Journal o f Immunological Methods, 23 (1978) 127--135 © Elsevier/North-Holland Biomedical Press
127
AN IMMUNOPRECIPITATION-DISSOCIATION TECHNIQUE FOR L A R G E SCALE ANTIBODY PURIFICATION AND AN ANTIGEN CONSUMPTION ELECTROIMMUNOASSAY FOR ANTIBODY QUANTITATION. A MODEL STUDY WITH ANTIBODIES TO PREGNANCY ZONE PROTEIN
J. F O L K E R S E N 1, B. TEISNER 1, p. S V E N D S E N 2, and S.-E. SVEHAG 1 1 Institute o f Medical Microbiology, Odense University, Odense and 2 Experimental Animal Unit, Odense University, Odense, Denmark
(Received 19 January 1978, accepted 24 March 1978) A simple immunoprecipitation---dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.
INTRODUCTION The basis for many immunological methods is the production of monospecific antibodies of high purity. Other contaminating proteins and nonreacting immunoglobulins can reduce the sensitivity and precision of RIAtests and affinity chromatographic techniques (Heinzel et al., 1976). Purification of immunoglobulins based on their physico-chemical properties often results in suboptimal reagents. The use of specific solid-phase immunosorbent principles for antibody purification (Cuatrecasas, 1969; Givol, 1970) are often necessary to obtain specific immunoglobulin preparations of satisfactory quality. The usefulness of solid-phase i m m u n o s o r b e n t techniques for antibody purification is limited, however, when labile antigens are destroyed during antibody elution. Furthermore, the effectiveness of purification is highly dependent upon the purity of the antigen preparation employed. Direct precipitation of the antibodies in monospecific antisera by soluble antigen in liquid phase systems is less demanding with regard to antigen purity and may be performed on a large scale after preliminary determination of the equilibrium point on a quantitative precipitation curve. The isolated immune precipitate can be dissociated by a variety of procedures including elevated temperatures (Korngold and Pressman, 1953), high
128 ionic strength (Heidelberger and Kabat, 1938) and changes in pH (Chow and Wu, 1936; Kabat, 1939; Lee and Wu, 1940; Singer and Campbell, 1955). Final separation of antigen and antibodies can be achieved by e.g. conventional column chromatography. In our work with pregnancy zone protein (PZP), which is labile, difficulties were encountered in finding an elution procedure which would retain the antigenicity of PZP as a ligand in solid-phase immunosorbent techniques to obtain monospecific anti-PZP antibodies of high purity. A liquid-phase immunosorbent precipitation technique was developed, therefore, for large scale purification of anti-PZP antibodies directly from immune serum. This m e t h o d and a new antigen-consumption electroimmunoassay for antibody quantitation are described in this report. MATERIALS AND METHODS
Partial purification of PZ-protein for immunization Sera were obtained from pregnant women in the 3rd trimester. One volume serum was diluted with 1.25 volumes of 0.03 M NaK--phosphate buffer (pH 7.2), aetacridine lactate was added to a final concentration of 0.4% w/v and pH was adjusted to 6.0. The mixture was incubated for 30 min and the resulting precipitate pelleted at 2500 × g for 15 min. The precipitate was redissolved in twice the original serum volume of 0.83 M NaC1 with the aid of a whirlmixer and end-over-end mixer and centrifuged at 1500 X g for 15 min. The supernatant was collected and dialysed overnight against 0.03 M NaK--phosphate buffer (pH 7.2). The dialysed sample was filtered on Sepharose 6BCL and fractions tested by fused rocket immunoelectrophoresis using a monospecific rabbit anti-human PZ-protein serum. PZ-protein containing fractions were concentrated, dialysed against PBS and sterile filtered.
PZ-protein determinations PZ-protein concentration was determined by rocket immunoelectrophoresis (Laurell, 1966) using a monospecific rabbit antiserum to human PZprotein. The electrophoresis was performed with 0.028 M barbital buffer, pH 8.6 at 1 V/cm gel for 18 h. PZ-protein concentration was expressed in arbitrary PZP units per ml (APU/ml). The lower limit of detection was ½ APU. In the range 6--80 APU, the coefficient of variation was less than 3%.
