- Email: [email protected]
Measurement of squirrel monkey serum IgG levels by a two-site sandwich radioimmunoassay with monoclonal antibodies
Journal oflmmunological Methods, 82 (1985) 295-301
295
Elsevier JIM 03603
Measurement of Squirrel Monkey Serum IgG Levels by a Two-Site Sandwich Radioimmunoassay with Monoclonal Antibodies J. Gysin *, C. Roussilhon and S. Pauillac Laboratoire d'lmmunoparasitologie, Institut Pasteur, 97306 Cayenne Cedex, French Guiana
(Received 26 March 1985, accepted 13 May 1985)
Monoclonal antibodies against the squirrel monkey Saimiri sciureus IgG have been produced for a more specific analysis of the antibody-related immunological aspects in experimental human or monkey malaria. Two monoclonal antibodies, 3D8/D5 and 3Fll/G10, out of 64 reacted with distinct epitopes on the IgG present throughout the complete population without interfering with each other. The 2 monoclonal antibodies were used to develop a highly specific, reliable and sensitive two-site sandwich radioimmunoassay for the measurement of the serum IgG levels in 83 animals. The antibodies also allowed us to produce by a simple immunoabsorbent technique a highly purified IgG standard easy to calibrate and store. The assay permits the detection of IgG levels as low as 0.48 ng/ml. The standard curve is linear between 3.9 and 125 ng protein/ml and allows by a simple mathematical equation an accurate measurement of the serum IgG levels. Key words: squirrel monkey- serum l g G -
monoclonal antibodies- immunoabsorbent-
radioim-
munoassay
I. Introduction Over the past years the squirrel m o n k e y Saimiri sciureus of different genetic backgrounds has been successfully used as a new experimental host for m o n k e y and h u m a n malaria (Deane et al., 1966; Rossan et al., 1972; Campbell et al., 1980; Gysin et al., 1980, 1982a; Gysin and Fandeur, 1983). Several studies have also shown its utility in the understanding of immunologically related aspects of the host-parasite relationship (Gysin and Nussenzweig, 1982; Gysin et al., 1982b; F a n d e u r et al., 1984). Exhaustive studies have highlighted the importance of antibodies, particularly IgG, in the development of a protective i m m u n e response against different stages of malaria parasites (Cohen et al., 1961; Diggs et al., 1972; Reeves and Motyl, 1979; Yoshida et al., 1980; Dubois et al., 1984). In this regard the Saimiri malaria model
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
296
has suffered by the lack of a specific approach, necessary for investigations dealing with antibody-related immunological aspects. For this reason we felt that an important prerequisite for further immunological studies was the development of monoclonal antibodies (MAb) against Saimiri IgG which have an evident advantage over the traditional use of anti-human or -monkey Ig polyclonal antisera. The aim of the present paper is to show that monoclonal antibodies recognizing different epitopes on the monkey IgG can be used for their purification by a simple immunoabsorbent technique and also allow the development of an accurate and highly specific radioimmunoassay for the measurement of the IgG serum level.
