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Antigen density in ELISA; Effect on avidity dependency
Journal oflmmunological Methods, 36 (1980) 63--70
63
© Elsevier/North-Holland Biomedical Press
ANTIGEN DENSITY IN ELISA; EFFECT ON AVIDITY DEPENDENCY
O.-P. LEHTONEN 1 and M.K. VILJANEN
Department of Medical Microbiology, Turku University, Turku, Finland (Received 3 December 1979, accepted 12 March 1980)
Two different densities of an antigen (bovine serum albumin, BSA) were used on paper disks in an enzyme-linked immunosorbent assay (ELISA) for chicken IgG anti-BSA antibodies. Disks of different antigen density but containing the same amount of the antigen showed a difference in immunoreactivity. Samples containing large amounts of low avidity antibodies gave higher absorbance when disks of lower antigen density were used. This p h e n o m e n o n was not due to a competition between antibodies of various immunoglobulin classes. These findings may indicate that antigen density affects the sensitivity of ELISA to antibodies of various avidities.
INTRODUCTION
Antibody assays in which antigen is coupled to a solid phase, e.g. the solid-phase radioimmunoassay (SPRIA) and the enzyme-linked immunosorbent assay (ELISA), have recently gained popularity. For these techniques to be quantitative and properly standardized the amount of coupled antigen should be quantified. This parameter has, however, been ignored in most of the work reported in this field. Brash and Lyman (1969) and Pesce et al. (1977) have shown that albumin may be coupled to polystyrene at surface concentrations of about 1 pg/cm 2. Antibodies do not necessarily reach all the determinants of such an antigen surface. Such high antigen densities may well be attained by the coupling concentrations which have been commonly used in ELISA (Engvall and Perlmann, 1972; Pesce et al., 1977). A heavily coupled antigen phase may affect the binding of antibodies in the assay owing to surface interactions, as suggested by Pesce et al. (1978). Also some ELISA studies indicate that the antigenic sites on the solid phase are utilized more efficiently by low than by high antibody concentrations (Bruins et al., 1978; Sippel et al., 1978). It is obvious that ELISA systems differ in avidity dependency (Engvall and Perlmann, 1972; Ahlstedt et al., 1974; Butler et al., 1978). Hitherto, this has been explained by an interfering effect of secondary antibody1 Correspondence to: Dr. O.-P. Lehtonen, Department of Medical Microbiology, Turku University, Kiinamyllynkatu 10, SF-20520 Turku 52, Finland.
64 enzyme complexes on the attachment of the primary antibodies (Butler et al., 1978). The dose response curves of different sera in ELISA may reach plateaus at different levels even in the same assay (Engvall and Carlsson, 1976; Carlsson and Lindberg, 1977; Gripenberg et al., 1978; Sippel et al., 1978). This indicates that the antigen has a different maximum binding degree with different sera, which is contrary to classical antibody binding kinetics. The purpose of this study was to look for differences in immunoreactivity of solid phase antigen at different densities. Stable and easily adjustable antigen phases were produced using cyanogen bromide activated paper (Lehtonen and Viljanen, 1980). A further aim was to test whether the avidity dependency observed in ELISA might be linked to antigen density. MATERIALS AND METHODS
Antigen Crystalline bovine serum albumin, BSA, {fraction V, Miles Laboratories Ltd., England) was used throughout the study. 12SI-labelled BSA was prepared according to Hunter and Greenwood (1962). The specific activity obtained was 3--4 pCi/pg of BSA. The fraction of total iodide which was protein b o u n d was 96--97% as determined by precipitation with 20% trichloracetic acid.
Anti-BSA antisera Five to 6-week-old inbred White Leghorn chickens were immunized intraperitoneally with 1.0 mg BSA in physiological saline. Twenty chickens were immunized at a time and three donors were chosen randomly at each time point to provide pooled serum samples. To obtain sera of high anti-BSA titer a repeated immunization procedure of 8 weekly injections was carried out.
Preparation o f the enzyme antiglobulin conjugate Sheep anti-chicken-~ was prepared as described by Viljanen et al. (1975) and its purity tested by immunodiffusion. Labelling of the anti-chicken-~ with alkaline phosphatase (Sigma t y p e VII, Sigma Chemical Company, St. Louis., MO, U.S.A.) was performed according to Avrameas {1969). The preparation was purified on a Sepharose 6B column (Pharmacia).
