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A new solid-phase radioimmunoassay to measure IgG secreted by cultured human lymphocytes
Journal of Immunological Methods, 35 (1980) 189--199
189
© Elsevier/North-Holland Biomedical Press
A NEW SOLID-PHASE RADIOIMMUNOASSAY TO MEASURE IgG SECRETED BY CULTURED HUMAN LYMPHOCYTES
STEFANO MARIOTTI 1, JOEL J.-F. OGER 2, PHILIPPE F R A G U s, JACK P. ANTEL, HAN-HWA KUO and LESLIE J. DeGROOT
(S.M., P.F. and L.J. DeG.) University of Chicago, Pritzker school of Medicine, Department of Medicine (Thyroid Study Unit), and (J.J.-F.O., J.P.A. and H.-H.K.) Department of Neurology, Chicago, IL 60637, U.S.A. (Received 5 December 1979, accepted 4 March 1980)
We describe a simple solid-phase radioimmunoassay (RIA) to detect IgG based on competitive binding between radiolabeled and unlabeled IgG for anti-IgG antibody physically adsorbed to the wells of polyvinyl microtiter plates. The assay is sensitive (1 ng), rapid, and is particularly suited for studies of in vitro IgG secretion by human peripheral blood lymphocytes, since such studies require large numbers of cultures. Conditions which permit measurement, by means of this assay, of helper and suppressor T cell effects on IgG production by human B cells in culture are described.
INTRODUCTION
Immunoglobulin (Ig) secretion in vitro by human peripheral blood lymphocytes is a convenient model to study mechanisms involved in immunoregulation (Waldman et al., 1974; Choi, 1977; Saxon et al., 1977; Fauci, 1979). Both synthesis and secretion of Ig are mediated by B lymphocytes, but cooperation by a helper T cell subset is required (Saxon et al., 1977). Ig production can also be suppressed by a different subset of T lymphocytes (suppressor T cells) (Waldman et al., 1978) and by other circulating non-T cells (Stobo, 1977; Rice et al., 1979). To utilize the Ig production system, mononuclear cells (MNCs) must be prepared in a manner which reduces pre-existent Ig adherent to the lymphocytes. To assess helper and suppressor functions B and T cell enriched cell fractions are recombined in varying proportions and this requires large numbers of cultures. Only small amounts of Ig are secreted into the culture medium by B cells, even when they are stimulated by pokeweed mitogen (PWM). For this reason 1 Dr. Mariotti is on leave from Cattedra di Patologia Medica 2, Universit~ di Pisa, Italy. 2 Dr. Oger is on leave from INSERM France. Address reprint requests to: J. Oger, M.D., The University of Chicago, Pritzker School of Medicine, Department of Neurology, 950 East 59th Street, Chicago, IL 60637, U.S.A. s Dr. Fragu is on leave from Institut Gustave-Roussy, Villejuif, France.
190
sensitive assay techniques have been developed to detect Ig. In one technique the incorporation of radiolabeled amino acids into Ig molecules is measured (Choi, 1977). This technique does n o t give absolute values of Ig. Hemolytic plaque-forming assay methods have been utilized, but these are cumbersome for large scale studies and again do not provide absolute values of Ig (Fauci and Pratt, 1976). Direct determination of Ig in culture supernatants by radioimmunoassay (RIA) would be the preferred assay system if one could develop an RIA system which combines high sensitivity and reproducibility with short incubation time and ease of performance. Such a system would permit rapid assessment of a great number of samples. In the present study, we describe a new solid-phase RIA for human IgG which meets the requirements outlined and indicate its applicability to the study of immunoregulation. MATERIALS AND METHODS
Preparation and radioiodination of human IgG Human IgG was prepared from pooled sera of normal subjects by ammonium sulfate precipitation and DEAE-cellulose chromatography (Sober and Peterson, 1958). This material gave a single precipitation band when tested by immunoelectrophoresis (Scheidegger, 1955) against a rabbit antiserum raised against whole human serum. IgG was iodinated with 12sI by the lactoperoxidase method (Marchalonis, 1969) at a specific activity ranging from 7.0 to 8.0 pCi/pg of protein. [12sI]IgG was separated from free iodine by Sephadex G-25 chromatography and then aggregates removed by ultracentrifugation at 250,000 × g for 60 min. The labeled IgG was divided into aliquots and kept frozen at --70°C for up to 2 months.
A n risera The results reported in this study were obtained using a commercially available rabbit anti-human IgG, Fc fragment specific antiserum (Cappel Laboratories, Inc., Cochranville, PA), b u t other anti-IgG antisera gave similar results. In most experiments, a crude gamma-globulin fraction obtained by precipitation of the antiserum with 33% saturated ammonium sulfate was employed. In some cases the original antiserum or purified anti-IgG antibody was used. Purified antibody was obtained b y affinity chromatography on Sepharose 4B to which human IgG was coupled (Cuatrecasas, 1970), following a procedure described elsewhere (Pinchera et al., 1977) and iodinated b y the same procedure employed for human IgG.
Preparation of the solid-state anti-IgG antibody This was performed following the procedure described by Catt (1969). U-shaped plastic (polyvinyl flexible) Microtiter ® plates (Cook Engineering
191 Co., Alexandria, VA) were filled with the anti-IgG solution in carbonatebicarbonate buffer 0.05 M, pH 9.5 and incubated for 6 h at r o o m temperature. Three control wells on each plate were filled with buffer alone. Following incubation, excess antibody was removed by aspiration and the cups filled with phosphate-buffered saline (PBS) at pH 7.6 containing 1.0% bovine serum albumin and 0.02% NaN3 (PBS-BSA). After standing f0r 30 rain at r o o m temperature, PBS-BSA was removed and the wells were washed twice with PBS. The anti-IgG coated plates were either used immediately or kept at 4°C for up to 1 m o n t h without significant loss of antib o d y activity.
Standard radioirnmunoassay procedure Immediately before the assay, the anti-IgG coated plates were rinsed with PBS, following which 100 pl of l y m p h o c y t e culture medium containing 0--2000 ng/ml of human IgG or 100 /~l of the u n k n o w n samples were added into the wells. Each sample was assayed in duplicate or triplicate. Wells precoated with buffer alone (blank} were filled with 100 pl of culture medium. After standing for 2 h at 37°C in a moist atmosphere, 15,000 cpm (-~1 ng) of [12sI]IgG diluted in fetal calf serum (FCS) were added and the plates incubated overnight at r o o m temperature. U n b o u n d material was then removed by aspiration, the plates were washed extensively under tap water, and individual wells cut o u t and counted in a gamma counter. Maximal binding (Bo) was defined as binding of [12sI]IgG to anti-IgG in the absence of added cold IgG minus binding in the blank. Results were expressed as the ratio of binding of [125I]IgG in the presence of added cold IgG minus binding in the blank (B) divided by Bo × 100. This gave percent B/Bo. Results were plotted on a logit-log scale.