Materials and Methods
Production of hybridoma antibodies Hybrid cell-secreting antibodies against Saimiri IgG were produced by following the methods of Kohler and Milstein (1975), Hales (1977) and Galfre and Milstein (1981) with minor modifications. The hybrid cell lines used in this study were derived from the fusion of mouse myeloma cell line P3U1 (gift from Professor R.S. Nussenzweig, New York University Medical Center) with immune spleen cells from BALB/cByJ. Ico mice (Iffa Credo, I'Arbresle, France), immunized by 2 consecutive intravenous injections of 50 #g affinity chromatography-purified monkey IgG, (Gysin and Nussenzweig, 1982; Gysin et al., 1982 a,b) at 15-day intervals. Hybridoma cultures producing anti-monkey IgG antibodies detected by a conventional radioimmunoassay were cloned 2 times by limiting dilutions in the presence of feeder cells and subsequently grown as intraperitoneal ascites in Pristane-primed BALB/c mice. Purification of monoclonal antibodies 3D8/D5 and 3 F l l / G 1 0 of isotype IgGl-x derived from ascitic fluids were isolated by a combination of (NH 4)zSO4 precipitation and affinity chromatography on protein A-Sepharose 4B (Pharmacia, France) following the procedure described by Uotila et al. (1981) with minor modifications. After washing the column with the 0.05 M Tris-HCl buffer, pH 8.0, the bound proteins were eluted with 0.1 M citrate phosphate buffer at pH 5.0 and dialyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.2 containing 0.1% sodium azide and 10% glycerol as stabilizer. Saimiri IgG standard Monkey IgG were isolated from a pool of fresh serum on immunoabsorbent prepared by coupling 10 mg of purified MAb 3D8/D5 per ml of swollen activated CNBr-Sepharose 4B (Pharmacia, France) as suggested by the manufacturer. The remaining reactive sites of the gel were blocked by reaction with 1 M ethanolamine, pH 8.0. After completing the washing cycle, 7 ml of gel was poured into a 1 x 10 cm column. The serum was diluted 1 : 5 in 0.1 M phosphate buffer pH 8.3 containing 0.15 M NaC1 and 5 ml passed through the column at a flow rate of 1 ml/10 min. After washing with the same buffer until the OD was below 0.005, the bound
297 proteins were eluted with 0.2 M glycine-HCl buffer, pH 2.3. The eluates were dialyzed against water, pooled and lyophilized.
Protein assays Protein estimation of affinity chromatography-purified MAb or monkey IgG was performed with the Bio-Rad protein assay (Bio-Rad, Touzart et Matignon, France) following the manufacturer's instructions. To check their purity, monkey IgG and MAb preparations were analyzed by electrophoresis in 10% SDS-polyacrylamide gels (SDS-PAGE) according to standard methods. Radiolabeling of MAb 25/~1 (25 #g) of MAb 3 F l l / G 1 0 or 3 D 8 / D 5 was radiolabeled with 1-2 mCi of 125I-IMS 300 (Amersham, France) using Iodogen (Pierce Chemical Co., Rockford, IL) based on the procedure suggested by the manufacturer. The free t25I was removed by passing the sample through a Sephadex G-25 PD-10 column (Pharmacia, France) followed by extensive dialysis against PBS, pH 7.2. The specific activities of the radiolabeled MAb ranged between 2.5 and 3.2 x 107 cpm//~g protein. Competitiue binding radioimmunoassay between MAb Microtiter plates MIC-2000 (Dynatech, France) were coated overnight at 4°C with 50 /~1 monkey IgG at a concentration of 10 /~g/ml in 0.01 M PBS pH 7.2 containing 0.01% sodium azide. Wells of antigen-coated plates were washed 4 times with the same buffer containing 5% bovine serum albumin (BSA) (Pentex, Miles Scientific, Naperville, IL) and 0.05% Tween-20 and kept in the same buffer for additional hour at room temperature. Wells of antigen-coated plates were incubated at room temperature with 30/tl of cold MAb at a concentration of 50 ~tg/ml in the same buffer used for washing. After 1 h of incubation 20/~1 of one of the 125I-labeled MAb at 5 x 104-1 × 105 cpm was added and incubated for an additional hour. Then the wells were washed 4 times under identical conditions, dried and counted in an LKB 1260 gamma-counter. Used as controls: unrelated antigens, BSA, human IgG and unrelated MAb anti-P, vivax. Two-site sandwich radioimmunoassay Wells of identical microtiter plates were coated overnight at 4°C with 100/~1 of MAb 3D8/D5 at 1 /~g protein/ml in the same coupling buffer. 50 /~1 of various concentrations of monkey standard IgG or unknown monkey serum samples at appropriate dilutions were then added to the wells, followed immediately by adding 50/~1 of the MAb 3 F l l / G 1 0 radiolabeled by ~25I at 1 x 105 cpm. All dilutions were performed with the previously described buffer. After 2 h of incubation at room temperature the plates were washed 4 times, dried and counted. The following controls were used: unrelated antigens, BSA, IgG-depleted monkey sera and unrelated MAb as solid phase antibody.
