Journal of Immunological Methods, 135 (1990) 181-189

181

Elsevier JIM05785

A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens B. A r u n a c h a l a m , G.P. T a l w a r a n d Raj R a g h u p a t h y National Institute of lmmunology, Shahid Jeet Singh Marg, New Delhi-110067, India

(Received 1 June 1990, revisedreceived21 August 1990, accepted 10 September1990)

This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens. This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants. The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents. In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (slg), this method involves a blocking step consisting of unlabelled secondary antibodies. Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay. This assay is simple, reproducible and sensitive. The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens. This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens. Key words: ELISA, cellular; Anti-HLA antibody; Anti-ABO blood group antibody; Monoclonal antibody; Epstein-Barr virus

transformation

Introduction

The advent of hybridoma technology has made possible the development of antibodies specific for a variety of important cell-surface antigens such as those coded by the major histocompatibility complex (MHC), molecules of the CD series: CD3, CD4, CD8 etc.; adhesion molecules: LFA-1, LFA-2; immunoglobulins; T cell receptors, etc. (Betts and McKenzie, 1982; Hosking and Georgiou, 1982). These antibody probes have permitted researchers to elucidate the role of cell-surface molecules in the regulation of immune responses Correspondence to: R. Raghupathy, National Institute of Immunology, Shahid Jeet Singh Marg, New Delhi-110067, India.

at the molecular level and to delineate leucocytes of different subsets and at different stages of lymphocyte maturation (Barnstable et al., 1978; Spangrude et al., 1988). Several assays have been described for the screening of antibodies to cell-surface antigens; these include cytotoxicity, immunofluorescence and agglutination assays. Although these assays have some advantages, they suffer from occasional problems such as prolonged assay times, lack of reproducibility, inflexibility in the detection of different classes and subclasses of antibodies, high subjectivity, high background:signal ratios etc. Furthermore, assays such as cytotoxicity and immunofluorescence are unsuitable for the screening of large numbers of samples that are usually generated in routine hybridoma production. The cel-

0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

182 lular enzyme-linked immunosorbent assay (CELISA) has many of these desirable features (Effros et al., 1985). This paper describes a procedure for a simplified CELISA for the detection of antibodies to HLA and blood group antigens. This assay can be adapted for the detection of antibodies to other cell-surface antigens on any other cell type.

Materials and methods

Reagents Phosphate-buffered saline (PBS, 50 mM phosphate, 150 mM NaC1, pH 7.4); washing buffer, PBS with 0.05% Tween 20; blocking buffer I, PBS with 1% milk powder containing 16 g of protein per 100 g of milk powder (Lactogen, Food Specialities, New Delhi, India); blocking buffer II, PBS with 1% rabbit serum and appropriate dilution of murine monoclonal anti-human /~ or 3' heavy chain antibodies; dilution buffer I, PBS with 1% milk powder and 0.05% Tween 20; dilution buffer II, PBS with 1% rabbit serum and 0.05% Tween 20; substrate solution, citrate phosphate buffer (22.1 mM citric acid, 51.4 mM disodium hydrogen phosphate, pH 5.0) containing 5 mg of o-phenylene diamine (OPD) (Sigma Chemical Company, St. Louis, MO) and 10/xl of hydrogen peroxide per 10 ml of buffer.

Target cells For the assay of anti-HLA antibodies, B lymphoblastoid cell lines (LCLs) were used as the target antigen-bearing cells. LCLs were prepared from peripheral blood mononuclear cells (PBL) of donors as follows. PBL were separated by density gradient centrifugation using Histopaque-1077 (Sigma Diagnostics, St. Louis, MO) and were either transformed with Epstein-Barr virus (EBV) or frozen for subsequent EBV transformation which was carried out by incubating 5 × 106 cells with 1 ml of EBV containing culture supernatant of B95-8 cell line for 4 h at 37 ° C. Cells were then washed and cultured at a concentration of 5 × 105/ml in RPMI 1640 (Flow Labs., Irvine, Scotland) containing 10% FCS (Flow Labs., Irvine, Scotland). After 2-3 weeks, transformed lymphocytes were visible as growing clumps of cells; these were then expanded. Lymphoblastoid cell lines (LCLs) grown

in bulk were either frozen or used immediately for coating ELISA plates. In addition to this panel of cell lines generated in our laboratory, we also used well established HLA-typed LCLs from a panel available with the American Society for Histocompatibility and Immunogenetics. For the assay of anti-ABO antibodies, heparinized blood was collected from individuals of different blood groups. RBCs were separated by centrifugation, washed thrice with PBS and then used for coating ELISA plates and for the agglutination assay.