Production of monospecific goat antiserum to human PZ-protein Goats were given weekly multiple subcutaneous injections (100 APU/ injection) of the partially purified PZ-protein in Freund's complete adjuvant. The antiserum was adsorbed with normal human male serum for 2 h at room temperature and overnight at 4°C. The serum mixture was centrifuged at 110,000 X g for 1 h and the absorbed serum was tested for monospecificity by crossed immunoelectrophoresis against undiluted pregnancy serum {Fig. 1).
129 l
Z
Fig. 1. Crossed immunoelectrophoresis with goat-anti-PZP-antiserum diluted 1 : 100 in the second dimension gel. Ten microlitres of a PZP-containing human serum was applied in the well.
Quantitation of antibodies to PZ-protein An antigen-consumption electroimmunoassay (ACE) was developed for antibody quantitation. One antigen consumption unit (ACU) was defined as the a m o u n t of antibody which reduced the PZ-protein c o n t e n t in a standard preparation by one APU as determined by quantitative rocket immunoelectrophoresis. The specific antibody activity was expressed in ACU/ml total protein. Two antigen standards A (150 APU/ml) and B (75 APU/ml) and one antibody standard (62 ACU/ml) were included in all ACE-assays. The sample to be tested for antibody activity was diluted 2-fold using 300 pl/ tube, and 300 pl of antigen standard A was added to each tube. An additional tube received 300 gl of standard A plus 300 pl of antibody standard. These tubes and standard B were incubated overnight at 4°C in an end-over-end mixer and were then examined by quantitative rocket immunoelectrophoresis (Fig. 2). The concentration of free PZ-protein in APU/ml was determined for all samples. The antibody activity in the test sample was determined by the use of the formula: (RD - R n ) X fX k = ACU/ml where Rb = APU/ml in standard B, Rn = APU/ml in the first tube in the dilution series where APU/ml exceeded 6, f = the final dilution factor of test
130
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Fig. 2. E x a m p l e o f design used in t h e ACE-assay for d e t e r m i n a t i o n of a n t i b o d y activity. S t a n d a r d B c o n t a i n s 75 A P U a n d t h e r o c k e t c o r r e s p o n d i n g to a s e r u m d i l u t i o n of 8 c o n t a i n e d 25 APU. T h e r e f o r e , t h e a n t i b o d y activity was ( 7 5 - - 2 5 ) x 8 x 1 = 4 0 0 ACU, p r o v i d e d t h e c o r r e c t i o n f a c t o r was 1.
sample in tube n, and k is a correction factor (RAc/R1Ac) where RAC = APU/ml in standard A + C and R~AC = ~ of APU/ml in standard A + C measured in 10 tests performed on different days.
Exposure o f goat an tibodies at low p H The effect of exposure at low pH on the antigen binding capacity of goat antibodies to PZ-protein was evaluated in the ACE-assay. Immune serum diluted I : 16 in 0.9% NaC1 was exposed to pH 2.3 for 5 h followed by pH 3.1 for 19 h. Samples were collected at several time intervals, the pH was adjusted to neutrality and the activity of the samples was tested by the ACEassay.
Purification o f antibodies to PZ-protein by immunoprecipitation and recovery o f the antibody fraction by exclusion chromatography at low pH Pregnancy serum, collected in the 3rd trimester, was centrifuged at 110,000 X g for 1 h and the supernatant was mixed with an a m o u n t of monospecific goat antiserum to PZ-protein corresponding to the equilibrium point on a quantitative PZ--anti-PZ precipitation curve. The mixture was incubated for 2 h at room temperature and overnight at 4°C and then centrifuged at 110,000 X g for 1 hour. The precipitate was washed twice with PBS and resuspended in 2 volumes of HCl---glycine buffer (pH 2.3), with a whirlmixer. The pH was readjusted to pH 2.3 after 15 min and dissociation allowed to proceed for 5 h at room temperature with magnetic stirring. The suspension was then centrifuged at 8,000 X g for 15 min and the
131
Fig. 3. Test for antibody identity using crossed immunoelectrophoresis with intermediate gel. Upper 2nd dimension gel contains reference rabbit-anti-human PZP-antiserum diluted 1 : 100 and intermediate gel contains goat anti-human PZP-serum diluted 1 : 100. Ten microlitres of a PZP containing human serum was applied in the well.
supernatant was applied to a Sepharose 6BCL (50/100) column equilibrated with HC1--glycine buf f e r (pH 3.1). The flow rate was 95 ml/h, corresponding to a total time of a n t i b o d y exposure at this pH of about 10 h. The fractions were collected in test tubes containing sufficient NaOH--glycine buffer pH 10 to bring the pH back to a b o u t 7. All protein-containing fractions were tested by rocket i m m u n o e l e c t r o p h o resis against rabbit anti-whole h u m a n serum, rabbit anti-goat 7-globulin serum, rabbit anti-whole goat serum and rabbit anti-human PZ-protein serum. Fractions containing goat 7-globulin were tested for ant i body activity by means o f the ACE assay.