Coupling o f the antigen to paper Cyanogen bromide activated paper was used as the solid phase for BSA in the ELISA. Activation of the paper was according to Ceska and Lundqvist {1972).
65 A b o u t 200 cm 2 (both sides included) of the activated paper was placed in a beaker containing 400 ml of the coupling solution which contained 1.0 or 5.0 pg of BSA per ml of 0.1 M phosphate buffer with 0.15 saline, pH 7.4 (PBS). At these coupling concentrations the final surface concentrations achieved were 220--310 and 550--790 ng/cm 2, respectively. ~2SI-labelled BSA was added to the coupling solution in sufficient a m o u n t to produce a specific activity of about 10 cpm/ng of BSA. Coupling was performed for 20--24 h at room temperature. After washing with PBS any reactive groups remaining on the paper were blocked with 0.05 M ethanolamine in 0.1 M NaHCO3 for 4--6 h at r o o m temperature. The paper was then washed once with 0.1 M NaHCO3, 5 times with 0.9% NaC1 containing 0.05% Tween (NaC1-Tw) and once with PBS. Depending on the surface concentrations obtained with the different coupling solutions, disks of different size were punched out of each paper strip in order to give the same a m o u n t of antigen on each disk. Usually paper disks of 0.362 cm 2 and 0.176 cm 2 were used for surface concentrations of 250 ng/cm 2 and 550 ng/cm 2, respectively. Before the ELISA procedure each disk was checked for its individual a m o u n t of antigen and disks showing greater deviation than 15% of the mean were discarded.
The ELISA procedure Paper disks of the two different surface concentrations were put into stoppered r o u n d - b o t t o m e d plastic tubes. The serum samples were diluted in the assay buffer (0.05 M borate buffer with 0.08 M NaC1 (pH = 9.0) containing 0.1% gelatin and Tween 20) and 1.0 ml of the dilution pipetted into each tube. The tubes were rolled for 8--14 h at r o o m temperature. Two kinds of controls were included in each experiment. Plain assay buffer was pipetted into some of the tubes to determine nonspecific a t t a c h m e n t of the conjugate. Total nonspecific binding was determined using BSA to inhibit specific binding to the solid phase antigen. Maximum inhibition was achieved with 0.5% BSA. After primary incubation, the disks were washed twice with NaC1-Tw and once with PBS. The anti~chicken-~ alkaline phosphatase conjugate was added in a volume of 1.0 ml. The conjugate was diluted with PBS containing 0.1% gelatin and Tween 20. The optimal dilution was determined separately for each batch of the conjugate. The disks were rolled for 8--14 h at room temperature and thereafter washed as before. The disks were picked out into disposable polystyrene cuvettes adapted to a 9 channel p h o t o m e t e r (FP-9 analyzer, Finnpipette ®, Helsinki, Finland). 250 gl of para-nitrophenylphosphate (Orion Diagnostica, Helsinki, Finland) diluted to 1 mg/ml with a diethanolamine-MgC12 buffer (Orion Diagnostica) was added to each cuvette. The cuvettes were incubated at 37°C for 50 min. The enzyme reaction was stopped by adding 250 pl of 1.0 M NaOH. The a m o u n t of the end product, para-nitrophenolate, was measured using a p h o t o m e t e r at a wavelength of 405 nm.
66
Isolation o f IgG anti-BSA antibodies A volume of 200 or 500 pl of the sample was run through a column of sheep anti-chickenff coated Sepharose 4B beads. The column was rinsed with 0.1 M borate buffer (pH = 8.2) containing 0.5 M NaC1. The attached IgG-class antibodies were eluted with 3.0 M NaI and dialyzed immediately against the borate buffer and, if necessary, concentrated by dialyzing against a 30% solution of polyethylene glycol 20,000 (m.w. >17,000). The concentration of IgM anti-BSA antibodies in the eluate was about 50 times less than t h a t in the sample before the absorption.