298
Results
Using a competitive radioimmunoassay we first selected, out of 64 monoclonal antibodies, 2 which did not interfere with each other in the binding to the monkey IgG molecules. MAb 3D8/D5 and 3F1 l / G 1 0 had the required characteristics and presented also the best dose-response curves. The use of polyclonal human IgG as unrelated control antigen indicated that MAb 3D8/D5 recognizes a Saimiri-specific epitope; in contrast MAb 3F11/G10 cross-reacted with the human IgG giving similar values of counts per minute for an identical concentration, compared to the monkey IgG standard. A 50% decrease in counts per minute was observed when the same MAb as the radiolabeled MAb had been used for coating (results not shown). Therefore we used MAb 3D8/D5 as solid phase antibody and 3 F l l / G 1 0 as radiolabeled second antibody for the two-site sandwich radioimmunoassay. The immunoabsorbent with 3D8/D5 or 3F11/G10 used for the purification of monkey IgG showed that both MAb had an exclusive specificity for the IgG molecules, as shown in Fig. 1, and recognized epitopes represented throughout the whole population. This observation was confirmed by the PAGE-SDS analysis of the unbound proteins to the immunoabsorbents (results not shown) indicating a complete depletion of the serum IgG. We next studied whether there was competition for the IgG
.I-I
.I..
I Fig. l. SDS-PAGE at 10% Tris-buffer pH 8.3 of immunoaffinity-purified Saimiri IgG. Lane 1, fraction bound to MAb 3 D 8 / D 5 and lane 2 to MAb 3 F l l / G 1 0 in a Sepharose 4B column. (H) heavy chain; (L) light chain.
299
3.2.
m oJ
n U
n:08 y sO.03x
U
7.1
lS.li
)~
62.6
4.0.18
t2S no/ml
Fig. 2. Standard curve for Saimiri IgG. The experimental procedure is outlined in Materials and Methods. Mean + 1 SD is depicted for varying standards from 3.9 to 125 ng IgG/ml.
molecule between the solid phase MAb 3D8/D5 and the radiolabeled 3 F l l / G 1 0 when they were allowed to react simultaneously. It was found that no significant differences occurred for the dose-response curves when the classical successive steps or simultaneous incubation procedure was performed. The standard curve computed from 4 different experiments over a period of 1 week indicated a sensitivity of 0.48 ng IgG/ml with a linearity (Fig. 2) between 3.9 and 125 ng IgG/ml for the IgG standard (r = 0.986) with P < 0.001. Experiments were performed to make sure that other serum components did not interfere in the assay by using IgG-depleted sera after adding predetermined amounts of standard IgG. The observed results did not indicate significant differences with the expected values (y=0.029, x=0.162, P < 0.001). The accurate measurement of the serum IgG levels can be obtained by a simple mathematical equation. The relationship between IgG concentration found in the diluted serum samples and dilutions was roughly linear between dilutions 1 : 100,000 and 1 : 400,000. Occasional sera containing less than 3.9 or more than 125 ng IgG/ml at a given dilution were differently diluted for more precise determination. The within- and between-assay coefficients of variation (CV) were evaluated with the same serum sample (mean concentration of lgG 6.5 mg/ml) in 12 and 24 assays, respectively. The within-CV was 4.29% and the between-CV 4.39%. IgG levels were determined in sera (Fig. 3) from 83 animals of different ages and both sexes. Mean values and standard deviation were 8.70 + 2.33 mg IgG/ml. This is similar to that described for apes or other monkey species (Damian et al., 1972; Voormolen-Kolova et al., 1974a,b; Cole and Bowen, 1976).
300
20
sample U
: 83
m mean
£
B.D.
g
: B.7 :2.3
m
5
m
5
15
10
IgG
m g / m l
N
;q 1'5
Fig. 3. Distribution of lgG levels in 83 monkeys. The interval between each class is 0.23 mg lgG/ml corresponding to one-tenth of the estimated population standard deviation. Raw data ranged from 3.6 to 15.9 mg IgG/ml.