Crossed immunoelectrophoresis Crossed immunoelectrophoresis with intermediate gel (Axelsen, 1973a) was used to com pa r e the reactivity o f the goat ant i body preparations towards PZ-protein with a reference antiserum kindly supplied by Bo von Schoultz, Ume~, Sweden. The prepared a n t i b o d y preparation showed a reaction o f identity when tested against the reference antiserum (Fig. 3). Protein determinations Th e protein concentrations were det er mi ned s p e c t r o p h o t o m e t r i c a l l y by reference to a standard curve based on bovine serum albumin.
132 RESULTS
Stability of goat anti-PZ antibodies and PZ-protein during prolonged exposure to low pH Goat immune serum and PZ-protein were exposed to low pH for varied times to establish the dissociation conditions to be used in large scale purification of the antibodies by the immunoprecipitation--dissociation technique. Goat immune serum to PZ-protein exposed to pH 2.3 for 5 h followed by 10 h at pH 3.1 (conditions corresponding to those in subsequent antibody purification experiments), lost at most 15% of the original PZ-binding capacity as indicated by the ACE-assay (Fig. 4). PZ-protein antigenicity, however, was undetectable after lowering the pH of a PZ-containing serum to 2.3 for less than half an hour.
Antigen-consumption electroimmunoassay for antibody quantitation The antigen-consumption electroimmunoassay (ACE-assay) was used to determine the antibody activity in the antisera used and to monitor the fractions obtained during large scale purification of antibodies to PZ-protein. The immune sera to PZP from the four goats used for the antibody production contained 260--620 ACU/ml. The coefficient of variation for the determination was 4%.
Large scale antibody purification by immunoprecipitation--dissociation technique Goat immune serum monospecific for PZ-protein was reacted with a Relot ive oss of antibod aclivily
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133
volume o f pregnancy serum corresponding to the equilibrium point on the quantitative precipitation curve. No PZ-protein or antibody activity was demonstrable in the supernatant after incubation and centrifugation of the mixture. The antibody activity in AC units of the re-neutralized samples was determined at different time intervals during dissociation of the immunoprecipitate at pH 2.3. A relatively constant level of ACU was observed during
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Fig. 5. U l t r a v i o l e t - e l u t i o n profile for f r a c t i o n a t i o n o f dissociated and precentrifuged i m m u n e c o m p l e x e s on a S e p h a r o s e 6 B C L c o l u m n . H a t c h e d c o l u m n s indicate a n t i b o d y purification factor. Eluted fractions were tested b y r o c k e t i m m u n o e l e c t r o p h o r e s i s using antiserum to w h o l e goat serum ( A ) and t o w h o l e h u m a n serum (B). The proteins d e t e c t e d o n plate A had the m o b i l i t y o f 7-globulins.
