A Microradioimmunoassay for Antibodies to Tumor-Associated Antigens J. C.-C. Huang, 3, 4 I. Berczi,3 G. Froese,5 H. M. Tsay,3 and A. H. Sehon SUMMARY-A versatile microradioimmunoassay for the detection of antibodies to tumor-associated and other tissue antigens was described. The method involved: a) the preparation of solid-phase antigen with cultured (already adhered) or noncultured cells (sedimented by centrifugation) fixed to MicroTest plates with neutral buffered formaldehyde or absolute methanol; b) the incubation of the antigen with test or control sera; and c) the incubation of the antigen with radioiodinated antiglobulin antibody_ The nonspecific background of radioactivity was reduced to an acceptable level by the fixed cells being precoated in the wells with 0.5% bovine serum albumin in phosphate-buffered saline which wa·s also used for the dilution of sera and labeled antiglobulin antibody_ Tumor cells in primary cultures gave a high background, as compared to long-term cultures, which was due to the presence of immunoglobulins (most likely tumor-specific antibody). The specific antibody response to a syngeneic mouse tumor was demonstrated by this technique.-J Natl Cancer Inst 55: 879-886, 1975.

Radioimmunoassays (RIA) for the detection and quantitation of an increasing variety of molecules, including tumor antigens and their corresponding antibodies, have been described and many have been routinely applied to clinical laboratory practice (1-11). Although the direct assay methods are extremely sensitive, they require highly purified antigens or antibodies that are difficult to obtain. By contrast, the isotopic antiglobulin test, originally designed by Harder and McKhann (4) for the estimation of antibodies to chemically induced tumors, has the advantage of not requiring the use of purified antigens and/or antibodies. This assay has been used extensively for the evaluation of serologic responses against H-2 (5), cell-surface (6, 7), membrane (8), and tumor antigens (8, 10,11).

A modification of the isotopic antiglobulin test for the detection of antibodies attached to tumor cells cultured as adherent monolayers has been described by Burdick and co-authors (12-14). During initial experimentation with this method in this laboratory, the following difficulties were encountered: a) Some loosely attached cells lost during the washing procedure made results irreproducible; b) the MicroTest polystyrene tissue culture plates adsorbed proteins, including immunoglobulins, which could not be eliminated by repeated washings and thus gave rise to a high nonspecific background count; c) not all tumors could be cultured, which limited the usefulness of this method; and d) the preparation of fresh monolayer cultures for each investigation was laborious, inconsistent, and inconvenient. The modification described here eliminates these problems. Thus loosely attached or nonculturable tumor cells were fixed to the microplate by formaldehyde or methanol, which also served as a preservative. We greatly reduced the nonspecific background of radioactivity due to the adsorption of immunoglobulins to the plastic plate by precoating the wells with phosphatebuffered saline (PBS; pH 7.2), which contained 0.5% bovine serum albumin (PBSA), and diluting the antisera

3, 6

and the iodinated antiglobulin serum in PBSA. The assay is fast, sensitive, and convenient for the routine determinations of antibodies to tumor and other tissue antigens. MATERIALS AND METHODS

