Bio-Gel P-6 (0.5 ml when packed).
+
+ +
+ , Weak precipitin line; + + , strong precipitin line; + + + . very strong precipitin line; , no precipitation
carbonate/(ala3/b4 IgG)adsorbent, the PBS eluate retained anti-b4 antibody activity indicating that not all of the applied antibody bound to the column. The acid eluate also contained anti-b4 antibody activity. The PBS eluate from the microcrystalline cellulose carbonate/(ala3/b4 IgG) column did not contain any anti-b4 antibody activity detectable by immunodiffusion therefore all anti-b4 IgG was bound to the adsorbent (Figure 1). The presence of anti-b4 antibody activity was demonstrated in the glycine/HC1 eluate.
a2/b4 serum (RB 913 a2/b4) with an equal volume of saturated a m m o n i u m sulphate solution as described previously. An aliquot of the IgG solution (1.0 ml: absorbance of 0.47 mg at 280 n m = 0.680) was applied to the macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG) adsorbent, which was washed with PBS to obtain unbound material [b4-negative (2 chain) IgG and unbound b4 IgG]. Bound b4 (h') lgG was eluted with glycine/HCl buffer (0.1 M, pH 2.2). The PBS eluate was concentrated by ultrafiltration to 1.3 ml. The glycine/HCl eluate was also concentrated to 1.3 ml, then dialysed against PBS overnight.
Microcrystalline cellulose trans-2,3-carbonate/(anti-b4 l qG). Rabbit a2/b4 serum (RB 913 a2/b4; 1.0 ml containing 9.0 mg IgG) was applied to the microcrystalline cellulose trans-2,3-carbonate/(anti-b4 IgG) adsorbent. A large peak, due to unbound b4 IgG and other serum proteins, was obtained by washing the column with PBS. Bound b4 (h) IgG was obtained by elution with glycine/HC1 (0.1 M, pH 2.2). The PBS eluate was concentrated by ultrafiltration to 2.3 ml. The glycine/HCl eluate was concentrated, then dialysed against Tris/HCl (0.2 M, pH 8.0), giving a final volume of 2.6 ml.
Chemical couplin,q of rabbit anti-b4 IgG antibody to cellulose carbonates Macroporous cellulose trans-2,3-carbonate. Rabbit anti-b4 IgG antibody, purified on the macroporous cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent column (0.8 ml, 0.3 rag), was mixed with phosphate buffer (0.1 M, pH 6.8; 2.0 ml). Macroporous cellulose carbonate (DS 0.25; 27.3 mg) was added and the mixture shaken for 24 h at 2 0 C . The product was centrifuged, then washed with PBS (4 ×2 ml). The macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG) adsorbent was suspended in PBS then packed in a Pasteur pipette above a layer of Bio-Gel P-6 (0.75 ml when settled).
Microcrystalline cellulose trans-2,3-carbonate. Rabbit anti-b4 IgG antibody purified on the microcrystalline cellulose carbonate/(ala3/b4 IgG) immunoadsorbent column (18.7 ml, 8.6 mg) was taken and the volume made up to 19.0 ml with phosphate buffer (0.1 M, pH 6.8). Microcrystalline cellulose trans-2,3-carbonate (DS 0.22,
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Int. J. Biol. Macromol., 1980, Vol 2, June
Figure i
(at Isolation of rabbit anti-b4 antibody on the
CC/ala3/b4 IgG} immunoadsorbent. Note the precipitin line obtained with the glycine/HCl eluate (anti-b4 antibody; well 3) with rabbit b4 lgG (1 mg/ml; well I) forming a line of identity with rabbit anti-b4 serum control (well 2), and the absence of anti-b4 antibody activity in the PBS eluate (non-bound material; well 4). (b) Isolation of rabbit b4 tl,) lgG on the CC/(anti-b4 IgGl immunoadsorbent. Outer w e l l s doubling dilutions of glycine/HCl eluate (b4 (h) lgG). Centre well rabbit ala2/b5b6 anti-b4 antiserum
Production of immunoadsorbents.: John F. Kennedy et al. Since [he PBS eluate was subsequently found to contain b4 (~c) IgG by immunodiffusion, the unbound material was re-applied to the column four times, eluting first with PBS and secondly with glycine/HCl buffer (0.1 M, pH 2.2) each time. The PBS and acid eluates were concentrated after every application and the acid peak dialysed against Tris/HCl buffer (0.2 M, pH 8.0). The concentrated eluates were tested each time for the presence of b4 (K) IgG by immunodiffusion. The amount of protein in the pooled acid eluates was determined by absorbance measurement at 280 nm (Table 2). The protein content of the final PBS eluate was determined from absorbance at 280 nm after precipitation with an equal volume of saturated ammonium sulphate solution and dialysis against phosphate buffer (0.01 M, pH 7.1) to obtain the 7-globulin fraction as described previously. The amount of b4-negative (2) IgG isolated is indicated in Table 2 (0.6 mg).