Discussion
The recent development of the human malaria/Saimiri sciureus model as an experimental approach for the understanding of antibody-related immunological aspects (Gysin et al., 1982a, b; Fandeur et al., 1984) suffered in many regards from the lack of an accurate and specific estimation of the antibody-mediated immune response. In this regard the development of MAb-recognizing epitopes on Saimiri IgG molecules was one of the possible solutions. There is already ample evidence that MAb are extremely helpful in the estimation of Ig levels in sera in man and different animal species. The present study also shows that such antibodies can be utilized as reagents in a highly specific and sensitive radioimmunoassay or be useful for the purification of monkey IgG by immunoaffinity chromatography. The results obtained by using a two-site sandwich radioimmunoassay directly derived from a similar ELISA technique described by Uotila et al. (1981) permit not only a substantial gain in the manipulation time but also appear to be reproducible. Its specificity is based on 2 MAb reacting with different epitopes represented throughout the whole IgG population. We felt that the availability of immunoaffinity-purified Saimiri IgG as highly purified standard, easy to calibrate and store would also present a more convenient solution than the use of calibrated sera taken as references.
301
Acknowledgements The skilful technical assistance of Mrs. M. Ribal is gratefully acknowledged. This study was supported by the Minist6re de la Recherche et de la Technologie, France.
References Campbell, C.C., H.C. Spencer, W. Chin and W. Collins, 1980, Trans. R. Soc. Trop. Med. Hyg. 74, 548. Cohen, S., I.A. MacGregor and S.C. Carington, 1961, Nature (London) 192, 733. Cole, M.F. and W.H. Bowen, 1976, Infect. Immun. 13, 1354. Damian, R.T. and N.D. Greene, 1972, in: Pathology of Simian Primates, ed. T.W. Fiennes (S. Karger, Basel). Deane, L.M., J.F. Neto and I.P.S. Silveira, 1966, Trans. R. Soc. Trop. Med. Hyg. 60, 811. Diggs, C.L., B.T. Wellde, J.S. Anderson, R.M. Weber and E. Rodriguez, 1972, Proc. Helminthol. Soc. Wash. 39, 449. Dubois, P., J.P. Dedet, T. Fandeur, C. Roussilhon, M. Jendoubi, S. Paulliac, O. Mercereau-Puijalon and L. Pereira da Silva, 1984, Proc. Natl. Acad. Sci. U.S.A. 81,229. Fandeur, T., P. Dubois, J. Gysin, J.P. Dedet and L. Pereira da Silva, 1984, J. Immunol. 132, 432. Galfre, G. and C. Milstein, 1981, in: Methods in Enzymology, Vol. 73, eds. J.J. Langone and H. Van Vunakis (Academic Press, New York) p. 3. Gysin, J. and R.S. Nussenzweig (eds.) 1982, Mol. Biochem. Parasitol. Suppl., 94. Gysin, J. and T. Fandeur, 1983, Am. J. Trop. Med. Hyg. 32, 461. Gysin, J., M. Hommel and L. Pereira da Silva, 1980, J. Parasitol 66, 1003. Gysin, J., T. Fandeur and L. Pereira da Silva, 1982a, Ann. Immunol. 133D, 95. Gysin, J., P. Dubois and L. Pereira da Silva, 1982b, Parasite Immunol. 4, 421. Hales, A., 1977, Somatic Cell Genet. 3, 227. Kohler, G. and C. Milstein, 1975, Nature (London) 256, 495. Reeves, R.T. and M.R. Motyl, 1979, J. Immunol. 123, 1894. Rossan, R.N., M.D. Young and D.C. Baerg, 1972, Proc. Helminthol. Soc. Wash. 39, 21. Uotila, M., E. Ruoslahti and E. Engvall, 1981, J. lmmunol. Methods 42, 11. Voormolen-Kolova, M., P. Van der Berg and J. Radl, 1974a, Med. Primatol. 3, 335. Voormolen-Kolova, M., P. Van der Berg and J. Radl, 1974b, Med. Primatol. 3, 343. Yoshida, N., P. Potocnjak, M. Aikawa, V. Nussenzweig and R.S. Nussenzweig, 1980, Science 207, 71.
