Immunodetection of cell-bound antigens using both mouse and human monoclonal antibodies

Journal of Immunological Methods, 81 (1985) 115-122

115

Elsevier JIM 03542

Immunodetection of Cell-Bound Antigens Using Both Mouse and Human Monoclonal Antibodies Mark C Glassy 1,2 and Charles D. Surh 3 U.C. San Diego Cancer Center, T-OI 1, Department of Medicine, Division of Hematology/ Oncology, University of California at San Diego, San Diego, CA 92103, U.S.A.

(Received 5 December 1984, accepted 15 March 1985)

A micro enzyme-linkedimmunoassay (EIA) has been developed for the rapid and sensitive detection of either human or mouse monoclonal antibodies reactive with cell bound antigens. Whole intact cells are immobilizedonto 96-well flat bottom microtiter plates by drying in an oven at 37°C overnight prior to the start of the assay. This method of attachment was suitable for all cell types tested, regardless of origin, size and chromosomal content. The dried cells were then rehydrated, incubated with the appropriate test hybridoma supernatant, followed with subsequent analysis by EIA. The plates can be stored at 4°C up to 1 month for future EIA analysis. This assay offers high sensitivity, requires only small amounts of target cells and test hybridoma supematant, and can be completed within 3 h. This EIA is well suited for the rapid screening of large numbers of hybridoma supernatants and can also be adapted to include cells of any species, providing the appropriate antibody reagents are available. Key words: human monoclonal antibodies - enzyme immunoassay - cell surface antigens - human sperm -

human tumor-associated antigens

Introduction T h e c o m b i n a t i o n of enzyme i m m u n o a s s a y s (Engvall a n d Pearlman, 1971) a n d m o n o c l o n a l a n t i b o d y ( M o A b ) technology (K6hler a n d Milstein, 1975) has provided investigators a n o p p o r t u n i t y to qualitate a n d q u a n t i t a t e the a n t i g e n - a n t i b o d y reaction with easy a n d simple methodology (for a recent review, see Glassy a n d H a n d l e y , 1985). Since m a n y laboratories p r o d u c e large n u m b e r s of M o A b s , emphasis m u s t b e placed on rapid a n d facile assays to identify the M o A b s of choice. F u r t h e r m o r e , with the i n t r o d u c t i o n of h u m a n M o A b s (Olssen a n d K a p l a n , 1980; Glassy et al.,

1 Recipient of an NIH New Investigator Research Award. 2 Address correspondence to: Mark C Glassy, Ph.D., U.C. San Diego Cancer Center, T-011, San Diego, CA 92103, U.S.A. Telephone: (619) 294-3906. 3 Present address: University of California at Davis, Department of Immunology, Davis, CA, U.S.A. Abbreviations: PBS, phosphate-buffered saline; MoAb, monoclonal antibody; HRP, horseradish peroxidase; EIA, enzyme immunoassay; OPD, o-phenylenediamine. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

116 1983a,b; Glassy et al., 1985b) new methods must be developed to identify those which are immunoreactive with the desired antigen. Of particular concern to the tumor immunologist is the identification of novel cell surface antigens, some of which may be clinically relevant. Large batteries of murine MoAbs have been produced against antigens on human tumor cells (Reisfeld et al., 1984). Recently, human MoAbs reactive to human tumor-associated antigens have been described (see Glassy et al., 1985b for a recent review). This new field presents several interesting problems, one of which is the nature of the antigen eliciting the antibody response. Unlike murine MoAbs, which are produced from xenogeneic immunizations, human MoAbs to human tumors are produced from lymphocytes of cancer patients without prior intentional immunizations. As such, unique and rare antigens may be identified. Therefore, detecting the specificity of these human MoAbs would require highly sensitive methods, ideally with the same preferred speed and accuracy utilized in analyzing murine MoAbs (Handley et al., 1982). In this report, we describe a rapid, simple, and sensitive method for the detection of cell-bound antigens recognized by mouse or human MoAbs.