Detection of b4 allotypic determinants in the PBS and glycine/HCl eluates by immunodiffusion Macroporous cellulose trans-2,3-carbonate/(anti-b4 lgG). After 24 h the PBS eluate (unbound material) gave precipitin lines with sheep anti-rabbit IgG antiserum (Z67) and rabbit anti-b4 antiserum (RB 9376). A faint line due to interaction of the glycine/HCl eluate with sheep anti-rabbit IgG antiserum (Z67) was observed (Table 3).
M icrocrystalline cellulose trans-2,3-carbonate/(anti-b4 log ). The PBS and glycine/HCl eluates were tested after each application to the adsorbent for the presence of b4 IgG by precipitation with rabbit anti-b4 antiserum (RB 9376; Table 4, Figure 1). After the fifth application to the column the PBS eluate was also tested for the presence of rabbit IgG. A precipitin line was observed after 24 h against sheep anti-rabbit IgG antiserum (Z67). It was noted that the PBS peak showed no b4 activity after the fifth application to the column and all b4 activity was assumed to have been removed leaving only b4-negative ()0 IgG (Table 4). The acid eluate, after each application of the PBS eluate to the column, was shown to contain b4 (K) IgG by the presence ofa precipitin line against rabbit antib4 antiserum (RB 9376; Table 4).
carbonate was 0.25 and 0.22, respectively. However, the percentage of ala3/b4 IgG coupled was much higher for microcrystalline cellulose carbonate (Table 5), which may be accounted for by the greater external surface area per unit volume of the microcrystalline derivative. Rabbit anti-b4 antiserum was applied to the cellulose trans-2,3-carbonate/(ala3/b4 IgG)immunoadsorbents~ From Table 3 it may be seen that a relatively greater amount of anti-b4 IgG antibody was eluted with glycine/HCl buffer from the microcrystalline cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent. The ratio of anti-b4 IgG eluted (mg) to al a3/b4 IgG (rag) coupled to the cellulose trans-2,3-carbonate was calculated for both the macroporous and microcrystalline supports (1:10 and 1:7, respectively). However, it must be noted that the latter value is a minimum estimate since the binding capacity of the adsorbent was not exceeded. The eluant was the same for both adsorbents, therefore the microcrystalline cellulose trans-2,3-carbonate/(ala3/b4 IgG) immunoadsorbent was the more efficient for the purification and isolation of rabbit anti-b4 antibody, and the antigenic sites were probably less sterically hindered in the microcrystalline derivative. Immunodiffusion results (Table 3) for the glycine/HC1 and PBS eluates from the macroporous immunoadsorbent indicated that not all of the rabbit anti-b4 IgG antibody applied to the column was adsorbed on first passag e, but the immunodiffusion results for the corresponding eluates from the microcrystalline immunoadsorbent (Table 4) indicated that with this matrix all of the anti-b4 IgG antibody applied to the column had been adsorbed as no residual anti-b4 antibody activity was detectable in the PBS eluate. The purified anti-b4 IgG antibody eluted from the macroporous and microcrystalline immunoadsorbents was coupled to macroporous cellulose trans-2,3-carbonate and microcrystalline cellulose trans-2,3-carbonate, respectively. The ratio of anti-b4 IgG (mg) to cellulose trans-2,3carbonate (mg) used in the coupling reaction was 1:91 for the macroporous derivative and 1:23 for the microcrystal-
Discussion