295
Elsevier JIM 03603
Measurement of Squirrel Monkey Serum IgG Levels by a Two-Site Sandwich Radioimmunoassay with Monoclonal Antibodies J. Gysin *, C. Roussilhon and S. Pauillac Laboratoire d'lmmunoparasitologie, Institut Pasteur, 97306 Cayenne Cedex, French Guiana
(Received 26 March 1985, accepted 13 May 1985)
Monoclonal antibodies against the squirrel monkey Saimiri sciureus IgG have been produced for a more specific analysis of the antibody-related immunological aspects in experimental human or monkey malaria. Two monoclonal antibodies, 3D8/D5 and 3Fll/G10, out of 64 reacted with distinct epitopes on the IgG present throughout the complete population without interfering with each other. The 2 monoclonal antibodies were used to develop a highly specific, reliable and sensitive two-site sandwich radioimmunoassay for the measurement of the serum IgG levels in 83 animals. The antibodies also allowed us to produce by a simple immunoabsorbent technique a highly purified IgG standard easy to calibrate and store. The assay permits the detection of IgG levels as low as 0.48 ng/ml. The standard curve is linear between 3.9 and 125 ng protein/ml and allows by a simple mathematical equation an accurate measurement of the serum IgG levels. Key words: squirrel monkey- serum l g G -
monoclonal antibodies- immunoabsorbent-
radioim-
munoassay
I. Introduction Over the past years the squirrel m o n k e y Saimiri sciureus of different genetic backgrounds has been successfully used as a new experimental host for m o n k e y and h u m a n malaria (Deane et al., 1966; Rossan et al., 1972; Campbell et al., 1980; Gysin et al., 1980, 1982a; Gysin and Fandeur, 1983). Several studies have also shown its utility in the understanding of immunologically related aspects of the host-parasite relationship (Gysin and Nussenzweig, 1982; Gysin et al., 1982b; F a n d e u r et al., 1984). Exhaustive studies have highlighted the importance of antibodies, particularly IgG, in the development of a protective i m m u n e response against different stages of malaria parasites (Cohen et al., 1961; Diggs et al., 1972; Reeves and Motyl, 1979; Yoshida et al., 1980; Dubois et al., 1984). In this regard the Saimiri malaria model
* To whom correspondence should be addressed. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
296
has suffered by the lack of a specific approach, necessary for investigations dealing with antibody-related immunological aspects. For this reason we felt that an important prerequisite for further immunological studies was the development of monoclonal antibodies (MAb) against Saimiri IgG which have an evident advantage over the traditional use of anti-human or -monkey Ig polyclonal antisera. The aim of the present paper is to show that monoclonal antibodies recognizing different epitopes on the monkey IgG can be used for their purification by a simple immunoabsorbent technique and also allow the development of an accurate and highly specific radioimmunoassay for the measurement of the IgG serum level.