134
the first 4 h, but this increased about 4-fold from the 5th hour to the time of centrifugation. No antigen activity in APU were detected in the supernatant and the redissolved precipitate contained only a few per cent of the total antibody-ACU recovered in the supernatant. When the antibody-containing supernatant was gel filtered on an acid Sepharose 6BCL column the ACU-activity was recovered in the fractions corresponding to the major protein peak (Fig. 5). Rocket immunoelectrophoresis with rabbit antisera to whole goat serum and goat 7-globulin showed that this peak contained primarily goat IgG (Fig. 5). Further immunoelectrophoretic analysis of the fractions by the use of rabbit antiwhole human serum showed traces of unidentified human proteins in the same region. When the fractions with the highest ACU-activity were pooled and dialysed against PBS a slight precipitate was formed. Removal of the precipitate by centrifugation increased the specific antibody activity (ACU/mg protein) in the supernatant by 40--50%. The finally concentrated goat IgG antibody preparation showed an average 60-fold (range 50--64) increase in ACU/mg protein when compared to the original immune serum. The total recovery in ACU varied from 28% to 38% for the immune sera from 4 goats used in the purification experiments. The purified antibody preparation showed no loss of activity when stored at -20°C for 2 months. In subsequent work, the antibody preparations have been coupled to cyanogen bromide activated Sepharose 4B. Immunosorbent columns prepared from these preparations have been kept at 4°C and used in PZ-protein purification studies over a period of 6--7 months without significant loss of PZ-protein binding capacity. DISCUSSION
The 2-step method for purification of goat immunoglobulin to PZ-protein is expeditious and simple and may be used on a large scale since the only limiting factor is the column capacity in step 2. No pre-purification of the 3,-globulin fraction is necessary. The average recovery of antibody activity, measured in ACU, exceeded 30% which was considered satisfactory; the purification factor was about 60. The final antibody preparation was stable on storage and has been found well suited for use in solid phase immunosorbent techniques and in a radioimmunoassay for PZP-determination. The choice of dissociation conditions in the purification procedure was based solely on results obtained when testing the antigen-binding capacity of the various fractions in the ACE-assay. Possible reversible or irreversible alterations of the immunogIobulin structure not registered in this assay were not evaluated. The antigen-binding activity {in ACU) of goat-IgG antibodies showed great stability to exposure of low pH over several hours provided the experiments
135
were performed on diluted (1 : 16) serum. A new assay system for determining the antigen-binding activity of the immune serum and antibody-containing fractions was developed based on the electroimmunoassay principle. The recently described 'reversed rocket immunoelectrophoresis' (Axelsen and Svendsen, 1973b) was n o t considered satisfactory as a fully quantitative assay for the antigen-binding capacity of immunoglobulin containing samples. The limitation of this technique is related to the finding that immunoglobulin preparations of different bleedings from the same animal can have varying average net charges resulting in different heights of the rockets in spite of the fact that the preparations have similar antigen binding capacity, (Brogren, 1977). The ACU-test m e t h o d is simple and the only facilities required are those for rocket immunoelectrophoresis. The coefficient of variation was 4% when the incubation step for the antiserum--antigen mixture was 24 h at 4°C. Shortening of the incubation to 4 h at 4°C increased the coefficient of variation to 8%. In the present study using PZ-protein as model antigen, it was noted that the optimal Ag/Ab-ratio in the ACE-assay was 2.3 times higher than the Ag/Ab-ratio at the point of equilibrium on the classical precipitation curve. This could be explained by the fact that the immune-complexes in the ACE-assay were formed in antigen excess. ACKNOWLEDGEMENTS
This study was supported by the Danish Medical Research Council (Project Nos. 512-5685 and 512-7208). The skilful technical assistance of Mrs. Marianne Kj~er and Mrs. Jette Brandt is gratefully acknowledged. REFERENCES Axelsen, N.H., 1973a, Scand. J. Immunol. Suppl. 1, 71. Axelsen, N.H. and P.J. Svendsen, 1973b, Scand. J. Immunol. Suppl. 1,155. Brogren, C.H., 1977, Immunochemical Dpt., Kommunehospitalet, Copenhagen, Denmark. Personal Communications. Chow, B.F. and H. Wu, 1936, Science 84, 316. Cuatrecasas, P., 1969, Biochem. Biophys. Res. Commun. 35,531. Givol, D., Y. Weinstein, M. Gorecki, M. Wilcheck, 1970, Biophys. Res. Commun. 38,826. Heidelberger, M. and E.A. Kabat, 1938, J. Exp. Med. 2,181. Heinzel, W., I. Rahimi-Laridjani and H. Grimmiger, 1976, J. Immunol. Methods 9,337. Kabat, E.A., 1939, J. Exp. Med. 69, 103. Korngold, L. and D. Pressman, 1953, J. Immunoi. 1, 1. Laurell, C.B., 1966, Analyt. Biochem. 15, 45. Lee, K.H. and H. Wu, 1940, Proc. Soc. Exp. Biol. Med. 43, 65. Singer, S.-J. and D.H. Campbell, 1955, J. Am. Chem. Soc. 73, 3504.