Antibody reagents The anti-HLA reagents used were murine monoclonal monomorphic antibodies: W6/32, anti-class I (Parham et al., 1979); L243, anti-DR; B7/21, anti-DP; Genox, anti-DQ (Brodsky et al., 1979) and human HLA allele-specific polyclonal antibodies (Biotest Diagnostics, Frankfurt Main, F.R.G.; Pel-Freeze, Brown Deer, WI 53221). Murine monoclonal anti-blood group A (Edelman et al., 1981) and group B (Rouger et al., 1983) were generous gifts of Dr. L. Edelman and Dr. G. Dighiero of Institut Pasteur, Paris, France.

Generation of murine hybridomas Spleen ceils from mice immunized with appropriate RBCs and showing an optimal antibody response were collected and fused with SP2/0 myeloma cells under the influence of PEG 1500 (Serva Chemicals, Feinbiochemica, Heidelberg, F.R.G.). Hybrids were selected using HAT-selective medium and screened for their ability to secrete antigen-specific antibodies by CELISA. Results were further confirmed by a haemagglutination assay. These hybrids were cloned thrice by limiting dilution analysis and finally grown as ascites for bulk antibody production.

Processing of monoclonal antibodies for the preparation of blocking buffer Murine hybridomas: ATCC HB-57 cells secreting anti-human /~ heavy chain monoclonal antibodies and ATCC HB-43 cells secreting anti-human 3' heavy chain monoclonal antibodies were used in this study. Monoclonal antibodies were purified by 35-40% ammonium sulphate saturation from peritoneal ascites in which the hy-

183

bridomas had been propagated. Precipitated antibodies were further purified by chromatography on DEAE Affi-Blue gel (Bio-Rad Labs., California, U.S.A). Partially purified antibodies were freeze-dried and stored at - 7 0 ° C until further use. For the preparation of blocking buffer, appropriate dilutions of these antibodies were made in PBS containing 1% rabbit serum.

Detection antibodies Murine monoclonal anti-human /~ chain and "r chain antibodies purified by ion-exchange chromatography, were conjugated with horseradish peroxidase (HRP) as described by Nakane and Kawaoi (1978). Goat anti-mouse Ig-HRP conjugate was prepared by the same method. Rabbit anti-human Ig-HRP conjugate (code P212) was purchased from Dakopatts, Denmark. Appropriate dilutions of these conjugates were made in dilution buffer for use in the assay. Coating ELISA plates with cells LCLs (in the case of anti-HLA antibodies), or RBC (in the case of anti-blood group antibodies) were washed three times in phosphate-buffered saline (PBS) pH 7.4 and then resuspended at an optimal density that was worked out first by testing a range of cell concentrations. 50/~1 of this cell suspension were added to the wells of fiat-bottomed ELISA plates and the plates were tapped gently to ensure the even distribution of the cells within the wells. The plates were left at room temperature (24 ° C) overnight in front of an airblower and allowed to dry. The drying time varied somewhat depending on the target cell type, and ranged from 6 to 12 h. Once the plates were completely dry, they were sealed and then stored at 4°C until required. CELISA All steps were carried out at room temperature. The procedures for the anti-ABO antibody assay and for the human anti-HLA antibody assay were essentially the same except that blocking buffer I and dilution buffer I were used for assaying antiRBC antibodies and also with most of the LCLs. Blocking buffer II and dilution buffer II were used with some of the LCLs. Wells coated with cells were rehydrated by the addition of 100/xl of PBS

in each well. 10 min later the wells were emptied and the plate was gently tapped on a thick pad of absorbent paper. 50 /~l of blocking buffer were then dispensed into each well and the plates incubated for 45 min. Blocking buffer was then flicked off and wells were washed three times with washing buffer. Different dilutions of the samples to be tested (culture supernatants, ascitic fluid of murine hybridomas and standard human antiHLA sera) were prepared using the appropriate dilution buffer. Appropriate controls such as culture supernatant or ascitic fluid containing human or murine monoclonal antibodies directed against unrelated antigens were also included. These suspensions were dispensed into the wells (50/~l/well) and the plates incubated for 45 min. After washing three times with washing buffer 50 /~l of the appropriate antibody-enzyme conjugate were added to each well. After a 45 min incubation, the wells were emptied and then washed three times with washing buffer. This was followed by the addition of 100 /~l of freshly prepared substrate