Cell suspension preparations Venous blood from y o u n g healthy donors was drawn into syringes containing preservative-free heparin. MNCs were isolated on Ficoll-Hypaque (specific density 1.076) and washed 3 times with Hank's balanced salt solution (HBSS). Aliquots of MNCs were removed and washed 6 additional times in HBSS. The remainder of the MNCs were rosetted with neuraminidasetreated sheep red blood cells (sRBC) for 1 h at r o o m temperature. Rosetted cells (E ÷) were separated from non-rosetted cells (E-) by centrifugation over a second Ficoll-Hypaque gradient. Cells at the interface (E-) were washed 5 times and suspended at 106/ml in culture medium (RPMI 1640, Microbiological Associates) supplemented with gentamycin (10 mg/ml), glutamine (2 mM, Flow Laboratories) and 20% heat-inactivated fetal bovine serum. Cells in the pellet (E ÷) were washed and treated for 10 min at 37°C with a m m o n i u m chloride (0.83%), washed an additional 3 times and suspended at 4 X 104 in culture medium. Overall, each cell fraction was washed 9 times prior to being p u t into culture.
192
Cell markers The E- cells contained > 4 0 % cells with m e m b r a n e b o u n d Ig (B lymphocytes), > 4 0 % peroxidase ÷ cells (Kaplow, 1975) and < 1 0 % sRBC rosetting cells (T cells). The E ÷ cell fraction contained ~ 2 % cells with m e m b r a n e b o u n d Ig and < 1 % peroxidase* cells.
Culture conditions 106 cells/ml were cultured in 12 m m × 75 m m plastic tubes (Falcon) and incubated at 37°C in a 5% CO2 humidified incubator for 1 or 7 days. Supernatants o f 1 day cultures served as a cont r ol for the effectiveness of washing. To some 7 day cultures pokew e e d mitogen (Gibco) at a final dilution of 1 : 100 was added at the beginning of the culture period. Other 7 day cultures were maintained in m e di um alone. When subpopulations of cells were c o ~ u l t u r e d the total volume of the culture was kept at 1 ml. All supernatants were harvested by centrifugation at 400 × g for 10 rain. All data points were on paired samples. RESULTS
Adsorption o f anti-IgG antibody on the plastic surface To evaluate the a m o u n t of a n t i b o d y adsorbed ont o the wells, a small aliquot o f 12SI-purified anti-IgG a n t i b o d y was added (10,000 cpm) to the wells which were then filled with different dilutions of anti-IgG gammaglobulin. After standing for 6 h at r o o m t e m p e r a t u r e , the plates were aspirated and the wells submitted to the same series of washings and incubations described in the Materials and Methods section for the standard radioimmunoassay p r o ce dur e, but excluding the presence of [~2sI]IgG. The following day the radioactivity f o u n d in the incubation mixtures and that fixed to the wells were each c o u n t e d and the a m o u n t of a n t i b o d y adsorbed to the wells calculated. The results obtained are given in Fig. 1. The a m o u n t of antiIgG gamma-globulin b o u n d to the wells increased from 69 to 354 ng as the protein c o n c e n t r a t i o n of the coating solution was increased from 0.5 to 4.0 pg/ml. The quant i t y of anti-IgG which detached from the solid phase was minimal; it never exceeded 5% of the b o u n d fraction at any concentration o f anti-IgG used for coating.
Determination of the best conditions for the radioimmunoassay Preliminary experiments showed t hat incubation times ranging from 12 to 48 h or incubation at 4°C versus r o o m t e m p e r a t u r e did n o t change the results o f the assay. Accordingly an overnight incubation at r o o m temperature was selected as standard procedure. The use of a disequilibrium
193 500-
400-
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~= 2ooo
k IOO-
.5
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410
Protein Concentration of the Coating Solution (/~g lint) Fig. 1. Amount of anti-IgG gamma-globulin bound to the wells of plastic microtiter plates using different concentrations for the coating solution (; ;). The amount of antiIgG detached during the standard series of washings and incubations is also shown (o .... o).
technique (Hales and Randle, 1963), i.e., a preincubation with unlabeled antigen before adding the radiolabeled IgG gave sensitive and reproducible results and was subsequently used as standard procedure. To determine the optimal dilution to coat the plastic wells a wide range of concentrations of anti-IgG antibody was tested. Binding of [~2sI]IgG in the presence of different concentrations of unlabeled IgG was then determined. Results are shown in Fig. 2. Maximal binding of the tracer in absence of unlabeled antigen increased from 32.1% to 78.5% as the concentration of anti-IgG used for coating was increased from 1.0 to 32.0 ttg/ml. Non-specific fixation was constantly <0.1%. The inhibition of binding of [125I]IgG obtained in the presence of 3 representative concentrations (111, 333 and 1000 ng/ml) of unlabeled IgG using different concentrations of anti-IgG for coating is also shown in Fig. 2. Based on the results of this experiment, a concentration of 2.0 pg/ml of anti-IgG (this gave a maximal binding of -~50%) was selected as the standard anti-IgG concentration to be used for coating. Similar results were obtained when the plates were coated with the original anti-IgG serum or with the purified anti-IgG antibody, although higher variability was observed when serum was used. The results using salt fractionated gammaglobulin were comparable to those obtained with affinity chromatography purified antibody. Thus, in the final assay, the salt fractionated gamma-
194 Anti-lg G concemrotions: = ~o-----o
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Fig. 2. Specific binding o f r a d i o i o d i n a t e d h u m a n IgG ([12sI]IgG) to plastic wells of a microtiter plate c o a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f anti-IgG ~'-globulin in presence o f d i f f e r e n t a m o u n t s o f cold IgG (111--1000 pg/ml). T h e results are e x p r e s s e d as p e r c e n t o f the total radioactivity added ( 1 5 , 0 0 0 c p m 2 1 ng) after subtraction o f the non-specific binding (< 0.1%}. Each p o i n t represents the m e a n o f a quadruplicate assay ±S.D.