The ammonium sulfate precipitation assay The Farr assay (Farr, 1958) was used to estimate the avidities of the IgG anti-BSA antibodies. The samples were diluted in quadruplicate 1 : 10 or 1 : 100 with 0.1 M borate buffer (pH = 8.2) containing 0.5 M NaC1 and 10% normal chicken serum. The nonspecifically precipitated a m o u n t of antigen was taken into account as Minden and Farr (1973) have described. The binding data was plotted using the Langmuir plot (the reciprocal of free antigen on the abscissa) according to Nisonoff and Pressman (1958). The concentration of the a n t i b o d y binding sites was determined by extrapolation of the binding curve to the ordinate axis. In this plot, homogeneous a n t i b o d y gives a straight line. A preponderance of low avidity antibodies results in downward curvature whereas dominance of high avidity antibodies gives upward curvature. RESULTS Chicken IgG antibodies against BSA were assayed by ELISA at two antigen densities on paper disks. Paper disks at both antigen densities were cut so that they contained the same a m o u n t of antigen. To study the role of antigen density when ELISA is used to measure antibodies developing during an immune response chickens were immunized with 1.0 mg BSA intraperitoneally and thereafter bled randomly at intervals of 3 days, and the pooled serum samples assayed (Fig. 1). After 3 days (Fig. l a ) there was a slight difference between results with the two antigen phases. The difference was marked in the sample obtained 6 days after injection. In the sample 9 days after immunization (Fig. l c ) this difference has disappeared and is likewise n o t demonstrable either 12 or 15 days after immunization (Fig. l d and e). The difference is demonstrable, however, in a serum pool obtained after a repeated immunization procedure of 8 weekly injections of 1.0 mg BSA (Fig. l f ) . This effect was observable both with borate buffer (pH = 9.0) and phosphate buffer (pH = 7.4), although the latter showed a lesser effect (data n o t shown). Immunoglobulins of IgG class were separated by sheep anti-chicken-~
67
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Fig. 1. ELISA measurements of chicken IgG anti-BSA antibodies at two antigen densities: 220 n g / c m 2 (o) and 550 ng/cm 2 (o). The sera were obtained (a) 3 days, (b) 6 days, (c) 9 days, (d) 12 days and (e) 15 days after immunization. Serum (f) is from a bird repeatedly injected. Borate buffer, pH = 9.0 was used in the primary incubation. The bars represent the standard deviation of 6 parallel determinations. * Buffer control (no serum). ** Serum diluted 1 : 20 with the buffer containing 0.5% BSA.
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Fig. 2. ELISA measurements of chicken IgG anti-BSA antibodies at two antigen densities: 310 ng/cm 2 (o) and 600 ng/cm 2 (0). The assay results of tWo native sera (a and c) and their IgG-fractions (b and d, respectively) are compared. Serum (a) was obtained 7 days after injection and serum (c) after repeated immunization. The bars represent the standard deviation of 6 parallel determinations. * Buffer control (no serum). ** Lowest dilution of serum used mixed with 0.5% BSA.
68
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Fig. 3. Langmuir plots of IgG fractions of anti-BSA sera determined by the ammonium sulfate precipitation assay. The sera are obtained 6 days (©), 9 days (o), 12 days (A), 15 days (A) after immunization or after repeated immunization (x).
serum coated Sepharose 4B beads. IgG preparations of two serum pools which showed the difference prior to purification were tested. The same e f f ect was observed with purified IgG antibodies (Fig. 2). These IgG fractions were assayed for anti-BSA antibodies by a m m o n i u m sulfate precipitation assay. The Langmuir plots of the purified IgG antibodies obtained by a m m o n i u m sulfate precipitation assay are shown in Fig. 3. The IgG fractions of the sera obtained 9, 12 and 15 days after the i m m u n i z a t i o n gave nearly linear relationships between the reciprocals of b o u n d and free antigen concentrations. This indicates limited het erogenei t y of avidity of the antibodies in these samples. In contrast, the t w o o t h e r samples show downward curvature in the Langmuir plot due to a preponderance o f low rather than high avidity antibodies. DISCUSSION In the present work a difference in i m m unoreact i vi t y between two antigen phases at different antigen density was observed. This difference was dem onstrable only with some sera. Purified IgG antibodies showed the effect as well as the native sera. Large a m ount s of low avidity antibodies seemed to be associated with the p h e n o m e n o n . Various explanations may be proposed. Although the antigen phases initially contained the same a m o u n t of antigen there might have been leaching o f antigen f r o m the solid phase during the assay and this leakage could have been greater in t he high density phase. Such antigen leakage has been observed when plastic surfaces have been used (Engvall et al., 1971; Zollinger et al., 197.6; Salonen and Vaheri, 1979). However, in the present work there