STD. ANTI-A Abs

~

Control

0.8

0.6

A 4 9 0

0.4

n In

0.2

----~--~ 0 2S6

~(_

~

-~

.~

i

I

I

i

I

i

i

I

i

I

i

12a0

640

a20

160

a0

40

20

l0

S

2.5

L25

CELL

CONCENTRATION

.~

X 1/1000

PER

WELL

Fig. 1. Determination of the opt i mum concentration of RBC for CELISA. Murine monoclonal anti-A RBC antibodies at a dilution of 1 / 1 0 0 were assayed on group A RBC coated at different concentrations.

184

Ill

W6/32

W6/32

~

L 243

20

]0

--,X-- C o n t r o l

-+- L243 -~- Control 1.6 ].4 1,2

1.5

A

A 4

9 0

I

94 0.8 o i1 m 0.6

1

tt m

11,4

0.5

0.2

640 320 160

80

40

20

10

5

v 2.S L25 0.6250.312

640 320 160

80

40

5

2.5 1,25 .625 ,312

CELL CONCENTRATIONX 1/1000 PER WELL

CELL CONCENTRATIONX 1/1000 PER WELL

IV

W6/32

+

+

L243

Control

W6/32

- 4 - - L243

---X-- C o n t r o l

1.6 1.4 1.2

1.5

1 A 4 9 0.8

A 4

9 0

l

n m 0.6 0.4

0.5

0.2~ 0

i

i

640 320 160

i

i

i

i

I

80

40

20

10

5

T

T

T

T

2.5 1.25 .625 .312

CELL CONCENTRATIONX Ill000 PER WELL

640 320 160

80

40

20

]0

5

2.5 1.25 .625 .312

CELL CONCENTRATIONX 111000 PER WELL

Fig. 2. D e t e r m i n a t i o n of the o p t i m u m c o n c e n t r a t i o n of l y m p h o b l a s t o i d cells for C E L I S A using m u r i n e antibodies. M u r i n e m o n o c l o n a l a n t i - H L A class I ( W 6 / 3 2 ) a n d a n t i - D R (L243) a n t i b o d i e s in ascitic fluid at a dilution of 104 w e r e a s s a y e d on four different B l y m p h o b l a s t o i d lines, n a m e l y R A J I ( I ) , A K I B A ( / / ) , K A S O l l (III), S W E I G (IV) c o a t e d at d i f f e r e n t c o n c e n t r a t i o n s .

185 s o l u t i o n a n d i n c u b a t i o n in the d a r k until the colo u r developed. T h e e n z y m e - s u b s t r a t e r e a c t i o n was s t o p p e d b y a d d i n g 5 0 / t l of 5 N sulphuric acid to each well. A b s o r b a n c e values were m e a s u r e d at 490 n m in a n E L I S A r e a d e r ( U V M a x K i n e t i c M i c r o p l a t e R e a d e r , M o l e c u l a r Devices C o r p o r a tion, California, U.S.A.).

1

w i t h bloclt,buf,

~

without block.but,

0.3

0.25

0,2 A

Results

4 9

o 0.15

Preparation of CELISA plates T h e initial p r o b l e m s which were e n c o u n t e r e d in the d e v e l o p m e n t of this assay i n c l u d e d high v a r i a b i t i l i t y b o t h within a n d b e t w e e n assays a n d high b a c k g r o u n d noise. W e therefore set out to d e t e r m i n e the o p t i m a l m e t h o d s of cell coating a n d p l a t e washing. G e n e r a l l y I m m u l o n E I A plates a n d N u n c E I A plates gave the m o s t r e p r o d u c i b l e resuits. A 6 - 1 2 h d r y i n g p e r i o d was sufficient to allow the p l a t e s to d r y a n d for the cells to c o a t the

Anti-DR serum

+

Control

1,6

n m

0.1

0,05

m

i

I

GDI SWE1G Fig. 4. Effect of blocking buffer on the sensitivity of CELISA. Lymphoblastoid cell lines GD1 and SWEIG coated on EIA plates were either blocked with buffer containing anti-human Ig ~ chain or y chain antibodies respectively or not blocked with blocking buffer. Human antibodies specific to an unrelated antigen were used as primary antibodies followed by HRP conjugate of the same antibody used in the blocking buffer.