127
AN IMMUNOPRECIPITATION-DISSOCIATION TECHNIQUE FOR L A R G E SCALE ANTIBODY PURIFICATION AND AN ANTIGEN CONSUMPTION ELECTROIMMUNOASSAY FOR ANTIBODY QUANTITATION. A MODEL STUDY WITH ANTIBODIES TO PREGNANCY ZONE PROTEIN
J. F O L K E R S E N 1, B. TEISNER 1, p. S V E N D S E N 2, and S.-E. SVEHAG 1 1 Institute o f Medical Microbiology, Odense University, Odense and 2 Experimental Animal Unit, Odense University, Odense, Denmark
(Received 19 January 1978, accepted 24 March 1978) A simple immunoprecipitation---dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.
INTRODUCTION The basis for many immunological methods is the production of monospecific antibodies of high purity. Other contaminating proteins and nonreacting immunoglobulins can reduce the sensitivity and precision of RIAtests and affinity chromatographic techniques (Heinzel et al., 1976). Purification of immunoglobulins based on their physico-chemical properties often results in suboptimal reagents. The use of specific solid-phase immunosorbent principles for antibody purification (Cuatrecasas, 1969; Givol, 1970) are often necessary to obtain specific immunoglobulin preparations of satisfactory quality. The usefulness of solid-phase i m m u n o s o r b e n t techniques for antibody purification is limited, however, when labile antigens are destroyed during antibody elution. Furthermore, the effectiveness of purification is highly dependent upon the purity of the antigen preparation employed. Direct precipitation of the antibodies in monospecific antisera by soluble antigen in liquid phase systems is less demanding with regard to antigen purity and may be performed on a large scale after preliminary determination of the equilibrium point on a quantitative precipitation curve. The isolated immune precipitate can be dissociated by a variety of procedures including elevated temperatures (Korngold and Pressman, 1953), high
128 ionic strength (Heidelberger and Kabat, 1938) and changes in pH (Chow and Wu, 1936; Kabat, 1939; Lee and Wu, 1940; Singer and Campbell, 1955). Final separation of antigen and antibodies can be achieved by e.g. conventional column chromatography. In our work with pregnancy zone protein (PZP), which is labile, difficulties were encountered in finding an elution procedure which would retain the antigenicity of PZP as a ligand in solid-phase immunosorbent techniques to obtain monospecific anti-PZP antibodies of high purity. A liquid-phase immunosorbent precipitation technique was developed, therefore, for large scale purification of anti-PZP antibodies directly from immune serum. This m e t h o d and a new antigen-consumption electroimmunoassay for antibody quantitation are described in this report. MATERIALS AND METHODS
Partial purification of PZ-protein for immunization Sera were obtained from pregnant women in the 3rd trimester. One volume serum was diluted with 1.25 volumes of 0.03 M NaK--phosphate buffer (pH 7.2), aetacridine lactate was added to a final concentration of 0.4% w/v and pH was adjusted to 6.0. The mixture was incubated for 30 min and the resulting precipitate pelleted at 2500 × g for 15 min. The precipitate was redissolved in twice the original serum volume of 0.83 M NaC1 with the aid of a whirlmixer and end-over-end mixer and centrifuged at 1500 X g for 15 min. The supernatant was collected and dialysed overnight against 0.03 M NaK--phosphate buffer (pH 7.2). The dialysed sample was filtered on Sepharose 6BCL and fractions tested by fused rocket immunoelectrophoresis using a monospecific rabbit anti-human PZ-protein serum. PZ-protein containing fractions were concentrated, dialysed against PBS and sterile filtered.
PZ-protein determinations PZ-protein concentration was determined by rocket immunoelectrophoresis (Laurell, 1966) using a monospecific rabbit antiserum to human PZprotein. The electrophoresis was performed with 0.028 M barbital buffer, pH 8.6 at 1 V/cm gel for 18 h. PZ-protein concentration was expressed in arbitrary PZP units per ml (APU/ml). The lower limit of detection was ½ APU. In the range 6--80 APU, the coefficient of variation was less than 3%.
Production of monospecific goat antiserum to human PZ-protein Goats were given weekly multiple subcutaneous injections (100 APU/ injection) of the partially purified PZ-protein in Freund's complete adjuvant. The antiserum was adsorbed with normal human male serum for 2 h at room temperature and overnight at 4°C. The serum mixture was centrifuged at 110,000 X g for 1 h and the absorbed serum was tested for monospecificity by crossed immunoelectrophoresis against undiluted pregnancy serum {Fig. 1).