globulin fraction of the anti-IgG serum was used for coating. Fig. 3 shows the average of 13 standard curves obtained from consecutive assays with a coating solution containing 2 pg/ml of anti-IgG. The minimum detectable level of IgG was consistently < 1 0 . 0 ng/ml (i.e., < 1 . 0 ng/100 pl in the assay); the average coefficient of variation within the assay was 10%; between assays it was 11% at a concentration of IgG of 31.2 ng/ml, 16% at 125 ng/ml and 19% at 1 0 0 0 ng/ml. One serum initially diluted to contain 1 mg/ml of IgG was further serially diluted and the percent B/Bo determined for each sample. The results paralleled the standard curve (Fig. 3). The calculated concentrations agreed with results obtained by radial immunodiffusion. When two PWM stimulated lymphocyte supernatants containing high levels of IgG were serially diluted and the B/Bo percentage of each sample determined, these results also closely paralleled the results obtained for the standard curve (Fig. 4). Purified IgM obtained from sera of subjects with multiple myeloma (kindly provided by Dr. J. Hopper, Department of Medicine, University of
195
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Fig. 3. Average s t a n d a r d curve o b t a i n e d f r o m 13 c o n s e c u t i v e assays (¢, v,). R e s u l t s are e x p r e s s e d as p e r c e n t o f t h e m a x i m a l b i n d i n g in a b s e n c e o f u n l a b e l e d IgG, a f t e r s u b t r a c t i o n o f t h e n o n - s p e c i f i c b i n d i n g ( B / B o % ) . As s h o w n , t h e B / B o % o f o n e a p p r o p r i a t e l y d i l u t e d h u m a n s e r u m (A A) parallels t h e result o b t a i n e d w i t h t h e s t a n d a r d curve.
Illinois), showed <0.1% of cross-reactivity. To study regulation of IgG secretion by lymphocytes in culture, the a m o u n t of IgG transferred to cultures along with the cells has to be deter95", 90' %'%.%%
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Fig. 4. T h e B / B o % o b t a i n e d f o r d i f f e r e n t d i l u t i o n s o f PWM s t i m u l a t e d c u l t u r e supern a t a n t s c o n t a i n i n g high level o f IgG is s h o w n (¢, o, x ×). T h e c u r v e o b t a i n e d f o r t h o s e d i f f e r e n t d i l u t i o n s parallels t h e s t a n d a r d curve ( . . . . . . ).
196 TABLE 1 A m o u n t o f IgG found in the washing solutions o f 106 MNCs washed in 5 ml of HBSS -result of a typical experiment. Number of washes
Total IgG content of the washing suspension (ng/ml)
3 6 9 12
200 180 46 20
mined. Preliminary experiments showed that the IgG content of the washing s o l u t i o n d e c r e a s e d w i t h r e p e a t e d c e l l w a s h i n g ( T a b l e 1). As a r o u t i n e w e w a s h e d t h e cel l s 9 t i m e s as d e s c r i b e d in t h e M e t h o d s a n d h a r v e s t e d c u l t u r e s at 24 h to e v a l u a t e the a m o u n t of a d s o r b e d IgG e l u t e d at 37°C. In our hands, 24 h cultures always contained <200 ng IgG. In studies o f u n f r a c t i o n a t e d MNCs, o n e m u s t c o m p a r e IgG s e c r e t i o n by unstimulated and PWM stimulated MNCs. After 7 days of culture, unstimu l a t e d M N C s d o s e c r e t e s o m e I g G ( T a b l e 2). I g G p r o d u c t i o n is m a r k e d l y TABLE 2 Secretion of IgG (ng/ml) in 7-day PWM stimulated and unstimulated MNC c u l t u r e s - comparison with E + + E- cultures at 1 : 1 ratio. --, data show the results for 11 individuals studied. MNC No PWM
E + + EWith PWM
240 162 150 20 64 76 530 740 50 1250 920 610 455 9200 495 6400 338 5800 250 2000 88 125 Mean ± S.E.M. 325 1580 ±79 ±1141
PWM response
No PWM
PWM
PWM response
Expected IgG secretion a
T cell helper effect b
---+ + -+ + + + --
160 150 48 96 64 980 381 445 339 222 145
800 127 195 380 8880 1250 15470 9000 4800 4000 1100
+ -+ + + + + + + + +
(172) (75) (60) (295) (125) (538) (201) (891) (311) (307) (105)
627 52 135 85 8755 712 15269 8108 4489 3692 995
275 ±80
4182 ~1506
a Expected IgG secretion is calculated by adding one-half the amount of IgG found in E- cultures to one-half the amount of IgG found in E + cultures. b T helper cell effect is calculated as amount of IgG in E++ E- cultures minus the expected amount o f IgG a
197 TABLE 3 Suppression of IgG secretion by increasing T : B cell ratio. The number of B cells is maintained constant at 0.5 × 106 cells. Results are expressed as mean percent of the maximum response -+ S.E.M. for 5 experiments. The results of a single typical experiment are also shown. T : B ratio
% of maximal response Typical experiment
1:1
2:1
5:1
8:1
91 ± 8.9% 100
68 ± 15.2% 80
35 _+12.5% 55
12 _+4.2% 5
i n c r e a s e d in PWM s t i m u l a t e d c u l t u r e s f r o m s o m e , b u t n o t all individuals. In o u r h a n d s , o n l y 7 o f 11 c o n t r o l s s t u d i e d s h o w e d s u c h increased I g G p r o d u c t i o n triggered b y PWM. Mean p r o d u c t i o n was 1 5 8 0 + 1141 ng f o r t h e g r o u p as a w h o l e . B y using c o c u l t u r e s o f E * a n d E - subsets in v a r y i n g p r o p o r t i o n s , o n e can d e t e r m i n e t h e B : T cell r a t i o s w h i c h result in m a x i m a l I g G p r o d u c t i o n . With a c o n s t a n t n u m b e r o f E - cells p r e s e n t in t h e c u l t u r e (0.5 X 106), m a x i m a l I g G s e c r e t i o n is usually o b t a i n e d at a 1 : 1 r a t i o o f E ÷ : E - (Table 3). A t this 1 : 1 r a t i o , E÷--E - r e c o n s t i t u t e d c u l t u r e s s h o w e d a m e a n p r o d u c t i o n o f 4 1 8 0 + 1 5 0 6 ng o f I g G a f t e r 1 w e e k in t h e p r e s e n c e o f PWM. This 1 : 1 r a t i o