69
was covalent bonding between the antigen and the solid phase. Leaching from this type of solid phase has been shown to be equal in both antigen densities and amounts to only 13% of total (Lehtonen and Viljanen, 1980). Thus antigen leaching cannot account for the different behavior of the two antigen phases. Since native sera were initially used the phenomenon could be due to competition between different immunoglobulin classes for antigenic sites on the solid phase as demonstrated by Keren (1979). IgM antibodies are larger than IgG antibodies, and might therefore block more antigenic determinants than IgG molecules. The resulting diminished availability of antigenic determinants would thus lead to decreased IgG binding, especially with the high density phase. The present work shows, however, that the phenomenon is present also with purified IgG fractions and thus could not be solely due to competition between various immunoglobulin classes. The low density phase has a greater total surface area and this could result in increased nonspecific binding. This effect was in fact observed with the BSA-inhibited controls for some samples. However, the observed difference is evidently too small to explain the phenomenon. Since the phenomenon was not detected in all samples, qualitative differences between the IgG anti-BSA antibodies might be a valid explanation. A preponderance of low avidity antibodies seems to be required. Thus it could be argued that the low density phase is more able to bind low avidity antibodies than the high density phase. More information is needed regarding antibody binding to a solid phase coupled antigen. This could be achieved by studying purified antibodies of given affinity in assays at different antigen densities. ACKNOWLEDGEMENTS
The technical assistance of Mrs. Marjo Ingman and Mrs. Seija Laanti is gratefully acknowledged. This work was supported by the Research and Science Foundation of L~i~ike Oy (Turku, Finland), and by a contract with the Association of Finnish Life Insurance Companies. REFERENCES Ahlstedt, S., J. Holmberg and L.A. Hanson, 1974, Int. Arch. Allergy 46,470. Avrameas, S., 1969, Immunochemistry 6, 43. Brash, J.L. and D.J. Lyman, 1969, J. Biomed. Mater. Res. 3, 175. Bruins, S.C., I. Ingwer, M.I. Zeckel and A.C. White, 1978, Infect. Immun. 21,721. Butler, J.E., T.L. Feldbush, P.L. McGivern and N. Steward, 1978, Immunochemistry 15, 131. Carlsson, H.E. and A.A: Lindberg, 1977, in: Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2, ed. T. Ming Swi Chang (Plenum Press, New York) p. 97. Ceska, M. and U. Lundqvist, 1972, Immunochemistry 9, 1021. Engvall, E. and H.E. Carlsson, 1976, in: First International Symposium on Immuno-
70 enzymatic Techniques, INSERM Symposium No. 2, eds. G. Feldmann et al. (NorthHolland, Amsterdam) p. 135. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109,129. Engvall, E., K. Jonsson and P. Perlmann, 1971, Biochim. Biophys. Acta 251,427. Farr, R.S., 1958, J. Infect. Dis. 103,239. Gripenberg, M., E. Linder, P. Kurki and E. Engvall, 1978, Scand. J. Immunol. 7,151. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Keren, D.F., 1979, Infect. Immun. 24,441. Lehtonen, O.-P. and M.K. Viljanen, 1980, J. Immunol. Methods 34, 61. Minden, P. and R.S. Farr, 1973, in: Handbook in Experimental Immunology, Vol. 1, 2nd edition, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 15.4. Nisonoff, A. and D. Pressman, 1958, J. Immunol. 80,417. Pesce, A.J., D.J. Ford, M. Gaizutis and V.E. Pollak, 1977, Biochim. Biophys. Acta 492,399. Pesce, A.J., D.J. Ford and M.A. Gaizutis, 1978, in: Quantitative Enzyme Immunoassay, eds. E. Engvall and A.J. Pesce, Scand. J. Immunol. 8 (Suppl. 7), 1. Salonen, E.-M. and A. Vaheri, 1979, J. Immunol. Methods 30,209. Sippel, J.E., H.K.M.E. Weiss, S.W. Josef and W.J. Beasley, 1978, J. Clin. Microbiol. 7,372. Viljanen, M.K., K. Granfors and P. Toivanen, 1975, Immunochemistry 12, 699. Zollinger, W.D., J.M. Dalrymple and M.S. Artenstein, 1976, J. Immunol. 117, 1788.