1,4 plates satisfactorily. D i s p e n s i n g 50 /xl of cell susp e n s i o n a n d d r y i n g at r o o m t e m p e r a t u r e (24°C) resulted in firm b i n d i n g of cells to the plastic.

1.2

A 4 9 0 n m

1

Cell concentration

0,8 \

0,6 0.4 0.2:

640 320 160 80 40 20

10

5

2.5 1.25 .625 .312

CELL CONCENTRATION X 1/1000 PER WELL Fig.

3.

Determination

of

the

optimum

concentration

of

lymphoblastoid cells for CELISA using human antibodies. Anti-DR serum at a dilution of 1/25 was assayed on RAJI cells coated at different concentrations using rabbit anti-human Ig-HRP conjugate as the detection antibody.

R B C s of different A B O g r o u p s were c o a t e d at differing c o n c e n t r a t i o n s a n d used for assaying s t a n d a r d a n t i - A B O reagents. T h e results o f a representative test are shown in Fig. 1. A n R B C c o n c e n t r a t i o n of 2 0 - 4 0 × 10 4 ceils p e r well gave high o p t i c a l d e n s i t y ( O D ) values a n d low b a c k g r o u n d noise. T h e sensitivity of the assay decreased as the cell c o n c e n t r a t i o n p e r well was either i n c r e a s e d o r d e c r e a s e d b e y o n d this range. I n the a n t i - H L A assay, L C L s n a m e l y R A J I , A K I B A , K A S O l l a n d S W E I G were used at different c o n c e n t r a t i o n s to d e t e r m i n e the o p t i m u m c o n c e n t r a t i o n for use in this assay. B a c k g r o u n d noise was high at high cell c o n c e n t r a t i o n s whereas the sensitivity was very low at low c o n c e n t r a t i o n s

186 m

(Figs. 2 and 3). The optimal LCL concentration ranged from 2 × 1 0 4 to 16 × 1 0 4.

Blocking buffer

[~

B group RBC

AB g r o u p RBC

I~

0 g r o u p RBC

1.2

In the CELISA for murine anti-ABO antibodies and murine anti-HLA antibodies, the use of blocking buffer I gave optimal results regardless of the cells used in this study. However, when human anti-HLA antibodies were assayed on different LCLs the use of blocking buffer I and rabbit anti-human Ig-HRP conjugate as the detection antibody gave satisfactory results with most, but not all, of the LCLs tested. These LCLs, namely BM9, SWEIG, GD1, H004, H006 and H010, gave high background noise which could not be reduced significantly even by increasing the blocking protein (BSA or milk powder) to 5% or with gelatin or rabbit serum alone (unpublished observations). The background noise could be reduced when PBS with 1% rabbit serum and an appropriate dilution of anti-human Ig (bt or 3') antibodies was used as blocking buffer and HRP

m

A g r o u p RBC

Prot,A-HRP Conj.

[~

0.8 A 4 9

0.6

n tn 0.4

0.2

ANTI-A

ANTI-B

4e6

DI7

Fig. 6. Specificity of CELISA for the detection of monoclonal anti-ABO antibodies. Murine monoclonal anti-A and anti-B blood group antibodies were assayed on h u m a n A, B, AB and O group RBCs concentration of 20 × 104 cells/well.

Antl Hu /g-HRP Conj.

conjugates of these anti-human Ig antibodies were used as the detection antibodies (Fig. 4).

Detection antibodies In the human anti-HLA antibody assay, the performance of the protein A - H R P conjugate was compared with that of the rabbit anti-human IgH R P conjugate. The anti-Ig-HRP conjugate yielded higher values than did the protein A-HRP conjugate (Fig. 5).

A

08 0 n 0.6

m

Specificity of the assay Standard murine monoclonal anti-A and anti-B antibodies were tested together with the murine monoclonal anti-A (4C6) and anti-B (D17) antibodies generated in our laboratory (manuscript in preparation). Monoclonal anti-A and anti-B antibodies bound only to specific RBCs (Fig. 6).

0.4

0.2

0

,

ANTI-DR Ab|

,

,

,

ANTI-DR3 Ab|

,

,

CONTROL

Fig. 5. C o m p a r i s o n of protein A - H R P a n d r a b b i t a n t i - h u m a n I g - H R P conjugates. H u m a n a n t i - D R a n d a n t i - D R 3 sera were assayed at a dilution of 1 / 2 5 on R A J I cells coated at a c o n c e n t r a t i o n of 5 × 104 c e l l s / w e l l .