a p p e a r s t o be t h e o p t i m a l c o n d i t i o n f o r I g G s e c r e t i o n ( c o m p a r e to T a b l e 2). T o aid in i n t e r p r e t a t i o n o f c o c u l t u r e e x p e r i m e n t s , d e t e r m i n a t i o n o f I g G p r o d u c t i o n b y e a c h cell f r a c t i o n has p r o v e n useful. N o I g G s e c r e t i o n is d e t e c t e d in t h e E ÷ cell s u p e r n a t a n t s in t h e p r e s e n c e or a b s e n c e o f PWM. B e t w e e n d a y 1 a n d d a y 7, in t h e a b s e n c e of PWM, t h e E - c u l t u r e s c o n s i s t e n t l y s h o w a m o d e s t increase in t h e a m o u n t o f I g G p r e s e n t in t h e supern a t a n t ( 1 4 4 -+ 42 vs. 365 -+ 67, n = 11). This s e c r e t i o n is f u r t h e r i n c r e a s e d b y t h e a d d i t i o n o f PWM in o n l y a f e w individuals ( s t i m u l a t o r y indices r a n g i n g f r o m 1.9 t o 4.0). Such e n h a n c e m e n t a p p e a r e d t o r e f l e c t t h e influe n c e o f residual T cells in t h e E - cell p o p u l a t i o n . DISCUSSION In t h e p r e s e n t s t u d y , we d e s c r i b e a n e w s i m p l e solid-phase R I A f o r h u m a n IgG. This a s s a y is b a s e d o n t h e c o m p e t i t i v e b i n d i n g o f r a d i o l a b e l e d a n d u n l a b e l e d I g G t o a n t i - I g G a n t i b o d y p h y s i c a l l y a d s o r b e d t o t h e wells o f p o l y v i n y l m i c r o t i t e r plates. T h e s t a b i l i t y o f a d s o r p t i o n o f t h e a n t i - I g G using g r a d e d c o n c e n t r a t i o n s o f r a d i o i o d i n a t e d a n t i - I g G a n t i b o d y a p p e a r s to be e x c e l l e n t . T h e assay d e s c r i b e d h e r e is m o r e sensitive (1 n g / t e s t s a m p l e ) t h a n m o s t r e p o r t e d solid-phase m e t h o d s . This s i m p l e p r o c e d u r e a p p e a r s as effic i e n t as a n d less c u m b e r s o m e t h a n o t h e r c o u p l i n g r e a c t i o n s used in p u b l i s h e d solid-phase R I A f o r h u m a n I g G ( M a n n et al., 1 9 6 9 ; B o m b a r d i e r i a n d Chris-
198
tian, 1970; Eady et al., 1975). In our hands the a m o u n t of anti-IgG antibody bound to the wells is very close to t h a t reported by Herrman and Collins (1976) for rabbit IgG with the same plastic. In a previous report, Salmon et al. (1969) attempted to coat polystyrene tubes directly with anti-IgG serum but failed to obtain significant binding. The reason for the discrepancy between these results and ours is n o t clear since polystyrene and polyvinyl show similar affinity for rabbit Ig (Herrman and Collins, 1976) and the coating reaction was performed using a procedure similar to that employed by us. The m e t h o d has been successfully applied to the study of Ig secretion in PWM stimulated cultures. Certain potential pitfalls in studies of this kind have been signaled. These include: (1) the necessity for extensive washing to remove non-specific IgG, and (2) the need for a control with unstimulated cells since some 'spontaneous' Ig secretion is c o m m o n l y encountered. Having controlled for these variables the assay has been employed to analyze helper and suppressor effects in vitro. To this end E ÷ and E- cells have been separated and recombined in variousratios. Here it must be emphasized that the E- cells must be sufficiently depleted of T cells so that no IgG secretory response to PWM by E- cells cultured alone can be shown. A 1 : 1 ratio of E + to E- cells has been shown to favor helper effect. As E ÷ cell numbers are increased (E- cell numbers being held constant) suppressor effect is progressively augmented. All of the above steps and controls translate into a large number of cultures for each study. The solid-phase RIA described here has permitted rapid performance of this type of analysis. ACKNOWLEDGEMENTS
This work was supported in part by the Kroc Foundation, the Multiple Sclerosis Society (Grant No. RG 1130-C-16) and USPHS (Grant No. AM13377 and CA 19266). REFERENCES Bombardieri, S. and C.L. Christian, 1970, Proc. Soc. Biol. Med. 133, 1366. Catt, K.J., 1969, Acta Endocr. 63 (Suppl. 142), 222. Choi, Y.S., 1977, J. Immunol. Methods 14, 37. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Eady, R.P., J.C. Chapple, D.W. Hough and G.T. Stevenson, 1975, J. Immunol. Methods 7,179. Gilden, D. and T. Tachovsky, 1979, J. Neurosci. Methods 1 , 1 3 3 . Fauci, A.S., 1979, Immunol. Rev. 45, 93. Fauci, A.S. and K.R. Pratt, 1976, J. Exp. Med. 144, 674. Hales, C.N. and P.J. Randle, 1963, Biochem. J. 8 8 , 1 3 7 . Haynes, B.F. and A.S. Fauci, 1977, J. Immunol. 118, 2281. Herrman, J.E. and M.F. Collins, 1976, J. Immunol. Methods 10,363. Kaplow, L.S., 1975, Am. J. Clin. Pathol. 6 3 , 4 5 1 .
199 Mann, D., M. Granger and J.L. Fahey, 1969, J. Immunol. 102, 618. Marchalonis, J.J., 1969, Biochem. J. 113, 299. Pinchera, A., S. Mariotti, P. Vitti, M. Tosi, L. Grasso, F. Pacini, R. Buti and L. Baschieri, 1977, J. Clin. Endocr. Metab. 45, 1077. Rice, L., A.H. Laughter and J.J. Twomey, 1979, J. Immunol. 122,991. Salmon, S.E., G. Mackey and H.H. Fudenberg, 1969, J. Immunol. 103, 129. Saxon, A., R.H. Stevens and R.F. Ashman, 1977, J. Immunol. 118, 1872. Scheidegger, J.J., 1955, Int. Arch. Allergy Appl. Immunol. 7, 103. Sober, H.A. and E.A. Peterson, 1958, Fed. Proc. 17, 1116. Stobo, J.D., 1977, J. Immunol. 119,918. Waldman, T.A., S. Broder, R.M. Blease, M. Durm, M. Blackman and W. Stober, 1974, Lancet ii, 609. Waldman, T.A., R.M. Blease, S. Broder and R.S. Krakauer, 1978, Ann. Intern. Med. 88,226. Zollinger, W.D., J.M. Dalrymple and M.S. Artenstein, 1976, J. Immunol. 117, 1788.