63
© Elsevier/North-Holland Biomedical Press
ANTIGEN DENSITY IN ELISA; EFFECT ON AVIDITY DEPENDENCY
O.-P. LEHTONEN 1 and M.K. VILJANEN
Department of Medical Microbiology, Turku University, Turku, Finland (Received 3 December 1979, accepted 12 March 1980)
Two different densities of an antigen (bovine serum albumin, BSA) were used on paper disks in an enzyme-linked immunosorbent assay (ELISA) for chicken IgG anti-BSA antibodies. Disks of different antigen density but containing the same amount of the antigen showed a difference in immunoreactivity. Samples containing large amounts of low avidity antibodies gave higher absorbance when disks of lower antigen density were used. This p h e n o m e n o n was not due to a competition between antibodies of various immunoglobulin classes. These findings may indicate that antigen density affects the sensitivity of ELISA to antibodies of various avidities.
INTRODUCTION
Antibody assays in which antigen is coupled to a solid phase, e.g. the solid-phase radioimmunoassay (SPRIA) and the enzyme-linked immunosorbent assay (ELISA), have recently gained popularity. For these techniques to be quantitative and properly standardized the amount of coupled antigen should be quantified. This parameter has, however, been ignored in most of the work reported in this field. Brash and Lyman (1969) and Pesce et al. (1977) have shown that albumin may be coupled to polystyrene at surface concentrations of about 1 pg/cm 2. Antibodies do not necessarily reach all the determinants of such an antigen surface. Such high antigen densities may well be attained by the coupling concentrations which have been commonly used in ELISA (Engvall and Perlmann, 1972; Pesce et al., 1977). A heavily coupled antigen phase may affect the binding of antibodies in the assay owing to surface interactions, as suggested by Pesce et al. (1978). Also some ELISA studies indicate that the antigenic sites on the solid phase are utilized more efficiently by low than by high antibody concentrations (Bruins et al., 1978; Sippel et al., 1978). It is obvious that ELISA systems differ in avidity dependency (Engvall and Perlmann, 1972; Ahlstedt et al., 1974; Butler et al., 1978). Hitherto, this has been explained by an interfering effect of secondary antibody1 Correspondence to: Dr. O.-P. Lehtonen, Department of Medical Microbiology, Turku University, Kiinamyllynkatu 10, SF-20520 Turku 52, Finland.
64 enzyme complexes on the attachment of the primary antibodies (Butler et al., 1978). The dose response curves of different sera in ELISA may reach plateaus at different levels even in the same assay (Engvall and Carlsson, 1976; Carlsson and Lindberg, 1977; Gripenberg et al., 1978; Sippel et al., 1978). This indicates that the antigen has a different maximum binding degree with different sera, which is contrary to classical antibody binding kinetics. The purpose of this study was to look for differences in immunoreactivity of solid phase antigen at different densities. Stable and easily adjustable antigen phases were produced using cyanogen bromide activated paper (Lehtonen and Viljanen, 1980). A further aim was to test whether the avidity dependency observed in ELISA might be linked to antigen density. MATERIALS AND METHODS
Antigen Crystalline bovine serum albumin, BSA, {fraction V, Miles Laboratories Ltd., England) was used throughout the study. 12SI-labelled BSA was prepared according to Hunter and Greenwood (1962). The specific activity obtained was 3--4 pCi/pg of BSA. The fraction of total iodide which was protein b o u n d was 96--97% as determined by precipitation with 20% trichloracetic acid.
Anti-BSA antisera Five to 6-week-old inbred White Leghorn chickens were immunized intraperitoneally with 1.0 mg BSA in physiological saline. Twenty chickens were immunized at a time and three donors were chosen randomly at each time point to provide pooled serum samples. To obtain sera of high anti-BSA titer a repeated immunization procedure of 8 weekly injections was carried out.
Preparation o f the enzyme antiglobulin conjugate Sheep anti-chicken-~ was prepared as described by Viljanen et al. (1975) and its purity tested by immunodiffusion. Labelling of the anti-chicken-~ with alkaline phosphatase (Sigma t y p e VII, Sigma Chemical Company, St. Louis., MO, U.S.A.) was performed according to Avrameas {1969). The preparation was purified on a Sepharose 6B column (Pharmacia).
Coupling o f the antigen to paper Cyanogen bromide activated paper was used as the solid phase for BSA in the ELISA. Activation of the paper was according to Ceska and Lundqvist {1972).