Materials and Methods
Production of hybridoma antibodies Hybrid cell-secreting antibodies against Saimiri IgG were produced by following the methods of Kohler and Milstein (1975), Hales (1977) and Galfre and Milstein (1981) with minor modifications. The hybrid cell lines used in this study were derived from the fusion of mouse myeloma cell line P3U1 (gift from Professor R.S. Nussenzweig, New York University Medical Center) with immune spleen cells from BALB/cByJ. Ico mice (Iffa Credo, I'Arbresle, France), immunized by 2 consecutive intravenous injections of 50 #g affinity chromatography-purified monkey IgG, (Gysin and Nussenzweig, 1982; Gysin et al., 1982 a,b) at 15-day intervals. Hybridoma cultures producing anti-monkey IgG antibodies detected by a conventional radioimmunoassay were cloned 2 times by limiting dilutions in the presence of feeder cells and subsequently grown as intraperitoneal ascites in Pristane-primed BALB/c mice. Purification of monoclonal antibodies 3D8/D5 and 3 F l l / G 1 0 of isotype IgGl-x derived from ascitic fluids were isolated by a combination of (NH 4)zSO4 precipitation and affinity chromatography on protein A-Sepharose 4B (Pharmacia, France) following the procedure described by Uotila et al. (1981) with minor modifications. After washing the column with the 0.05 M Tris-HCl buffer, pH 8.0, the bound proteins were eluted with 0.1 M citrate phosphate buffer at pH 5.0 and dialyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.2 containing 0.1% sodium azide and 10% glycerol as stabilizer. Saimiri IgG standard Monkey IgG were isolated from a pool of fresh serum on immunoabsorbent prepared by coupling 10 mg of purified MAb 3D8/D5 per ml of swollen activated CNBr-Sepharose 4B (Pharmacia, France) as suggested by the manufacturer. The remaining reactive sites of the gel were blocked by reaction with 1 M ethanolamine, pH 8.0. After completing the washing cycle, 7 ml of gel was poured into a 1 x 10 cm column. The serum was diluted 1 : 5 in 0.1 M phosphate buffer pH 8.3 containing 0.15 M NaC1 and 5 ml passed through the column at a flow rate of 1 ml/10 min. After washing with the same buffer until the OD was below 0.005, the bound
297 proteins were eluted with 0.2 M glycine-HCl buffer, pH 2.3. The eluates were dialyzed against water, pooled and lyophilized.
Protein assays Protein estimation of affinity chromatography-purified MAb or monkey IgG was performed with the Bio-Rad protein assay (Bio-Rad, Touzart et Matignon, France) following the manufacturer's instructions. To check their purity, monkey IgG and MAb preparations were analyzed by electrophoresis in 10% SDS-polyacrylamide gels (SDS-PAGE) according to standard methods. Radiolabeling of MAb 25/~1 (25 #g) of MAb 3 F l l / G 1 0 or 3 D 8 / D 5 was radiolabeled with 1-2 mCi of 125I-IMS 300 (Amersham, France) using Iodogen (Pierce Chemical Co., Rockford, IL) based on the procedure suggested by the manufacturer. The free t25I was removed by passing the sample through a Sephadex G-25 PD-10 column (Pharmacia, France) followed by extensive dialysis against PBS, pH 7.2. The specific activities of the radiolabeled MAb ranged between 2.5 and 3.2 x 107 cpm//~g protein. Competitiue binding radioimmunoassay between MAb Microtiter plates MIC-2000 (Dynatech, France) were coated overnight at 4°C with 50 /~1 monkey IgG at a concentration of 10 /~g/ml in 0.01 M PBS pH 7.2 containing 0.01% sodium azide. Wells of antigen-coated plates were washed 4 times with the same buffer containing 5% bovine serum albumin (BSA) (Pentex, Miles Scientific, Naperville, IL) and 0.05% Tween-20 and kept in the same buffer for additional hour at room temperature. Wells of antigen-coated plates were incubated at room temperature with 30/tl of cold MAb at a concentration of 50 ~tg/ml in the same buffer used for washing. After 1 h of incubation 20/~1 of one of the 125I-labeled MAb at 5 x 104-1 × 105 cpm was added and incubated for an additional hour. Then the wells were washed 4 times under identical conditions, dried and counted in an LKB 1260 gamma-counter. Used as controls: unrelated antigens, BSA, human IgG and unrelated MAb anti-P, vivax. Two-site sandwich radioimmunoassay Wells of identical microtiter plates were coated overnight at 4°C with 100/~1 of MAb 3D8/D5 at 1 /~g protein/ml in the same coupling buffer. 50 /~1 of various concentrations of monkey standard IgG or unknown monkey serum samples at appropriate dilutions were then added to the wells, followed immediately by adding 50/~1 of the MAb 3 F l l / G 1 0 radiolabeled by ~25I at 1 x 105 cpm. All dilutions were performed with the previously described buffer. After 2 h of incubation at room temperature the plates were washed 4 times, dried and counted. The following controls were used: unrelated antigens, BSA, IgG-depleted monkey sera and unrelated MAb as solid phase antibody.