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© Elsevier/North-Holland Biomedical Press
A NEW SOLID-PHASE RADIOIMMUNOASSAY TO MEASURE IgG SECRETED BY CULTURED HUMAN LYMPHOCYTES
STEFANO MARIOTTI 1, JOEL J.-F. OGER 2, PHILIPPE F R A G U s, JACK P. ANTEL, HAN-HWA KUO and LESLIE J. DeGROOT
(S.M., P.F. and L.J. DeG.) University of Chicago, Pritzker school of Medicine, Department of Medicine (Thyroid Study Unit), and (J.J.-F.O., J.P.A. and H.-H.K.) Department of Neurology, Chicago, IL 60637, U.S.A. (Received 5 December 1979, accepted 4 March 1980)
We describe a simple solid-phase radioimmunoassay (RIA) to detect IgG based on competitive binding between radiolabeled and unlabeled IgG for anti-IgG antibody physically adsorbed to the wells of polyvinyl microtiter plates. The assay is sensitive (1 ng), rapid, and is particularly suited for studies of in vitro IgG secretion by human peripheral blood lymphocytes, since such studies require large numbers of cultures. Conditions which permit measurement, by means of this assay, of helper and suppressor T cell effects on IgG production by human B cells in culture are described.
INTRODUCTION
Immunoglobulin (Ig) secretion in vitro by human peripheral blood lymphocytes is a convenient model to study mechanisms involved in immunoregulation (Waldman et al., 1974; Choi, 1977; Saxon et al., 1977; Fauci, 1979). Both synthesis and secretion of Ig are mediated by B lymphocytes, but cooperation by a helper T cell subset is required (Saxon et al., 1977). Ig production can also be suppressed by a different subset of T lymphocytes (suppressor T cells) (Waldman et al., 1978) and by other circulating non-T cells (Stobo, 1977; Rice et al., 1979). To utilize the Ig production system, mononuclear cells (MNCs) must be prepared in a manner which reduces pre-existent Ig adherent to the lymphocytes. To assess helper and suppressor functions B and T cell enriched cell fractions are recombined in varying proportions and this requires large numbers of cultures. Only small amounts of Ig are secreted into the culture medium by B cells, even when they are stimulated by pokeweed mitogen (PWM). For this reason 1 Dr. Mariotti is on leave from Cattedra di Patologia Medica 2, Universit~ di Pisa, Italy. 2 Dr. Oger is on leave from INSERM France. Address reprint requests to: J. Oger, M.D., The University of Chicago, Pritzker School of Medicine, Department of Neurology, 950 East 59th Street, Chicago, IL 60637, U.S.A. s Dr. Fragu is on leave from Institut Gustave-Roussy, Villejuif, France.
190
sensitive assay techniques have been developed to detect Ig. In one technique the incorporation of radiolabeled amino acids into Ig molecules is measured (Choi, 1977). This technique does n o t give absolute values of Ig. Hemolytic plaque-forming assay methods have been utilized, but these are cumbersome for large scale studies and again do not provide absolute values of Ig (Fauci and Pratt, 1976). Direct determination of Ig in culture supernatants by radioimmunoassay (RIA) would be the preferred assay system if one could develop an RIA system which combines high sensitivity and reproducibility with short incubation time and ease of performance. Such a system would permit rapid assessment of a great number of samples. In the present study, we describe a new solid-phase RIA for human IgG which meets the requirements outlined and indicate its applicability to the study of immunoregulation. MATERIALS AND METHODS
Preparation and radioiodination of human IgG Human IgG was prepared from pooled sera of normal subjects by ammonium sulfate precipitation and DEAE-cellulose chromatography (Sober and Peterson, 1958). This material gave a single precipitation band when tested by immunoelectrophoresis (Scheidegger, 1955) against a rabbit antiserum raised against whole human serum. IgG was iodinated with 12sI by the lactoperoxidase method (Marchalonis, 1969) at a specific activity ranging from 7.0 to 8.0 pCi/pg of protein. [12sI]IgG was separated from free iodine by Sephadex G-25 chromatography and then aggregates removed by ultracentrifugation at 250,000 × g for 60 min. The labeled IgG was divided into aliquots and kept frozen at --70°C for up to 2 months.
A n risera The results reported in this study were obtained using a commercially available rabbit anti-human IgG, Fc fragment specific antiserum (Cappel Laboratories, Inc., Cochranville, PA), b u t other anti-IgG antisera gave similar results. In most experiments, a crude gamma-globulin fraction obtained by precipitation of the antiserum with 33% saturated ammonium sulfate was employed. In some cases the original antiserum or purified anti-IgG antibody was used. Purified antibody was obtained b y affinity chromatography on Sepharose 4B to which human IgG was coupled (Cuatrecasas, 1970), following a procedure described elsewhere (Pinchera et al., 1977) and iodinated b y the same procedure employed for human IgG.
Preparation of the solid-state anti-IgG antibody This was performed following the procedure described by Catt (1969). U-shaped plastic (polyvinyl flexible) Microtiter ® plates (Cook Engineering
191 Co., Alexandria, VA) were filled with the anti-IgG solution in carbonatebicarbonate buffer 0.05 M, pH 9.5 and incubated for 6 h at r o o m temperature. Three control wells on each plate were filled with buffer alone. Following incubation, excess antibody was removed by aspiration and the cups filled with phosphate-buffered saline (PBS) at pH 7.6 containing 1.0% bovine serum albumin and 0.02% NaN3 (PBS-BSA). After standing f0r 30 rain at r o o m temperature, PBS-BSA was removed and the wells were washed twice with PBS. The anti-IgG coated plates were either used immediately or kept at 4°C for up to 1 m o n t h without significant loss of antib o d y activity.
Standard radioirnmunoassay procedure Immediately before the assay, the anti-IgG coated plates were rinsed with PBS, following which 100 pl of l y m p h o c y t e culture medium containing 0--2000 ng/ml of human IgG or 100 /~l of the u n k n o w n samples were added into the wells. Each sample was assayed in duplicate or triplicate. Wells precoated with buffer alone (blank} were filled with 100 pl of culture medium. After standing for 2 h at 37°C in a moist atmosphere, 15,000 cpm (-~1 ng) of [12sI]IgG diluted in fetal calf serum (FCS) were added and the plates incubated overnight at r o o m temperature. U n b o u n d material was then removed by aspiration, the plates were washed extensively under tap water, and individual wells cut o u t and counted in a gamma counter. Maximal binding (Bo) was defined as binding of [12sI]IgG to anti-IgG in the absence of added cold IgG minus binding in the blank. Results were expressed as the ratio of binding of [125I]IgG in the presence of added cold IgG minus binding in the blank (B) divided by Bo × 100. This gave percent B/Bo. Results were plotted on a logit-log scale.