65 A b o u t 200 cm 2 (both sides included) of the activated paper was placed in a beaker containing 400 ml of the coupling solution which contained 1.0 or 5.0 pg of BSA per ml of 0.1 M phosphate buffer with 0.15 saline, pH 7.4 (PBS). At these coupling concentrations the final surface concentrations achieved were 220--310 and 550--790 ng/cm 2, respectively. ~2SI-labelled BSA was added to the coupling solution in sufficient a m o u n t to produce a specific activity of about 10 cpm/ng of BSA. Coupling was performed for 20--24 h at room temperature. After washing with PBS any reactive groups remaining on the paper were blocked with 0.05 M ethanolamine in 0.1 M NaHCO3 for 4--6 h at r o o m temperature. The paper was then washed once with 0.1 M NaHCO3, 5 times with 0.9% NaC1 containing 0.05% Tween (NaC1-Tw) and once with PBS. Depending on the surface concentrations obtained with the different coupling solutions, disks of different size were punched out of each paper strip in order to give the same a m o u n t of antigen on each disk. Usually paper disks of 0.362 cm 2 and 0.176 cm 2 were used for surface concentrations of 250 ng/cm 2 and 550 ng/cm 2, respectively. Before the ELISA procedure each disk was checked for its individual a m o u n t of antigen and disks showing greater deviation than 15% of the mean were discarded.
The ELISA procedure Paper disks of the two different surface concentrations were put into stoppered r o u n d - b o t t o m e d plastic tubes. The serum samples were diluted in the assay buffer (0.05 M borate buffer with 0.08 M NaC1 (pH = 9.0) containing 0.1% gelatin and Tween 20) and 1.0 ml of the dilution pipetted into each tube. The tubes were rolled for 8--14 h at r o o m temperature. Two kinds of controls were included in each experiment. Plain assay buffer was pipetted into some of the tubes to determine nonspecific a t t a c h m e n t of the conjugate. Total nonspecific binding was determined using BSA to inhibit specific binding to the solid phase antigen. Maximum inhibition was achieved with 0.5% BSA. After primary incubation, the disks were washed twice with NaC1-Tw and once with PBS. The anti~chicken-~ alkaline phosphatase conjugate was added in a volume of 1.0 ml. The conjugate was diluted with PBS containing 0.1% gelatin and Tween 20. The optimal dilution was determined separately for each batch of the conjugate. The disks were rolled for 8--14 h at room temperature and thereafter washed as before. The disks were picked out into disposable polystyrene cuvettes adapted to a 9 channel p h o t o m e t e r (FP-9 analyzer, Finnpipette ®, Helsinki, Finland). 250 gl of para-nitrophenylphosphate (Orion Diagnostica, Helsinki, Finland) diluted to 1 mg/ml with a diethanolamine-MgC12 buffer (Orion Diagnostica) was added to each cuvette. The cuvettes were incubated at 37°C for 50 min. The enzyme reaction was stopped by adding 250 pl of 1.0 M NaOH. The a m o u n t of the end product, para-nitrophenolate, was measured using a p h o t o m e t e r at a wavelength of 405 nm.
66
Isolation o f IgG anti-BSA antibodies A volume of 200 or 500 pl of the sample was run through a column of sheep anti-chickenff coated Sepharose 4B beads. The column was rinsed with 0.1 M borate buffer (pH = 8.2) containing 0.5 M NaC1. The attached IgG-class antibodies were eluted with 3.0 M NaI and dialyzed immediately against the borate buffer and, if necessary, concentrated by dialyzing against a 30% solution of polyethylene glycol 20,000 (m.w. >17,000). The concentration of IgM anti-BSA antibodies in the eluate was about 50 times less than t h a t in the sample before the absorption.