298
Results
Using a competitive radioimmunoassay we first selected, out of 64 monoclonal antibodies, 2 which did not interfere with each other in the binding to the monkey IgG molecules. MAb 3D8/D5 and 3F1 l / G 1 0 had the required characteristics and presented also the best dose-response curves. The use of polyclonal human IgG as unrelated control antigen indicated that MAb 3D8/D5 recognizes a Saimiri-specific epitope; in contrast MAb 3F11/G10 cross-reacted with the human IgG giving similar values of counts per minute for an identical concentration, compared to the monkey IgG standard. A 50% decrease in counts per minute was observed when the same MAb as the radiolabeled MAb had been used for coating (results not shown). Therefore we used MAb 3D8/D5 as solid phase antibody and 3 F l l / G 1 0 as radiolabeled second antibody for the two-site sandwich radioimmunoassay. The immunoabsorbent with 3D8/D5 or 3F11/G10 used for the purification of monkey IgG showed that both MAb had an exclusive specificity for the IgG molecules, as shown in Fig. 1, and recognized epitopes represented throughout the whole population. This observation was confirmed by the PAGE-SDS analysis of the unbound proteins to the immunoabsorbents (results not shown) indicating a complete depletion of the serum IgG. We next studied whether there was competition for the IgG
.I-I
.I..
I Fig. l. SDS-PAGE at 10% Tris-buffer pH 8.3 of immunoaffinity-purified Saimiri IgG. Lane 1, fraction bound to MAb 3 D 8 / D 5 and lane 2 to MAb 3 F l l / G 1 0 in a Sepharose 4B column. (H) heavy chain; (L) light chain.
299
3.2.
m oJ
n U
n:08 y sO.03x
U
7.1
lS.li
)~
62.6
4.0.18
t2S no/ml
Fig. 2. Standard curve for Saimiri IgG. The experimental procedure is outlined in Materials and Methods. Mean + 1 SD is depicted for varying standards from 3.9 to 125 ng IgG/ml.
molecule between the solid phase MAb 3D8/D5 and the radiolabeled 3 F l l / G 1 0 when they were allowed to react simultaneously. It was found that no significant differences occurred for the dose-response curves when the classical successive steps or simultaneous incubation procedure was performed. The standard curve computed from 4 different experiments over a period of 1 week indicated a sensitivity of 0.48 ng IgG/ml with a linearity (Fig. 2) between 3.9 and 125 ng IgG/ml for the IgG standard (r = 0.986) with P < 0.001. Experiments were performed to make sure that other serum components did not interfere in the assay by using IgG-depleted sera after adding predetermined amounts of standard IgG. The observed results did not indicate significant differences with the expected values (y=0.029, x=0.162, P < 0.001). The accurate measurement of the serum IgG levels can be obtained by a simple mathematical equation. The relationship between IgG concentration found in the diluted serum samples and dilutions was roughly linear between dilutions 1 : 100,000 and 1 : 400,000. Occasional sera containing less than 3.9 or more than 125 ng IgG/ml at a given dilution were differently diluted for more precise determination. The within- and between-assay coefficients of variation (CV) were evaluated with the same serum sample (mean concentration of lgG 6.5 mg/ml) in 12 and 24 assays, respectively. The within-CV was 4.29% and the between-CV 4.39%. IgG levels were determined in sera (Fig. 3) from 83 animals of different ages and both sexes. Mean values and standard deviation were 8.70 + 2.33 mg IgG/ml. This is similar to that described for apes or other monkey species (Damian et al., 1972; Voormolen-Kolova et al., 1974a,b; Cole and Bowen, 1976).
300
20
sample U
: 83
m mean
£
B.D.
g
: B.7 :2.3
m
5
m
5
15
10
IgG
m g / m l
N
;q 1'5
Fig. 3. Distribution of lgG levels in 83 monkeys. The interval between each class is 0.23 mg lgG/ml corresponding to one-tenth of the estimated population standard deviation. Raw data ranged from 3.6 to 15.9 mg IgG/ml.