Cell suspension preparations Venous blood from y o u n g healthy donors was drawn into syringes containing preservative-free heparin. MNCs were isolated on Ficoll-Hypaque (specific density 1.076) and washed 3 times with Hank's balanced salt solution (HBSS). Aliquots of MNCs were removed and washed 6 additional times in HBSS. The remainder of the MNCs were rosetted with neuraminidasetreated sheep red blood cells (sRBC) for 1 h at r o o m temperature. Rosetted cells (E ÷) were separated from non-rosetted cells (E-) by centrifugation over a second Ficoll-Hypaque gradient. Cells at the interface (E-) were washed 5 times and suspended at 106/ml in culture medium (RPMI 1640, Microbiological Associates) supplemented with gentamycin (10 mg/ml), glutamine (2 mM, Flow Laboratories) and 20% heat-inactivated fetal bovine serum. Cells in the pellet (E ÷) were washed and treated for 10 min at 37°C with a m m o n i u m chloride (0.83%), washed an additional 3 times and suspended at 4 X 104 in culture medium. Overall, each cell fraction was washed 9 times prior to being p u t into culture.
192
Cell markers The E- cells contained > 4 0 % cells with m e m b r a n e b o u n d Ig (B lymphocytes), > 4 0 % peroxidase ÷ cells (Kaplow, 1975) and < 1 0 % sRBC rosetting cells (T cells). The E ÷ cell fraction contained ~ 2 % cells with m e m b r a n e b o u n d Ig and < 1 % peroxidase* cells.
Culture conditions 106 cells/ml were cultured in 12 m m × 75 m m plastic tubes (Falcon) and incubated at 37°C in a 5% CO2 humidified incubator for 1 or 7 days. Supernatants o f 1 day cultures served as a cont r ol for the effectiveness of washing. To some 7 day cultures pokew e e d mitogen (Gibco) at a final dilution of 1 : 100 was added at the beginning of the culture period. Other 7 day cultures were maintained in m e di um alone. When subpopulations of cells were c o ~ u l t u r e d the total volume of the culture was kept at 1 ml. All supernatants were harvested by centrifugation at 400 × g for 10 rain. All data points were on paired samples. RESULTS
Adsorption o f anti-IgG antibody on the plastic surface To evaluate the a m o u n t of a n t i b o d y adsorbed ont o the wells, a small aliquot o f 12SI-purified anti-IgG a n t i b o d y was added (10,000 cpm) to the wells which were then filled with different dilutions of anti-IgG gammaglobulin. After standing for 6 h at r o o m t e m p e r a t u r e , the plates were aspirated and the wells submitted to the same series of washings and incubations described in the Materials and Methods section for the standard radioimmunoassay p r o ce dur e, but excluding the presence of [~2sI]IgG. The following day the radioactivity f o u n d in the incubation mixtures and that fixed to the wells were each c o u n t e d and the a m o u n t of a n t i b o d y adsorbed to the wells calculated. The results obtained are given in Fig. 1. The a m o u n t of antiIgG gamma-globulin b o u n d to the wells increased from 69 to 354 ng as the protein c o n c e n t r a t i o n of the coating solution was increased from 0.5 to 4.0 pg/ml. The quant i t y of anti-IgG which detached from the solid phase was minimal; it never exceeded 5% of the b o u n d fraction at any concentration o f anti-IgG used for coating.
Determination of the best conditions for the radioimmunoassay Preliminary experiments showed t hat incubation times ranging from 12 to 48 h or incubation at 4°C versus r o o m t e m p e r a t u r e did n o t change the results o f the assay. Accordingly an overnight incubation at r o o m temperature was selected as standard procedure. The use of a disequilibrium
193 500-
400-
(.9
-? 3ooI I= 0 C
~= 2ooo
k IOO-
.5
1.0
2'O
410
Protein Concentration of the Coating Solution (/~g lint) Fig. 1. Amount of anti-IgG gamma-globulin bound to the wells of plastic microtiter plates using different concentrations for the coating solution (; ;). The amount of antiIgG detached during the standard series of washings and incubations is also shown (o .... o).
technique (Hales and Randle, 1963), i.e., a preincubation with unlabeled antigen before adding the radiolabeled IgG gave sensitive and reproducible results and was subsequently used as standard procedure. To determine the optimal dilution to coat the plastic wells a wide range of concentrations of anti-IgG antibody was tested. Binding of [~2sI]IgG in the presence of different concentrations of unlabeled IgG was then determined. Results are shown in Fig. 2. Maximal binding of the tracer in absence of unlabeled antigen increased from 32.1% to 78.5% as the concentration of anti-IgG used for coating was increased from 1.0 to 32.0 ttg/ml. Non-specific fixation was constantly <0.1%. The inhibition of binding of [125I]IgG obtained in the presence of 3 representative concentrations (111, 333 and 1000 ng/ml) of unlabeled IgG using different concentrations of anti-IgG for coating is also shown in Fig. 2. Based on the results of this experiment, a concentration of 2.0 pg/ml of anti-IgG (this gave a maximal binding of -~50%) was selected as the standard anti-IgG concentration to be used for coating. Similar results were obtained when the plates were coated with the original anti-IgG serum or with the purified anti-IgG antibody, although higher variability was observed when serum was used. The results using salt fractionated gammaglobulin were comparable to those obtained with affinity chromatography purified antibody. Thus, in the final assay, the salt fractionated gamma-
194 Anti-lg G concemrotions: = ~o-----o
80-
=
~
.I,
.1.
~
I p. g/mi 2 . ,'
4
16
.
32
,,
70-
# t9 600
•~
5040-
~ 30~ 20Q.
I0-
0
III
333
1,000
Ig G ( n g / m l )
Fig. 2. Specific binding o f r a d i o i o d i n a t e d h u m a n IgG ([12sI]IgG) to plastic wells of a microtiter plate c o a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f anti-IgG ~'-globulin in presence o f d i f f e r e n t a m o u n t s o f cold IgG (111--1000 pg/ml). T h e results are e x p r e s s e d as p e r c e n t o f the total radioactivity added ( 1 5 , 0 0 0 c p m 2 1 ng) after subtraction o f the non-specific binding (< 0.1%}. Each p o i n t represents the m e a n o f a quadruplicate assay ±S.D.
globulin fraction of the anti-IgG serum was used for coating. Fig. 3 shows the average of 13 standard curves obtained from consecutive assays with a coating solution containing 2 pg/ml of anti-IgG. The minimum detectable level of IgG was consistently < 1 0 . 0 ng/ml (i.e., < 1 . 0 ng/100 pl in the assay); the average coefficient of variation within the assay was 10%; between assays it was 11% at a concentration of IgG of 31.2 ng/ml, 16% at 125 ng/ml and 19% at 1 0 0 0 ng/ml. One serum initially diluted to contain 1 mg/ml of IgG was further serially diluted and the percent B/Bo determined for each sample. The results paralleled the standard curve (Fig. 3). The calculated concentrations agreed with results obtained by radial immunodiffusion. When two PWM stimulated lymphocyte supernatants containing high levels of IgG were serially diluted and the B/Bo percentage of each sample determined, these results also closely paralleled the results obtained for the standard curve (Fig. 4). Purified IgM obtained from sera of subjects with multiple myeloma (kindly provided by Dr. J. Hopper, Department of Medicine, University of
195
95' 90'
70~m 6o Iz 50 uJ 40 re' 3020" I0"
i
!