The ammonium sulfate precipitation assay The Farr assay (Farr, 1958) was used to estimate the avidities of the IgG anti-BSA antibodies. The samples were diluted in quadruplicate 1 : 10 or 1 : 100 with 0.1 M borate buffer (pH = 8.2) containing 0.5 M NaC1 and 10% normal chicken serum. The nonspecifically precipitated a m o u n t of antigen was taken into account as Minden and Farr (1973) have described. The binding data was plotted using the Langmuir plot (the reciprocal of free antigen on the abscissa) according to Nisonoff and Pressman (1958). The concentration of the a n t i b o d y binding sites was determined by extrapolation of the binding curve to the ordinate axis. In this plot, homogeneous a n t i b o d y gives a straight line. A preponderance of low avidity antibodies results in downward curvature whereas dominance of high avidity antibodies gives upward curvature. RESULTS Chicken IgG antibodies against BSA were assayed by ELISA at two antigen densities on paper disks. Paper disks at both antigen densities were cut so that they contained the same a m o u n t of antigen. To study the role of antigen density when ELISA is used to measure antibodies developing during an immune response chickens were immunized with 1.0 mg BSA intraperitoneally and thereafter bled randomly at intervals of 3 days, and the pooled serum samples assayed (Fig. 1). After 3 days (Fig. l a ) there was a slight difference between results with the two antigen phases. The difference was marked in the sample obtained 6 days after injection. In the sample 9 days after immunization (Fig. l c ) this difference has disappeared and is likewise n o t demonstrable either 12 or 15 days after immunization (Fig. l d and e). The difference is demonstrable, however, in a serum pool obtained after a repeated immunization procedure of 8 weekly injections of 1.0 mg BSA (Fig. l f ) . This effect was observable both with borate buffer (pH = 9.0) and phosphate buffer (pH = 7.4), although the latter showed a lesser effect (data n o t shown). Immunoglobulins of IgG class were separated by sheep anti-chicken-~
67
°~ a
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,
oil ~oa
c
l,,,oJ •
..'
11320t 120 11280 180
d
e 11280 1 80 i
~ F ~
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1
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I
•
il
l
I
11.320I
t 120
11280 180
"L
1~I0 1~o
11280 180 SAMPLE DILUTION
• .
,32oo~2oo
1128001800
Fig. 1. ELISA measurements of chicken IgG anti-BSA antibodies at two antigen densities: 220 n g / c m 2 (o) and 550 ng/cm 2 (o). The sera were obtained (a) 3 days, (b) 6 days, (c) 9 days, (d) 12 days and (e) 15 days after immunization. Serum (f) is from a bird repeatedly injected. Borate buffer, pH = 9.0 was used in the primary incubation. The bars represent the standard deviation of 6 parallel determinations. * Buffer control (no serum). ** Serum diluted 1 : 20 with the buffer containing 0.5% BSA.
1.5b
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•
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.
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I
}j:12801 1:80 . o.m. 1:160 1:10 1:5120 1:320 1:640 1:40 SAMPLE DILUTION
Fig. 2. ELISA measurements of chicken IgG anti-BSA antibodies at two antigen densities: 310 ng/cm 2 (o) and 600 ng/cm 2 (0). The assay results of tWo native sera (a and c) and their IgG-fractions (b and d, respectively) are compared. Serum (a) was obtained 7 days after injection and serum (c) after repeated immunization. The bars represent the standard deviation of 6 parallel determinations. * Buffer control (no serum). ** Lowest dilution of serum used mixed with 0.5% BSA.
68
5-0I o z ~
2.0-
S
f
o
J
×
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rn 1.0-
0.0
0.0
10 20 30 I ]FREE ANTIGEN10111/M
40
Fig. 3. Langmuir plots of IgG fractions of anti-BSA sera determined by the ammonium sulfate precipitation assay. The sera are obtained 6 days (©), 9 days (o), 12 days (A), 15 days (A) after immunization or after repeated immunization (x).
serum coated Sepharose 4B beads. IgG preparations of two serum pools which showed the difference prior to purification were tested. The same e f f ect was observed with purified IgG antibodies (Fig. 2). These IgG fractions were assayed for anti-BSA antibodies by a m m o n i u m sulfate precipitation assay. The Langmuir plots of the purified IgG antibodies obtained by a m m o n i u m sulfate precipitation assay are shown in Fig. 3. The IgG fractions of the sera obtained 9, 12 and 15 days after the i m m u n i z a t i o n gave nearly linear relationships between the reciprocals of b o u n d and free antigen concentrations. This indicates limited het erogenei t y of avidity of the antibodies in these samples. In contrast, the t w o o t h e r samples show downward curvature in the Langmuir plot due to a preponderance o f low rather