Sensitivity of the assay Ascitic fluid from a mouse with a hybridoma secreting an anti-B specific monoclonal antibody was titrated against RBC expressing various ABO

187

blood groups both in the Coombs test and in the CELISA. The results of CELISA are shown in Fig. 7. CELISA was much more sensitive than the Coombs test; the end point titer of the anti-B RBC ascitic fluid w a s 1 0 3 by Coombs test and 1 0 4 by CELISA. In the anti-HLA assay, murine monoclonal anti-HLA antibodies and human antiD R sera were titrated. Titre values of the ascitic fluid were in the range of 1 0 6 o r more (Fig. 8). Standard human anti-HLA antibodies were found to give optical densities three times higher than the control value even at a dilution of 1/400 (Fig. 9).

W6/32

~

L243

GENOX

~

Control

B7/21

2,5

2 A 4 9 0

1.5-

n rn 1

Discussion

A rapid, simple and reliable method is highly desirable for the screening of antibody-secreting hybridomas. The CELISA is one such method; once cell-coated plates have been prepared, the assay can be completed in a few hours and can be assessed visually. While we have adapted the

A group

RBC

AB g r o u p

RBC

--4--

B g r o u p RBC

-~-

O group

RBC

0.5

o -2

-3

-4 -5 -6 DILUTION OF ASCITES (log)

-7

-8

Fig. 8. Sensitivity of C E L I S A for the d e t e c t i o n of m u r i n e m o n o c l o n a l a n t i - H L A a n t i b o d i e s in ascitic fluid. Different dilutions of the ascitic fluid c o n t a i n i n g a n t i - H L A class I ( W 6 / 3 2 ) , a n t i - D R (L243), a n t i - D P ( B 7 / 2 1 ) a n d a n t i - D Q ( G e n o x ) a n t i b o d i e s were t i t r a t e d o n R A J I cells c o a t e d at a c o n c e n t r a t i o n of 5 × 104 c e l l s / w e l l . A s a c o n t r o l ascitic fluid containing murine monoclonal antibodies reacting with an u n r e l a t e d antigen were used at a d i l u t i o n of 1 / 1 0 0 .

2

1.5 A 4 9 0

1

n Ill

0.5

0 -2

t~ -3 -4 DILUTION OF ASCITE$ (log)

-5

Fig. 7. Sensitivity of CELISA for the detection of murine monoclonal anti-B RBC antibodies in ascitic fluid. Different dilutions of the ascitic fluid were assayed on A, B, AB and O group RBCs coated at 20 × 104 cells/well.

CELISA for the detection of anti-HLA antibodies and anti-blood group antibodies it can be modified appropriately for the detection of antibodies to other cell-surface antigens and for the detection of cellular antigens. The same basic method can be made more sensitive by the use of iodinated secondary antibody or iodinated protein A. The assay appears to be sufficiently sensitive for the screening of antibodies in hybridoma culture supernatants. The fact that B cells and RBC can be prepared in bulk and then coated onto plates to generate a stock for several months, is a definite advantage and the procedure obviates the need for viable cells for each test. We have screened plates that have been stored at 4 ° C for up to 6 months without any obvious effect on sensitivity. Other assays such as cytotoxicity (for the detection of anti-HLA antibodies), haemagglutination (for the detection of anti-blood group antibodies) and ira-

188 Anti-DR s e r u m

~

Control

1

0.8

A 0.6 4 9 0 n

na 0.4

0.2

/25

I

i

1/50

1/100 DILUTION

I

i

i

i

I

1/200 1/400 1/800 1/1600 1/3200 OF

ANTI-DR

SERUM

Fig. 9. Sensitivity of CELISA in the detection of human anti- DR antibodies in serum. Human anti-DR serum was titrated on RAJI cells coated at 5 × 104 cells/well and revealed with rabbit anti-human Ig-HRP conjugate. H u m a n monoclonal antibodies reacting with an unrelated antigen were used as control.