I()
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1,000
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Fig. 3. Average s t a n d a r d curve o b t a i n e d f r o m 13 c o n s e c u t i v e assays (¢, v,). R e s u l t s are e x p r e s s e d as p e r c e n t o f t h e m a x i m a l b i n d i n g in a b s e n c e o f u n l a b e l e d IgG, a f t e r s u b t r a c t i o n o f t h e n o n - s p e c i f i c b i n d i n g ( B / B o % ) . As s h o w n , t h e B / B o % o f o n e a p p r o p r i a t e l y d i l u t e d h u m a n s e r u m (A A) parallels t h e result o b t a i n e d w i t h t h e s t a n d a r d curve.
Illinois), showed <0.1% of cross-reactivity. To study regulation of IgG secretion by lymphocytes in culture, the a m o u n t of IgG transferred to cultures along with the cells has to be deter95", 90' %'%.%%
80. w° 70-
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~ z 50uJ o 40n,,.
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% "%'%%. %
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Fig. 4. T h e B / B o % o b t a i n e d f o r d i f f e r e n t d i l u t i o n s o f PWM s t i m u l a t e d c u l t u r e supern a t a n t s c o n t a i n i n g high level o f IgG is s h o w n (¢, o, x ×). T h e c u r v e o b t a i n e d f o r t h o s e d i f f e r e n t d i l u t i o n s parallels t h e s t a n d a r d curve ( . . . . . . ).
196 TABLE 1 A m o u n t o f IgG found in the washing solutions o f 106 MNCs washed in 5 ml of HBSS -result of a typical experiment. Number of washes
Total IgG content of the washing suspension (ng/ml)
3 6 9 12
200 180 46 20
mined. Preliminary experiments showed that the IgG content of the washing s o l u t i o n d e c r e a s e d w i t h r e p e a t e d c e l l w a s h i n g ( T a b l e 1). As a r o u t i n e w e w a s h e d t h e cel l s 9 t i m e s as d e s c r i b e d in t h e M e t h o d s a n d h a r v e s t e d c u l t u r e s at 24 h to e v a l u a t e the a m o u n t of a d s o r b e d IgG e l u t e d at 37°C. In our hands, 24 h cultures always contained <200 ng IgG. In studies o f u n f r a c t i o n a t e d MNCs, o n e m u s t c o m p a r e IgG s e c r e t i o n by unstimulated and PWM stimulated MNCs. After 7 days of culture, unstimu l a t e d M N C s d o s e c r e t e s o m e I g G ( T a b l e 2). I g G p r o d u c t i o n is m a r k e d l y TABLE 2 Secretion of IgG (ng/ml) in 7-day PWM stimulated and unstimulated MNC c u l t u r e s - comparison with E + + E- cultures at 1 : 1 ratio. --, data show the results for 11 individuals studied. MNC No PWM
E + + EWith PWM
240 162 150 20 64 76 530 740 50 1250 920 610 455 9200 495 6400 338 5800 250 2000 88 125 Mean ± S.E.M. 325 1580 ±79 ±1141
PWM response
No PWM
PWM
PWM response
Expected IgG secretion a
T cell helper effect b
---+ + -+ + + + --
160 150 48 96 64 980 381 445 339 222 145
800 127 195 380 8880 1250 15470 9000 4800 4000 1100
+ -+ + + + + + + + +
(172) (75) (60) (295) (125) (538) (201) (891) (311) (307) (105)
627 52 135 85 8755 712 15269 8108 4489 3692 995
275 ±80
4182 ~1506
a Expected IgG secretion is calculated by adding one-half the amount of IgG found in E- cultures to one-half the amount of IgG found in E + cultures. b T helper cell effect is calculated as amount of IgG in E++ E- cultures minus the expected amount o f IgG a
197 TABLE 3 Suppression of IgG secretion by increasing T : B cell ratio. The number of B cells is maintained constant at 0.5 × 106 cells. Results are expressed as mean percent of the maximum response -+ S.E.M. for 5 experiments. The results of a single typical experiment are also shown. T : B ratio
% of maximal response Typical experiment
1:1
2:1
5:1
8:1
91 ± 8.9% 100
68 ± 15.2% 80
35 _+12.5% 55
12 _+4.2% 5
i n c r e a s e d in PWM s t i m u l a t e d c u l t u r e s f r o m s o m e , b u t n o t all individuals. In o u r h a n d s , o n l y 7 o f 11 c o n t r o l s s t u d i e d s h o w e d s u c h increased I g G p r o d u c t i o n triggered b y PWM. Mean p r o d u c t i o n was 1 5 8 0 + 1141 ng f o r t h e g r o u p as a w h o l e . B y using c o c u l t u r e s o f E * a n d E - subsets in v a r y i n g p r o p o r t i o n s , o n e can d e t e r m i n e t h e B : T cell r a t i o s w h i c h result in m a x i m a l I g G p r o d u c t i o n . With a c o n s t a n t n u m b e r o f E - cells p r e s e n t in t h e c u l t u r e (0.5 X 106), m a x i m a l I g G s e c r e t i o n is usually o b t a i n e d at a 1 : 1 r a t i o o f E ÷ : E - (Table 3). A t this 1 : 1 r a t i o , E÷--E - r e c o n s t i t u t e d c u l t u r e s s h o w e d a m e a n p r o d u c t i o n o f 4 1 8 0 + 1 5 0 6 ng o f I g G a f t e r 1 w e e k in t h e p r e s e n c e o f PWM. This 1 : 1 r a t i o a p p e a r s t o be t h e o p t i m a l c o n d i t i o n f o r I g G s e c r e t i o n ( c o m p a r e to T a b l e 2). T o aid in i n t e r p r e t a t i o n o f c o c u l t u r e e x p e r i m e n t s , d e t e r m i n a t i o n o f I g G p r o d u c t i o n b y e a c h cell f r a c t i o n has p r o v e n useful. N o I g G s e c r e t i o n is d e t e c t e d in t h e E ÷ cell s u p e r n a t a n t s in t h e p r e s e n c e or a b s e n c e o f PWM. B e