The purification of rabbit b4 (~-) IgG using cellulose carbonate derived from macroporous cellulose and microcrystalline cellulose was effected in the following stages: (a) preparation of a cellulose trans-2,3-carbonate/(ala3/b4 IgG)immunoadsorbent; (b) application of rabbit anti-b4 IgG antiserum to the above immunoadsorbent and its elution as a purified antibody; (c) preparation of a cellulose trans-2,3-carbonate/(antib4 IgG) immunoadsorbent; (d) application of rabbit a2/b4 IgG to the adsorbent prepared in (c) and elution of bound b4 (~) IgG with glycine/HCl buffer, pH 2.2. The results of each stage, including amount of antigen or antibody coupled or bound, are given in Tables 1 and 2. The weight ratio of ala3/b4 IgG to cellulose trans-2,3carbonate used in the coupling procedure was similar for the macroporous and microcrystalline derivatives, and the degree of cyclic carbonate substitution for the macroporous cellulose carbonate and microcrystalline cellulose
Comparison of immunoadsorbents prepared using macroporous cellulose trans-2,3-carbonate and microcrystalline cellulose trans-2,3-carbonate Table 5
MCC Cellulose trans-2,3-carbonate/(ala3/b4 IgG) Ratio ala3/b4 IgG added (mg)/cellulose trans2,3-carbonate (mg) 1:23 IgG bound (%) 44.6 Ratio ala3/b4 IgG coupled (mg)/cellulosetrans-2,3-carbonate (mg) 1:51 Anti-b4 IgG antibody eluted (rag) 0.4 Ratio anti-b4 IgG eluted (mg)/coupled ala3/b4 IgG (mg) 1:10 Cellulose-trans-2,3-carbonate/[antl-b4 lgG) Ratio anti-b4 IgG added (mg)/cellulose trans2,3-carbonate (mg) 1:91 IgG bound (%) trace
CC
1:18 70.2 1:39 8.8 1:7" 1:23 23.3
* minimum value since binding capacity of column was not exceeded
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Production of immunoadsorbents: John F. Kennedy et al. line derivative (Table 5). Only a trace of anti-b4 antibody was successfully coupled to the macroporous cellulose trans-2,3-carbonate (since bound a2/b4 IgG was eluted with glycine/HCl after application to the immunoadsorbent column) but the amount of coupled antibody could not be determined accurately. However, 2 mg (23!~,) of the available anti-b4 IgG antibody was coupled to microcrystalline cellulose trans-2,3-carbonate, giving a ratio of antib4 antibody (mg) to cellulose trans-2,3-carbonate (mg) of 1:100. On application of rabbit a2/b4 lgG to the macroporous cellulose-2,3-carbonate/(anti-b4 IgG) immunoadsorbent, one peak was obtained on washing with PBS (unbound material) and a second, smaller peak was eluted with .glycine/HC1 pH 2.2. The peaks obtained were then tested for b4 activity by immunodiffusion (Table 3). The PBS eluate still reacted strongly with anti-b4 antiserum showing the presence of residual unbound b4 (K) IgG. The acid peak, containing ---0.5 mg protein, did not give a precipitin line with anti-b4 antiserum but a line was obtained against sheep anti-rabbit IgG antiserum, indicating that b4-negative rabbit immunoglobulin was present. Rabbit a2/b4 serum was also applied to a column of microcrystalline cellulose trans-2,3-carbonate/(anti-b4 IgG). Five applications were necessary to remove all of the b4 (~c)IgG, and the decrease in the b4 IgG content of the PBS eluate on each application to the column can be seen from the immunodiffisuon results (Table 4). Glycine/HC1 buffer pH 2.2 was used to elute the bound b4 (K) IgG each time. Originally, 9.0 mg of a2/b4 IgG were applied to the column, and totals of ~ 3 mg of b4 (t,) IgG and 0.6 mg of b4-negative (2) IgG