129 l
Z
Fig. 1. Crossed immunoelectrophoresis with goat-anti-PZP-antiserum diluted 1 : 100 in the second dimension gel. Ten microlitres of a PZP-containing human serum was applied in the well.
Quantitation of antibodies to PZ-protein An antigen-consumption electroimmunoassay (ACE) was developed for antibody quantitation. One antigen consumption unit (ACU) was defined as the a m o u n t of antibody which reduced the PZ-protein c o n t e n t in a standard preparation by one APU as determined by quantitative rocket immunoelectrophoresis. The specific antibody activity was expressed in ACU/ml total protein. Two antigen standards A (150 APU/ml) and B (75 APU/ml) and one antibody standard (62 ACU/ml) were included in all ACE-assays. The sample to be tested for antibody activity was diluted 2-fold using 300 pl/ tube, and 300 pl of antigen standard A was added to each tube. An additional tube received 300 gl of standard A plus 300 pl of antibody standard. These tubes and standard B were incubated overnight at 4°C in an end-over-end mixer and were then examined by quantitative rocket immunoelectrophoresis (Fig. 2). The concentration of free PZ-protein in APU/ml was determined for all samples. The antibody activity in the test sample was determined by the use of the formula: (RD - R n ) X fX k = ACU/ml where Rb = APU/ml in standard B, Rn = APU/ml in the first tube in the dilution series where APU/ml exceeded 6, f = the final dilution factor of test
130
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132 Sfondords
Fig. 2. E x a m p l e o f design used in t h e ACE-assay for d e t e r m i n a t i o n of a n t i b o d y activity. S t a n d a r d B c o n t a i n s 75 A P U a n d t h e r o c k e t c o r r e s p o n d i n g to a s e r u m d i l u t i o n of 8 c o n t a i n e d 25 APU. T h e r e f o r e , t h e a n t i b o d y activity was ( 7 5 - - 2 5 ) x 8 x 1 = 4 0 0 ACU, p r o v i d e d t h e c o r r e c t i o n f a c t o r was 1.
sample in tube n, and k is a correction factor (RAc/R1Ac) where RAC = APU/ml in standard A + C and R~AC = ~ of APU/ml in standard A + C measured in 10 tests performed on different days.
Exposure o f goat an tibodies at low p H The effect of exposure at low pH on the antigen binding capacity of goat antibodies to PZ-protein was evaluated in the ACE-assay. Immune serum diluted I : 16 in 0.9% NaC1 was exposed to pH 2.3 for 5 h followed by pH 3.1 for 19 h. Samples were collected at several time intervals, the pH was adjusted to neutrality and the activity of the samples was tested by the ACEassay.
Purification o f antibodies to PZ-protein by immunoprecipitation and recovery o f the antibody fraction by exclusion chromatography at low pH Pregnancy serum, collected in the 3rd trimester, was centrifuged at 110,000 X g for 1 h and the supernatant was mixed with an a m o u n t of monospecific goat antiserum to PZ-protein corresponding to the equilibrium point on a quantitative PZ--anti-PZ precipitation curve. The mixture was incubated for 2 h at room temperature and overnight at 4°C and then centrifuged at 110,000 X g for 1 hour. The precipitate was washed twice with PBS and resuspended in 2 volumes of HCl---glycine buffer (pH 2.3), with a whirlmixer. The pH was readjusted to pH 2.3 after 15 min and dissociation allowed to proceed for 5 h at room temperature with magnetic stirring. The suspension was then centrifuged at 8,000 X g for 15 min and the
131
Fig. 3. Test for antibody identity using crossed immunoelectrophoresis with intermediate gel. Upper 2nd dimension gel contains reference rabbit-anti-human PZP-antiserum diluted 1 : 100 and intermediate gel contains goat anti-human PZP-serum diluted 1 : 100. Ten microlitres of a PZP containing human serum was applied in the well.
supernatant was applied to a Sepharose 6BCL (50/100) column equilibrated with HC1--glycine buf f e r (pH 3.1). The flow rate was 95 ml/h, corresponding to a total time of a n t i b o d y exposure at this pH of about 10 h. The fractions were collected in test tubes containing sufficient NaOH--glycine buffer pH 10 to bring the pH back to a b o u t 7. All protein-containing fractions were tested by rocket i m m u n o e l e c t r o p h o resis against rabbit anti-whole h u m a n serum, rabbit anti-goat 7-globulin serum, rabbit anti-whole goat serum and rabbit anti-human PZ-protein serum. Fractions containing goat 7-globulin were tested for ant i body activity by means o f the ACE assay.