t w e e n d a y 1 a n d d a y 7, in t h e a b s e n c e of PWM, t h e E - c u l t u r e s c o n s i s t e n t l y s h o w a m o d e s t increase in t h e a m o u n t o f I g G p r e s e n t in t h e supern a t a n t ( 1 4 4 -+ 42 vs. 365 -+ 67, n = 11). This s e c r e t i o n is f u r t h e r i n c r e a s e d b y t h e a d d i t i o n o f PWM in o n l y a f e w individuals ( s t i m u l a t o r y indices r a n g i n g f r o m 1.9 t o 4.0). Such e n h a n c e m e n t a p p e a r e d t o r e f l e c t t h e influe n c e o f residual T cells in t h e E - cell p o p u l a t i o n . DISCUSSION In t h e p r e s e n t s t u d y , we d e s c r i b e a n e w s i m p l e solid-phase R I A f o r h u m a n IgG. This a s s a y is b a s e d o n t h e c o m p e t i t i v e b i n d i n g o f r a d i o l a b e l e d a n d u n l a b e l e d I g G t o a n t i - I g G a n t i b o d y p h y s i c a l l y a d s o r b e d t o t h e wells o f p o l y v i n y l m i c r o t i t e r plates. T h e s t a b i l i t y o f a d s o r p t i o n o f t h e a n t i - I g G using g r a d e d c o n c e n t r a t i o n s o f r a d i o i o d i n a t e d a n t i - I g G a n t i b o d y a p p e a r s to be e x c e l l e n t . T h e assay d e s c r i b e d h e r e is m o r e sensitive (1 n g / t e s t s a m p l e ) t h a n m o s t r e p o r t e d solid-phase m e t h o d s . This s i m p l e p r o c e d u r e a p p e a r s as effic i e n t as a n d less c u m b e r s o m e t h a n o t h e r c o u p l i n g r e a c t i o n s used in p u b l i s h e d solid-phase R I A f o r h u m a n I g G ( M a n n et al., 1 9 6 9 ; B o m b a r d i e r i a n d Chris-
198
tian, 1970; Eady et al., 1975). In our hands the a m o u n t of anti-IgG antibody bound to the wells is very close to t h a t reported by Herrman and Collins (1976) for rabbit IgG with the same plastic. In a previous report, Salmon et al. (1969) attempted to coat polystyrene tubes directly with anti-IgG serum but failed to obtain significant binding. The reason for the discrepancy between these results and ours is n o t clear since polystyrene and polyvinyl show similar affinity for rabbit Ig (Herrman and Collins, 1976) and the coating reaction was performed using a procedure similar to that employed by us. The m e t h o d has been successfully applied to the study of Ig secretion in PWM stimulated cultures. Certain potential pitfalls in studies of this kind have been signaled. These include: (1) the necessity for extensive washing to remove non-specific IgG, and (2) the need for a control with unstimulated cells since some 'spontaneous' Ig secretion is c o m m o n l y encountered. Having controlled for these variables the assay has been employed to analyze helper and suppressor effects in vitro. To this end E ÷ and E- cells have been separated and recombined in variousratios. Here it must be emphasized that the E- cells must be sufficiently depleted of T cells so that no IgG secretory response to PWM by E- cells cultured alone can be shown. A 1 : 1 ratio of E + to E- cells has been shown to favor helper effect. As E ÷ cell numbers are increased (E- cell numbers being held constant) suppressor effect is progressively augmented. All of the above steps and controls translate into a large number of cultures for each study. The solid-phase RIA described here has permitted rapid performance of this type of analysis. ACKNOWLEDGEMENTS
This work was supported in part by the Kroc Foundation, the Multiple Sclerosis Society (Grant No. RG 1130-C-16) and USPHS (Grant No. AM13377 and CA 19266). REFERENCES Bombardieri, S. and C.L. Christian, 1970, Proc. Soc. Biol. Med. 133, 1366. Catt, K.J., 1969, Acta Endocr. 63 (Suppl. 142), 222. Choi, Y.S., 1977, J. Immunol. Methods 14, 37. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Eady, R.P., J.C. Chapple, D.W. Hough and G.T. Stevenson, 1975, J. Immunol. Methods 7,179. Gilden, D. and T. Tachovsky, 1979, J. Neurosci. Methods 1 , 1 3 3 . Fauci, A.S., 1979, Immunol. Rev. 45, 93. Fauci, A.S. and K.R. Pratt, 1976, J. Exp. Med. 144, 674. Hales, C.N. and P.J. Randle, 1963, Biochem. J. 8 8 , 1 3 7 . Haynes, B.F. and A.S. Fauci, 1977, J. Immunol. 118, 2281. Herrman, J.E. and M.F. Collins, 1976, J. Immunol. Methods 10,363. Kaplow, L.S., 1975, Am. J. Clin. Pathol. 6 3 , 4 5 1 .
199 Mann, D., M. Granger and J.L. Fahey, 1969, J. Immunol. 102, 618. Marchalonis, J.J., 1969, Biochem. J. 113, 299. Pinchera, A., S. Mariotti, P. Vitti, M. Tosi, L. Grasso, F. Pacini, R. Buti and L. Baschieri, 1977, J. Clin. Endocr. Metab. 45, 1077. Rice, L., A.H. Laughter and J.J. Twomey, 1979, J. Immunol. 122,991. Salmon, S.E., G. Mackey and H.H. Fudenberg, 1969, J. Immunol. 103, 129. Saxon, A., R.H. Stevens and R.F. Ashman, 1977, J. Immunol. 118, 1872. Scheidegger, J.J., 1955, Int. Arch. Allergy Appl. Immunol. 7, 103. Sober, H.A. and E.A. Peterson, 1958, Fed. Proc. 17, 1116. Stobo, J.D., 1977, J. Immunol. 119,918. Waldman, T.A., S. Broder, R.M. Blease, M. Durm, M. Blackman and W. Stober, 1974, Lancet ii, 609. Waldman, T.A., R.M. Blease, S. Broder and R.S. Krakauer, 1978, Ann. Intern. Med. 88,226. Zollinger, W.D., J.M. Dalrymple and M.S. Artenstein, 1976, J. Immunol. 117, 1788.