Chemicals and materials.-All chemicals used were of Fisher's certified analytical grade; others included dextran T250 (Pharmacia, Uppsala, Sweden). Freund's complete adjuvant (FCA) (Difco Laboratories, Inc., Detroit, Mich.), DEAE cellulose (DE22) (Whatman, Maidstone, Kent, England), pepsin (Mann Research Laboratories, Inc., New York, N.Y.), Sephadex G-150 (Pharmacia), glutaraldehyde (Eastman Koadak Co., Rochester, N.Y.), bovine serum albumin (BSA; Calbiochem, San Diego, Calif.), and chloramine T (Eastman). All ingredients for tissue culture medium were purchased from Difco Laboratories, Inc., except for fetal calf serum (FCS; Gibco, Grand Island, N.Y.), Vibrio cholerae neuraminidase (#70500; General Biochemicals, Chagrin Falls, Ohio), BCG (Connaught Medical Research Laboratories, Toronto, Ontario), mitomycin C (Nutritional Biochemicals Corp., Cleveland, Ohio), 125 1 as NaI (New England Nuclear Corp., Boston, Mass.), MicroTest culture plates (# 3040; Falcon Plastics Oxnard, Calif.), and lids (# 3041; Falcon Plastics). Animals.-C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) and Wistar Furth (W /Fu) rats from Microbiological Associates (Bethesda, Md.). Colonies of inbred guinea pigs (strains 2 and 13) were bred at North American Laboratory Supplies, Gunton, Manitoba, from nuclei obtained from the National Institutes of Health (NIH; Bethesda) and Rockefeller University (New York, N.Y.), respectively. Rabbits and sheep were obtained from local dealers. Tumors.-Details of all the tumors, test sera, and corresponding iodinated antibodies are listed in table 1. A ntisera to tumors.-All antisera were produced against tumor cells freshly prepared from tumor-bearing animals, and care was taken to avoid antigenic tissue culture reagents (e.g., FCS) throughout the immunizing procedure. Rabbit antiserum to leukemia (L2C) of strain 2 guinea pigs: Leukemia cells were separated from the blood of leukemic guinea pigs, by flotation with dextran (15), washed thrice, resuspended in PBS, mixed with FCA, Received January 10, 1975; accepted May 23, 1975. Supported by grants MA4745 and MA5280 from the Medical Research Council of Canada, The National Cancer Institute of Canada, and Public Health Service grant CAl3192 from the National Cancer Institute. 3 Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3. 4 Present address: Laboratory Center for Disease Control, Ottawa, Ontario, Canada. 5 The Manitoba Cancer Research and Treatment Foundation, Winnipeg, Manitoba, Canada R3E OV9. 6 We are indebted to Drs. L. S. McMorris, T. K. Thorlakson, and R. H. Thorlakson for their cooperation in this stUdy and related ones, and to Mr. B. Blaskovic and Mrs. Hide Huang for their technical assistance. 1



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1, 2




I.-Tumor cell antigens, corresponding test sera, and iodinated antiglobulins used/or the micro-RIA

Solid-phase cell antigens

Tumor-bearing animals and their source

L 2C ___ _

Strain 2 guinea pigs from Dr. E. M. Shevach (NIH) 3-Methylcholanthrene-induced Strain 13 guinea pigs from sarcoma (MC-D) Dr. H. F. Oettgen (SloanKettering Institute for Cancer Research, New York, N.Y.) W /Fu rats from Dr. S. C. Polyoma virus-induced tumor Bansal (University of (PWI3) Washington, Seattle, Wash. Polyoma tumor (Po) ______________ _ C57BL/6J mice from K. Habel (Scripps Clinical Research Foundation, La Jolla, Calif.)