were isolated (Table 2), giving a final recovery of ~40°o. The presence of rabbit IgG was detected in the final PBS eluate (Table 4) in the absence of detectable b4 (K) determinants. This is consistent with the isolated material containing only b4-negative ().) IgG molecules. Losses would be expected to occur through dialysis, ultrafiltration and transference from one vessel to another. From Table 5 it can be seen that the trans-2,3carbonate derived from microcrystalline cellulose is superior to the macroporous derivative with respect to chemical coupling of rabbit ala3/b4 IgG antigen and binding of rabbit anti-b4 antibody. The microcrystalline cellulose carbonate is also more effective for the coupling of specific anti-b4 antibody and subsequent binding and elution of b4 (K) IgG. Use of the more efficient microcrystalline cellulose carbonate in the preparation of im-
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munoadsorbents for the purification of b4 (K) lgG provides a powerful technique for the separation of rabbit immunoglobulin expressing kappa light chain (90'};i of total immunoglobulin) from rabbit immunoglobulin expressing lambda light chain (10~0 of total immunoglobulin) thus enabling studies on immunological and chemical properties of the separate populations of molecules to be performed. The isolation of immunoglobulin expressing 2 light chain from normal rabbit immunoglobulin by immunoadsorption, as opposed to exclusive production of immunoglobulin with 2 light chain by K-suppression in rabbit, facilitates the study of )~ chain allotypic determinants in the normal situation and the production of specific antisera against these determinants. This work has demonstrated the use of allotypic specificities of rabbit immunoglobulin populations and may be extended to the use of different allotypic systems and antibody antigen populations.
References 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 19
Oudin, J. C.R. Acad. Sci. Paris 1956, 242, 2489 Oudin, J. C.R. Acad. Sci. Paris 1956, 242, 2606 Oudin, J. J. Exp. Med. 1960, 112, 125 Dray, S., Dubiski, S., Kelus, A., Lennox, E. S. and Oudin, J. Nature (London) 1962, 195, 785 Appella, E., Chersi, A, Rejnek, J. and Reisfeld, R. A. in 'Developmental Aspects of Antibody Formation and Structure' (Eds J. Sterzl and J. Riha) Academic Press, New York, 1970, Vol. 1, p. 327 Hood, L., Waterfield, M. D., Morris, J. and Todd, C. W. Ann. N Y Acad. Sci. 1971, 190, 26 Chersi, A., Mage, R. G. and Appella, E. Fed. Proc. 1971, 30, 1307 Nisonoff, A., Hopper, J. E. and Spring, S. B, 'The Antibody Molecule' Academic Press, New York, 1975 Kindt, T. J. Adv. lmmunol. 1975, 21, 37 Mole, L. E. Biochem. Soc. Trans. 1976, 4, 33 Barker, S. A., Cho, Tun H., Doss, S. H., Gray, C. J. and Kennedy, J. F. Carbohydr. Res. 1971, 17, 471 McLaughlan, P., Stanworth, D. R., Kennedy, J. F. and Cho Tun, H. Nature New Biol. 1971, 232, 345 Catty, D., Kennedy, J. F., Drew, R. L. and Cho Tun H. J. tmmunol. Methods 1973, 2, 353 Kennedy, J. F., Barker, S. A. and Rosevear, A. J. Chem. Soc. Perkin Trans. 1. 1973, 2293 Mancini, G., Carbonara, A. O. and Heremans, J. F. lmmunochemistry 1965, 2, 235 Ouchterlony, O. Progr. Allergy 1958, 5, 1 Graber, P. and Williams, C. A. Biochim. Biophys. Acta 1953, 10, 193 Grossberg, A. L., Radzimski, G. and Pressman, D. Biochemistry 1962, 1,391 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. d. Biol. Chem. 1951, 193, 265