Crossed immunoelectrophoresis Crossed immunoelectrophoresis with intermediate gel (Axelsen, 1973a) was used to com pa r e the reactivity o f the goat ant i body preparations towards PZ-protein with a reference antiserum kindly supplied by Bo von Schoultz, Ume~, Sweden. The prepared a n t i b o d y preparation showed a reaction o f identity when tested against the reference antiserum (Fig. 3). Protein determinations Th e protein concentrations were det er mi ned s p e c t r o p h o t o m e t r i c a l l y by reference to a standard curve based on bovine serum albumin.
132 RESULTS
Stability of goat anti-PZ antibodies and PZ-protein during prolonged exposure to low pH Goat immune serum and PZ-protein were exposed to low pH for varied times to establish the dissociation conditions to be used in large scale purification of the antibodies by the immunoprecipitation--dissociation technique. Goat immune serum to PZ-protein exposed to pH 2.3 for 5 h followed by 10 h at pH 3.1 (conditions corresponding to those in subsequent antibody purification experiments), lost at most 15% of the original PZ-binding capacity as indicated by the ACE-assay (Fig. 4). PZ-protein antigenicity, however, was undetectable after lowering the pH of a PZ-containing serum to 2.3 for less than half an hour.
Antigen-consumption electroimmunoassay for antibody quantitation The antigen-consumption electroimmunoassay (ACE-assay) was used to determine the antibody activity in the antisera used and to monitor the fractions obtained during large scale purification of antibodies to PZ-protein. The immune sera to PZP from the four goats used for the antibody production contained 260--620 ACU/ml. The coefficient of variation for the determination was 4%.
Large scale antibody purification by immunoprecipitation--dissociation technique Goat immune serum monospecific for PZ-protein was reacted with a Relot ive oss of antibod aclivily
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6 8 10 12 14 16 18 20 22 24 Hours of exposure
Fig. 4. Relative loss o f a n t i b o d y activity ( A C U / m l ) as a f u n c t i o n o f t i m e o f e x p o s u r e at low pH. T h e n o r m a l processing t i m e in r o u t i n e p u r i f i c a t i o n o f a n t i b o d y p r e p a r a t i o n s was 15 h.
133
volume o f pregnancy serum corresponding to the equilibrium point on the quantitative precipitation curve. No PZ-protein or antibody activity was demonstrable in the supernatant after incubation and centrifugation of the mixture. The antibody activity in AC units of the re-neutralized samples was determined at different time intervals during dissociation of the immunoprecipitate at pH 2.3. A relatively constant level of ACU was observed during
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A
B
Fig. 5. U l t r a v i o l e t - e l u t i o n profile for f r a c t i o n a t i o n o f dissociated and precentrifuged i m m u n e c o m p l e x e s on a S e p h a r o s e 6 B C L c o l u m n . H a t c h e d c o l u m n s indicate a n t i b o d y purification factor. Eluted fractions were tested b y r o c k e t i m m u n o e l e c t r o p h o r e s i s using antiserum to w h o l e goat serum ( A ) and t o w h o l e h u m a n serum (B). The proteins d e t e c t e d o n plate A had the m o b i l i t y o f 7-globulins.