Discussion
The recent development of the human malaria/Saimiri sciureus model as an experimental approach for the understanding of antibody-related immunological aspects (Gysin et al., 1982a, b; Fandeur et al., 1984) suffered in many regards from the lack of an accurate and specific estimation of the antibody-mediated immune response. In this regard the development of MAb-recognizing epitopes on Saimiri IgG molecules was one of the possible solutions. There is already ample evidence that MAb are extremely helpful in the estimation of Ig levels in sera in man and different animal species. The present study also shows that such antibodies can be utilized as reagents in a highly specific and sensitive radioimmunoassay or be useful for the purification of monkey IgG by immunoaffinity chromatography. The results obtained by using a two-site sandwich radioimmunoassay directly derived from a similar ELISA technique described by Uotila et al. (1981) permit not only a substantial gain in the manipulation time but also appear to be reproducible. Its specificity is based on 2 MAb reacting with different epitopes represented throughout the whole IgG population. We felt that the availability of immunoaffinity-purified Saimiri IgG as highly purified standard, easy to calibrate and store would also present a more convenient solution than the use of calibrated sera taken as references.
301
Acknowledgements The skilful technical assistance of Mrs. M. Ribal is gratefully acknowledged. This study was supported by the Minist6re de la Recherche et de la Technologie, France.
References Campbell, C.C., H.C. Spencer, W. Chin and W. Collins, 1980, Trans. R. Soc. Trop. Med. Hyg. 74, 548. Cohen, S., I.A. MacGregor and S.C. Carington, 1961, Nature (London) 192, 733. Cole, M.F. and W.H. Bowen, 1976, Infect. Immun. 13, 1354. Damian, R.T. and N.D. Greene, 1972, in: Pathology of Simian Primates, ed. T.W. Fiennes (S. Karger, Basel). Deane, L.M., J.F. Neto and I.P.S. Silveira, 1966, Trans. R. Soc. Trop. Med. Hyg. 60, 811. Diggs, C.L., B.T. Wellde, J.S. Anderson, R.M. Weber and E. Rodriguez, 1972, Proc. Helminthol. Soc. Wash. 39, 449. Dubois, P., J.P. Dedet, T. Fandeur, C. Roussilhon, M. Jendoubi, S. Paulliac, O. Mercereau-Puijalon and L. Pereira da Silva, 1984, Proc. Natl. Acad. Sci. U.S.A. 81,229. Fandeur, T., P. Dubois, J. Gysin, J.P. Dedet and L. Pereira da Silva, 1984, J. Immunol. 132, 432. Galfre, G. and C. Milstein, 1981, in: Methods in Enzymology, Vol. 73, eds. J.J. Langone and H. Van Vunakis (Academic Press, New York) p. 3. Gysin, J. and R.S. Nussenzweig (eds.) 1982, Mol. Biochem. Parasitol. Suppl., 94. Gysin, J. and T. Fandeur, 1983, Am. J. Trop. Med. Hyg. 32, 461. Gysin, J., M. Hommel and L. Pereira da Silva, 1980, J. Parasitol 66, 1003. Gysin, J., T. Fandeur and L. Pereira da Silva, 1982a, Ann. Immunol. 133D, 95. Gysin, J., P. Dubois and L. Pereira da Silva, 1982b, Parasite Immunol. 4, 421. Hales, A., 1977, Somatic Cell Genet. 3, 227. Kohler, G. and C. Milstein, 1975, Nature (London) 256, 495. Reeves, R.T. and M.R. Motyl, 1979, J. Immunol. 123, 1894. Rossan, R.N., M.D. Young and D.C. Baerg, 1972, Proc. Helminthol. Soc. Wash. 39, 21. Uotila, M., E. Ruoslahti and E. Engvall, 1981, J. lmmunol. Methods 42, 11. Voormolen-Kolova, M., P. Van der Berg and J. Radl, 1974a, Med. Primatol. 3, 335. Voormolen-Kolova, M., P. Van der Berg and J. Radl, 1974b, Med. Primatol. 3, 343. Yoshida, N., P. Potocnjak, M. Aikawa, V. Nussenzweig and R.S. Nussenzweig, 1980, Science 207, 71.