Materials and Methods

Reagents and buffers Affinity-purified horseradish peroxidase (HRP)-conjugated goat anti-human IgG, anti-human IgM, and anti-mouse IgG antibodies (Tago, Burlingame, CA) were used at a 1:1000 or 1:2000 dilution in FBS buffer (10% fetal calf serum, 1% bovine serum albumin and 0.3% gelatin (swine skin type I, Sigma, St. Louis, MO) in phosphate-buffered saline (PBS, pH 7.4, NaH2PO4, Na2HPO4, 0.9% saline)) with 0.01% Thimersol (Sigma). o-Phenylenediamine (OPD, Calbiochem, La Jolla, CA) was used at 400 # g / m l in 0.05 M citrate buffer, pH 5.0, with 60 ng/ml hydrogen peroxide (Mallinkrodt, Paris, KY) as substrate for the HRP enzyme. Cells were washed (500 x g/lO min per wash) in gelatin buffer (1% swine skin gelatin (Sigma) in PBS at pH 7.4) with 0.01% thimersol.

Cells The human cell hnes used in this study were T-293H, a carcinoma of the lung; T-84, a carcinoma of the colon; CaSki, a carcinoma of the cervix; and A431, a carcinoma of the vulva. All were grown in RPMI 1640 media with 10% fetal calf serum at 37°C in a 95% air/5% CO 2 humidified incubator. Sperm ejaculates obtained from healthy human volunteers were washed and incubated at 37°C in RPMI 1640 containing 1% fetal calf serum for 1 h at 5 × 10 6 sperm/ml. After incubation, sperm was frozen in freeze medium (RPMI 1640 containing 8% DMSO and 30% fetal calf serum) at - 1 5 ° C until ready to be used (Glassy et al., 1984).

Cell preparation Log-phase human adherent cells were harvested from tissue culture flasks with

117 0.25% EDTA + 0.25% trypsin in PBS and washed twice with 37°C PBS and resuspended to 4 x 106 cells/ml in PBS. The aliquoted frozen sperm was quickly thawed, washed twice in 37°C PBS, and resuspended to 2 × 106 cells/ml in PBS.

Monoclonal antibodies Both human and mouse MoAbs have been produced in our laboratory as previously described (Glassy et al., 1981, 1982, 1983a,b, 1984). Briefly, human lymphocytes from regional draining lymph nodes of cancer patients were fused with UC 729-6, a 6-thioguanine-resistant human lymphoblastoid B cell line (Glassy et al.,, 1983b). Lymphocytes from the spleen of a mouse immunized with human ejaculate sperm were fused with P3-NS1-Ag4-1 cells to generate murine MoAbs (Glassy et al., 1984). All fusions were done with 35% polyethylene glycol 1500 (lot no. 6229890; BDH Chemicals, Poole, England). SP7A7 is a murine IgG2a MoAb specific for human sperm (Glassy et al., 1984). VLN3G2 and VLN6H2 are human IgG1 MoAbs, whereas VLN1H12 is a human IgM MoAb. These human MoAbs are reactive with the A431, T-84, and T293H cell lines, respectively (see Table I). (Glassy et al., 1985 and manuscript in preparation).

Assay plates The 96-well Immulon Iflat bottom microtiter plates were prepared for the assay in the following manner. Fifty ~1 of the prepared cell suspension (see previous section) were dispensed into each of the wells of the plate and gently tapped to evenly coat the bottom of the wells with the liquid. Each plate was then incubated overnight in a 37°C drying oven to attach the cells to the assay plates. When removed from the oven, 200/~1 of FCS buffer with 0.1% NaN 3 were added to each well to rehydrate the cells and to block the non-specific binding sites. The plates can then either be used immediately or stored at 4°C for future use.

Assay procedure The 200/~1 of FCS buffer in the wells of the prepared plates (see above) were finger flicked out over a sink. Fifty/~1 of the test hybridoma supernatant were added to triplicate wells, incubated for 60 min at room temperature, and washed twice. Each wash consisted of adding 300 #1 of 37°C gelatin buffer to each well, incubating for 1 min, and forcefully flicked out over a sink. Each plate was then set at a 45 ° angle for 1 min to let the excess wash medium pool to one side of the wells and then shaken off by flicking the plate. After 2 such washes 50 /~1 of HRP-conjugated secondary antibody were added to the appropriate wells and incubated for 45 rain at room temperature. The wells were then washed 3 times as before and 200 ttl of citrate buffer containing OPD and hydrogen peroxide were added and incubated in the dark for 30 rain at room temperature. The enzyme reactions were stopped by adding 50 /~1 of 2.5 M H2SO4 to each well and were read at 490 nm using a microELISA reader (Dynatech; Alexandria, VA, model MR 600).