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the first 4 h, but this increased about 4-fold from the 5th hour to the time of centrifugation. No antigen activity in APU were detected in the supernatant and the redissolved precipitate contained only a few per cent of the total antibody-ACU recovered in the supernatant. When the antibody-containing supernatant was gel filtered on an acid Sepharose 6BCL column the ACU-activity was recovered in the fractions corresponding to the major protein peak (Fig. 5). Rocket immunoelectrophoresis with rabbit antisera to whole goat serum and goat 7-globulin showed that this peak contained primarily goat IgG (Fig. 5). Further immunoelectrophoretic analysis of the fractions by the use of rabbit antiwhole human serum showed traces of unidentified human proteins in the same region. When the fractions with the highest ACU-activity were pooled and dialysed against PBS a slight precipitate was formed. Removal of the precipitate by centrifugation increased the specific antibody activity (ACU/mg protein) in the supernatant by 40--50%. The finally concentrated goat IgG antibody preparation showed an average 60-fold (range 50--64) increase in ACU/mg protein when compared to the original immune serum. The total recovery in ACU varied from 28% to 38% for the immune sera from 4 goats used in the purification experiments. The purified antibody preparation showed no loss of activity when stored at -20°C for 2 months. In subsequent work, the antibody preparations have been coupled to cyanogen bromide activated Sepharose 4B. Immunosorbent columns prepared from these preparations have been kept at 4°C and used in PZ-protein purification studies over a period of 6--7 months without significant loss of PZ-protein binding capacity. DISCUSSION
The 2-step method for purification of goat immunoglobulin to PZ-protein is expeditious and simple and may be used on a large scale since the only limiting factor is the column capacity in step 2. No pre-purification of the 3,-globulin fraction is necessary. The average recovery of antibody activity, measured in ACU, exceeded 30% which was considered satisfactory; the purification factor was about 60. The final antibody preparation was stable on storage and has been found well suited for use in solid phase immunosorbent techniques and in a radioimmunoassay for PZP-determination. The choice of dissociation conditions in the purification procedure was based solely on results obtained when testing the antigen-binding capacity of the various fractions in the ACE-assay. Possible reversible or irreversible alterations of the immunogIobulin structure not registered in this assay were not evaluated. The antigen-binding activity {in ACU) of goat-IgG antibodies showed great stability to exposure of low pH over several hours provided the experiments
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were performed on diluted (1 : 16) serum. A new assay system for determining the antigen-binding activity of the immune serum and antibody-containing fractions was developed based on the electroimmunoassay principle. The recently described 'reversed rocket immunoelectrophoresis' (Axelsen and Svendsen, 1973b) was n o t considered satisfactory as a fully quantitative assay for the antigen-binding capacity of immunoglobulin containing samples. The limitation of this technique is related to the finding that immunoglobulin preparations of different bleedings from the same animal can have varying average net charges resulting in different heights of the rockets in spite of the fact that the preparations have similar antigen binding capacity, (Brogren, 1977). The ACU-test m e t h o d is simple and the only facilities required are those for rocket immunoelectrophoresis. The coefficient of variation was 4% when the incubation step for the antiserum--antigen mixture was 24 h at 4°C. Shortening of the incubation to 4 h at 4°C increased the coefficient of variation to 8%. In the present study using PZ-protein as model antigen, it was noted that the optimal Ag/Ab-ratio in the ACE-assay was 2.3 times higher than the Ag/Ab-ratio at the point of equilibrium on the classical precipitation curve. This could be explained by the fact that the immune-complexes in the ACE-assay were formed in antigen excess. ACKNOWLEDGEMENTS
This study was supported by the Danish Medical Research Council (Project Nos. 512-5685 and 512-7208). The skilful technical assistance of Mrs. Marianne Kj~er and Mrs. Jette Brandt is gratefully acknowledged. REFERENCES Axelsen, N.H., 1973a, Scand. J. Immunol. Suppl. 1, 71. Axelsen, N.H. and P.J. Svendsen, 1973b, Scand. J. Immunol. Suppl. 1,155. Brogren, C.H., 1977, Immunochemical Dpt., Kommunehospitalet, Copenhagen, Denmark. Personal Communications. Chow, B.F. and H. Wu, 1936, Science 84, 316. Cuatrecasas, P., 1969, Biochem. Biophys. Res. Commun. 35,531. Givol, D., Y. Weinstein, M. Gorecki, M. Wilcheck, 1970, Biophys. Res. Commun. 38,826. Heidelberger, M. and E.A. Kabat, 1938, J. Exp. Med. 2,181. Heinzel, W., I. Rahimi-Laridjani and H. Grimmiger, 1976, J. Immunol. Methods 9,337. Kabat, E.A., 1939, J. Exp. Med. 69, 103. Korngold, L. and D. Pressman, 1953, J. Immunoi. 1, 1. Laurell, C.B., 1966, Analyt. Biochem. 15, 45. Lee, K.H. and H. Wu, 1940, Proc. Soc. Exp. Biol. Med. 43, 65. Singer, S.-J. and D.H. Campbell, 1955, J. Am. Chem. Soc. 73, 3504.