munofluorescence require a constant supply of viable cells. These assays are also time-consuming and laborious especially if one is faced with the task of screening several plates of hybridoma supernatants. In the procedure that we have described here, intact ceils harvested from cultures or from fresh blood were used for coating the plates. This eliminates the very laborious and time-consuming procedure of purifying antigens from lysed cell preparations to be used for coating EIA plates (Bouillot et al., 1989; Buchs and Nydegger, 1989). Cellular ELISAs have also been reported for assaying antibodies against sperm (McArthur et al., 1981), platelets (Horai et al., 1981) and melanoma cells (Suter et al., 1980). In these assays either glutaraldehyde or formalin was used for fixing cells onto plastic plates. Glutaraldehyde has been reported to alter M H C antigens (Pancake and Nathanson, 1973; Gatti et al., 1974). Procedures that avoid fixatives generally rely on the centrifu-

gation of ceils into the wells of microtiter plates and the careful aspiration of supernatants at each step (Morris et al., 1982). Some protocols use poly L-lysine (PLL) to bind the cells to the plastic; the use of PLL often results in high background values even in the absence of target cells (Horai et al., 1981). An increase in the number of B cells/well results in an increase in background noise (Figs. 2 and 3). This could be due to an increase in surface immunoglobulin (slg) and Fc receptors. This problem was solved by a strategy of blocking slg with unlabelled anti-human /~ or 3' heavy chain antibodies followed by H R P conjugate of the same blocking antibody as the revealing reagent. Fc receptors can be blocked with rabbit Ig. Milk proteins have been reported to reduce non-specific binding of human Ig to plastic surfaces, and casein has been observed to be a more efficient blocking agent than BSA or gelatin (Kenna et al., 1985). In our study, 1% milk powder (containing 16 g of protein per 100 g of milk powder) in PBS served as an excellent blocking buffer with all groups of RBCs and with most of LCLs. An increase in the concentration of RBCs/well did not result in a significant increase in background noise. However, at higher concentrations there was some inhibition in the binding of antibodies to A and B antigens; this may have been due to the Hook effect (Pesce et al., 1983). The CELISA appears to be specific. Even at very high concentrations of ascitic fluid containing anti-B antibodies, the reaction with non-specific RBCs (A and O group RBCs) was negligible (Fig. 7). The use of protein A - H R P conjugate for the detection of antibodies to cell surface antigens was found to be unsatisfactory by one group (Morris et al., 1982) but satisfactory by another group (Effros et al., 1985). Our results are in agreement with the results of the latter authors. Background noise was lower with protein A - H R P conjugate than with anti-human Ig-HRP conjugate. However, use of the former would restrict the detection of antibodies to a few isotypes only and this would be a handicap in the generation of monoclonal antibodies, especially for human antibodies where EBV-transformed lines have been reported to secrete antibodies of the IgM class more often

189 t h a n I g G class ( C h a n et al., 1986). T h e s t r a t e g y o f b l o c k i n g F c r e c e p t o r s a n d s l g o n t a r g e t cells u s i n g blocking antibodies reduces the high background n o i s e w i t h s o m e L C L s . I n c o n t r a s t to E f f r o s et al. (1985), this p e r m i t s the use o f a n a n t i - h u m a n I g - H R P c o n j u g a t e f o r all f u r t h e r s c r e e n i n g o f h u m a n a n t i - H L A a n t i b o d i e s in h y b r i d o m a c u l t u r e supernatants. T h e C E L I S A has several a d v a n t a g e s . It is simple a n d r a p i d a n d s e n s i t i v e a n d c a n b e a d a p t e d for t h e d e t e c t i o n o f a n t i b o d i e s s p e c i f i c to d i f f e r e n t c e l l - s u r f a c e a n t i g e n s . D e v e l o p m e n t o f this a s s a y has f a c i l i t a t e d t h e g e n e r a t i o n o f h y b r i d o m a s s e c r e t i n g m o n o c l o n a l a n t i b o d i e s to d i f f e r e n t cells u r f a c e a n t i g e n s in o u r l a b o r a t o r y .

Acknowledgements T h i s w o r k was s u p p o r t e d b y a g r a n t to R a j Raghupathy from the Department of Biotechnology, G o v e r n m e n t o f I n d i a . B. A r u n a c h a l a m is a r e c i p i e n t of a S e n i o r R e s e a r c h F e l l o w s h i p f r o m the C o u n c i l o f S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h , Government of India. W e are g r a t e f u l to D r . S o o Y o u n g Y a n g o f Memorial Sloan-Kettering Cancer Institute, New York, U.S.A., and Dr. Jack Strominger of Harvard University, Cambridge, U.S.A., for their generous c o n t r i b u t i o n o f B l y m p h o b l a s t o i d cell lines. W e appreciate the excellent technical support of Mrs. Chandrakanta Gupta.

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A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens.

This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens...
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