Sperm

0.618 0.521 0.260 0.095 0.088 0.061

SP7A7

100 c 10 1 0.5 0.1 0.05

Background: 0.120

575 ¢ 115 23 4.6 1.15 0.575 0.29

VLN3G2

MoAb

0.549 0.290 0.207 0.165 0.153 0.149 0.127

A431

Cells b

Background: 0.043

100 c 20 4 0.8 0.2 0.1 0.05

VLN6H2

MoAb

0.247 0.138 0.085 0.068 0.056 0.052 0.049

T-84

Cells b

Background: 0.145

1450 c 290 58 11.6 2.9 1.45

VLNIH12

MoAb

0.328 0.175 0.170 0.144 0.145 0.150

T293H

Cells b

a Cells were used in the EIA at 2.0× 105 ceUs/well. SP7A7 is a murine IgG2a MoAb; VLN3G2 and VLN6H2 are human IgG1 MoAbs; VLNIH12 is a human IgM MoAb (see Materials and Methods). b The cells used with each MoAb were the ones which demonstrated high reactivity in our EIA. c Concentrations of the MoAbs are expressed in n g / m l .

Background: 0.045

Cells b

MoAb

TITRATION OF MoAbs A G A I N S T TARGET CELL B O U N D A N T I G E N S a

TABLE I

119 Results

To determine the concentration of the HRP-conjugated goat anti-Ig which optimizes the signal to noise ratio, reported as the reactivity index, several dilutions of both anti-mouse and anti-human IgG conjugates were titrated against MoAbs and known cell-bound target antigens. In Fig. 1A, the goat anti-murine IgG-HRP conjugate was titrated against 2.0 × 105 human sperm cell/well with 25 ng of the murine MoAb, SP7A7 per well. The optimal dilution of the H R P conjugate was determined to be 1 : 2000, with a reactivity index of 12. The reactivity index was calculated as the OD490 of the test MoAb divided by the O1)490 of the control or irrelevant MoAb. In Fig. 1A, the control MoAb was MPC-11, a murine IgG2a, same

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murine MoAb (A); reactivity index (m i; see Results for explanation). B: titration of HRP-goat anti-human IgG against A431 cells. VLN3G2 human MoAb (O O); irrelevant IgG control (/, A); reactivityindex (13 12; see Results for explanation).

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Fig. 2. Titration of target cells against MoAbs. HPR-conjugated goat anti-mouse or - h u m a n IgG was used at 1:2000. H u m a n VLN3G2 (28 ng/well) reactive with target A431 cells ( O O). Murine SP7A7 (25 ng/weU) reactive with target sperm cells (e 0). H u m a n IgG irrelevant control ( O - - - - - O ) . Murine IgG irrelevant control ( 0 - - - - - - e ) .

subtype as SP7A7 (Glassy et al., 1984), that was unreactive with human sperm. In Fig. 1B, the goat anti-human IgG-HRP conjugate was titrated against 2.0 × 105 target A431 cells/well with 28 ng of the human MoAb VLN3G2 per well. The optimal dilution of the H R P conjugate was in the range of I : 2000 to 1 : 4000, with a maximal reactivity index of 5.6. An irrelevant affinity purified human IgG was used as the control. The sensitivity of this assay is shown in Fig. 2. Here, we have utilized a 1 : 2000 dilution of the H R P conjugate (both anti-human and anti-mouse IgG) and 25 ng/well or 28 ng/well of either SP7A7 or VLN3G2. The target cells A431 and human ejaculated sperm were diluted from a maximum of 2.0 × 105/well to 1.0 x 104/well. Even at the lowest cell number we can still detect a greater than 2-fold reactivity over background. The maximum reactivity index for VLN3G2 and A431 cells was 6.4 at 2.0 × 105 cells/well; the maximum reactivity index for SP7A7 and sperm was 15.8 at 1.5 × 105 cells/well. Titrations of SP7A7, VLN3G2, VLN6H2, and V L N I H 1 2 MoAbs against target cell bound antigens are shown in Table I. The data presented here are the results of typical experiments. With a lower limit cutoff range of 2-fold over background, this EIA is sensitive enough to detect, using cell bound target antigens, approximately 1 n g / m l of murine IgG MoAb, 5-100 n g / m l o f human IgG MoAbs, and approximately 500 ng-1 # g / m l of human IgM MoAb.