than high avidity antibodies. DISCUSSION In the present work a difference in i m m unoreact i vi t y between two antigen phases at different antigen density was observed. This difference was dem onstrable only with some sera. Purified IgG antibodies showed the effect as well as the native sera. Large a m ount s of low avidity antibodies seemed to be associated with the p h e n o m e n o n . Various explanations may be proposed. Although the antigen phases initially contained the same a m o u n t of antigen there might have been leaching o f antigen f r o m the solid phase during the assay and this leakage could have been greater in t he high density phase. Such antigen leakage has been observed when plastic surfaces have been used (Engvall et al., 1971; Zollinger et al., 197.6; Salonen and Vaheri, 1979). However, in the present work there
69
was covalent bonding between the antigen and the solid phase. Leaching from this type of solid phase has been shown to be equal in both antigen densities and amounts to only 13% of total (Lehtonen and Viljanen, 1980). Thus antigen leaching cannot account for the different behavior of the two antigen phases. Since native sera were initially used the phenomenon could be due to competition between different immunoglobulin classes for antigenic sites on the solid phase as demonstrated by Keren (1979). IgM antibodies are larger than IgG antibodies, and might therefore block more antigenic determinants than IgG molecules. The resulting diminished availability of antigenic determinants would thus lead to decreased IgG binding, especially with the high density phase. The present work shows, however, that the phenomenon is present also with purified IgG fractions and thus could not be solely due to competition between various immunoglobulin classes. The low density phase has a greater total surface area and this could result in increased nonspecific binding. This effect was in fact observed with the BSA-inhibited controls for some samples. However, the observed difference is evidently too small to explain the phenomenon. Since the phenomenon was not detected in all samples, qualitative differences between the IgG anti-BSA antibodies might be a valid explanation. A preponderance of low avidity antibodies seems to be required. Thus it could be argued that the low density phase is more able to bind low avidity antibodies than the high density phase. More information is needed regarding antibody binding to a solid phase coupled antigen. This could be achieved by studying purified antibodies of given affinity in assays at different antigen densities. ACKNOWLEDGEMENTS
The technical assistance of Mrs. Marjo Ingman and Mrs. Seija Laanti is gratefully acknowledged. This work was supported by the Research and Science Foundation of L~i~ike Oy (Turku, Finland), and by a contract with the Association of Finnish Life Insurance Companies. REFERENCES Ahlstedt, S., J. Holmberg and L.A. Hanson, 1974, Int. Arch. Allergy 46,470. Avrameas, S., 1969, Immunochemistry 6, 43. Brash, J.L. and D.J. Lyman, 1969, J. Biomed. Mater. Res. 3, 175. Bruins, S.C., I. Ingwer, M.I. Zeckel and A.C. White, 1978, Infect. Immun. 21,721. Butler, J.E., T.L. Feldbush, P.L. McGivern and N. Steward, 1978, Immunochemistry 15, 131. Carlsson, H.E. and A.A: Lindberg, 1977, in: Biomedical Applications of Immobilized Enzymes and Proteins, Vol. 2, ed. T. Ming Swi Chang (Plenum Press, New York) p. 97. Ceska, M. and U. Lundqvist, 1972, Immunochemistry 9, 1021. Engvall, E. and H.E. Carlsson, 1976, in: First International Symposium on Immuno-
70 enzymatic Techniques, INSERM Symposium No. 2, eds. G. Feldmann et al. (NorthHolland, Amsterdam) p. 135. Engvall, E. and P. Perlmann, 1972, J. Immunol. 109,129. Engvall, E., K. Jonsson and P. Perlmann, 1971, Biochim. Biophys. Acta 251,427. Farr, R.S., 1958, J. Infect. Dis. 103,239. Gripenberg, M., E. Linder, P. Kurki and E. Engvall, 1978, Scand. J. Immunol. 7,151. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Keren, D.F., 1979, Infect. Immun. 24,441. Lehtonen, O.-P. and M.K. Viljanen, 1980, J. Immunol. Methods 34, 61. Minden, P. and R.S. Farr, 1973, in: Handbook in Experimental Immunology, Vol. 1, 2nd edition, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 15.4. Nisonoff, A. and D. Pressman, 1958, J. Immunol. 80,417. Pesce, A.J., D.J. Ford, M. Gaizutis and V.E. Pollak, 1977, Biochim. Biophys. Acta 492,399. Pesce, A.J., D.J. Ford and M.A. Gaizutis, 1978, in: Quantitative Enzyme Immunoassay, eds. E. Engvall and A.J. Pesce, Scand. J. Immunol. 8 (Suppl. 7), 1. Salonen, E.-M. and A. Vaheri, 1979, J. Immunol. Methods 30,209. Sippel, J.E., H.K.M.E. Weiss, S.W. Josef and W.J. Beasley, 1978, J. Clin. Microbiol. 7,372. Viljanen, M.K., K. Granfors and P. Toivanen, 1975, Immunochemistry 12, 699. Zollinger, W.D., J.M. Dalrymple and M.S. Artenstein, 1976, J. Immunol. 117, 1788.