121

Discussion The identification of novel cell-bound antigens, particularly those with clinical utility, is limited by the types of assays and techniques available to achieve this goal. In this report, we describe a solid-phase EIA capable of rapidly qualitating and quantitating cell-bound antigens using MoAbs as screening reagents. This EIA is capable of detecting antigens bound to haploid cells (sperm), diploid cells (A431), and tetraploid human-human hybridomas (unpublished observations), suggesting that the size and complexity of the cell is not critical. We have stored plates coated with target cells at 4°C for up to 1 month and have observed no loss of reactivity with either mouse or human MoAbs. Presumably, MoAbs from other species (e.g., rat) would behave in a gimilar manner. After the plates have been prepared, this EIA takes no more than 3 h to complete, offering the investigator a rapid means to screen large numbers of hybridoma supernatants against target cell-bound antigens, thereby avoiding constant culturing. In addition, the investigator may not have access to a large number of target cells, particularly in the case of autologous tumor tissue. This EIA can easily detect as few as 1.0 x 104 cells/well. Therefore, cell availability should not be an important factor in screening hybridoma supernatants with this EIA. Also, since these plates coated with cells can be stored for short periods of time, 'on demand' assays can be performed to assist the investigator in searching for the MoAb(s) of choice. For the most part, human hybridomas secrete less antibody than their murine counterparts (Glassy et al., 1983, 1984). Mouse hybrids have typically secreted about 5-20 #g of MoAb/106 cells/ml/day and human hybridomas typically secrete about 200 rig-5 #g of MoAb/106 cells/ml/day. Since the EIA described in this report requires very few cells and uses low nanogram levels of MoAbs, then detecting positive reactivity even with low antibody producing hybridomas does not present any major limitations. This is particularly valuable with human hybridomas. In summary, we have developed a 3 h EIA useful for detecting immunoreactive mouse and human MoAbs to cell-bound antigens. Target cells are dried down on 96-well microtiter plates that serve as a solid support facilitating the wash steps, a time consuming procedure in most conventional assays. As little as 1 ng of the test MoAb can detect as few as 1.0 × 10 4 target cells.

Acknowledgements The data presented here were partially funded by the National Institutes of Health (CA32047, PO1 CA 37497) and by a grant from the Arco Oil Foundation. We would like to thank Dr. Ivor Royston in whose laboratory this work was performed and Dorothy Kwiat for the preparation of this manuscript. References Engvall, E. and P. Perlman, 1971, Immunochemistry 8, 871. Glassy, M.C. and H.H. Handle),, 1985, in: Bioteclmology Handbook, eds. P.N. Cheremisinoff and R.P. Ouellete (Technomic Publishing Co., Lancaster, PA) p. 420.

122 Glassy, M.C., H. Handley, J.E. Seegmiller and 1. Royston, 1981, Fed. Proc. 40, 996. Glassy, M.C, H. Handley, H. Lowe, R.E. Sobol, J. Leonard and I. Royston, 1982, Fed. Proc. 41, 553. Glassy, M.C., H.H. Handley, P.H. Cleveland and I. Royston, 1983a, J. Immunol. Methods 58, 119. Glassy, M.C., H.H. Handley, H. Hagiwara and I. Royston, 1983b, Proc. Natl. Acad. Sci. U.S.A. 80, 6327. Glassy, M.C., C. Surh and S. Sarkar, 1984, Hybridoma 3, 363. Glassy, M.C., H. Handley and I. Royston, 1985, in: Human Hybridoma and Monoclonal Antibodies, eds. E.G. Engleman, S. Foung, J. Larrick and A. Raubitschek (Plenum, New York) in press. Handley, H., M.C. Glassy, P. Cleveland and I. Royston, 1982, J. Immunol. Methods 54, 291. K6hler, G. and C. Milstein, 1975, Nature (London) 256, 495. Olssen, L. and H.S. Kaplan, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 5429. Reisfeld, R.A., J.R. Harper and T.F. Bumol, 1984, CRC Crit. Rev. Biochem. 5, 27.