and injected (l08 cells) in a total volume of 1 ml into the four foot pads of each rabbit. A second dose of 108 ~C cells was administered iv 2 weeks later and the animals were bled 12 days after the second injection. The serum then was diluted ten times with PBS and absorbed with normal guinea pig tissues (10% wt/vol for 4 hr at 4° C each time) as follows: a) with pooled and homogenized tissues of brain, lung, heart, liver, and kidney two times; b) with pooled lymphoid cells of thymus, spleen, and lymph nodes once; c) with washed cells of peripheral blood two times; and .d) with insolubilized (16) normal guinea pig plasma three times. IgG was then separated (17) from the original and absorbed immune serum and tested on L 2C and normal guinea pig white blood cells for cytotoxicity by 51Cr release (18) and for antibody activity by RIA. Rabbit antiserum to MC-D: Ten million tumor cells in FCA were injected in a volume of 1 ml into the four foot pads of each rabbit and boosted biweekly three times with identically prepared antigen administered sc. The animals were bled 10 days after the last injection, and the serum was thoroughly absorbed with homogenized normal guinea pig tissues. As revealed by the mixed hemadsorption technique (19), the absorbed serum reacted strongly with MC-D cells (titer 31,000) and only weakly with cultured guinea pig testis cells (titer 250); there was no reaction with cultured guinea pig kidney cells. Mouse antiserum to Po: C57BL/6J mice received an ip injection of 0.12 mg BCG; 20 days later they were given ip 106 Po cells which had been treated with V. cholerae neuraminidase (25 U jl06 cells, 45 min, room temperature) and mitomycin C (20 ftgj 106 cells, 20 min, room temperature) sequentially. On day 27, the animals received ip in their backs an additional dose of 10 6 nontreated Po cells in FCA in a total volume of 0.2 ml. Serum was collected 10 days after the last injection. Antiglobulin sera.-The IgG fraction was isolated from rabbit, rat, mouse, guinea pig, and sheep sera by ion exchange chromatography with DEAE cellulose (17). F(ab'h fragments of these immunoglobulins were obtained by pepsin digestion followed by gel filtration on Sephadex G-150 (17). Antiglobulin antibodies were produced by three sc biweekly injections of IgG or their corresponding F(ab'h fractions incorporated in FCA (1 mg protein/ml emulsion): Rabbits were given 1, 1, and 2 mg antigen and sheep received three doses of 5 mg each. The specificity of antisera was tested by the immunoelectrophoresis technique against whole serum containing the relevant immunoglobulin antigen and by the double diffusion method against purified antigens (17). The Downloaded from https://academic.oup.com/jnci/article-abstract/55/4/879/932404 by Insead user on 05 March 2018

N onlabeled antiserum or antibody (test antibody)

125I-labeled IgG antiglobulin antibodies (indicator)

Rabbit antibody to L 2C (xenogeneic) Rabbit antiserum to guinea pig MC-D (xenogeneic)

Sheep antibody to rabbit IgG or corresponding F(ab'h Sheep antibody to rabbit F(ab'h

Surface Ig (syngeneic)

Rabbit antibody to rat IgG or corresponding F(ab'h

Mouse antiserum to Po (syngeneic)

Rabbit antibody to mouse IgG

IgG fraction of the antiglobulin sera was separated as in (17) and iodinated according to the modified chloramineT method (8); PBSA was used for the saturation of the Dowex column before separation of free iodine from the 125I-labeled antiglobulin antibodies. The concentration of IgG was determined by UV adsorption at 280 nm; for this calculation the optical density of 1.4 was considered to correspond to 1 mg protein/m!. The specific activity of 1251 conjugates was 10-12 ftCi/ ftg IgG. Tissue culture.-Eagle's minimum essential medium supplemented with nonessential amino acids, 20% heatinactivated (56 0 C, 30 min) FCS, 100 U penicillin/m!, and 100 f-tg streptomycin/ml (CMEM) was used for all cultures. Cell lines were derived from solid tumors by the method described in (20). For RIA, 104 tumor cells in 0.1 ml CMEM were delivered to each well of the MicroTest culture plate, which was covered with a lid, and cultured at 37° C in a water-saturated atmosphere containing 5% CO2 ; a monolayer developed within 24-48 hours. The culture medium was then aspirated and the plates containing the cells were dried at room temperature, fixed, and either used immediately or stored at 4° C. Preparation of noncultured cells in MicroTest or Microtiter plates.-L2 C cells obtained from the blood of leukemic guinea pigs were suspended in PBSA, and volumes of 0.1 ml containing 10 5 cells were placed in each well of the MicroTest or Micro-Titer plate. The plates were sealed and centrifuged (150xg, 10 min, 4 0 C) in carriers (# 14-245-33; Fischer Scientific Co., Fairlawn, N.J.), and the supernatant was carefully aspirated. Airdried plates were fixed and used immediately or stored at 4° C until use. General procedure of RIA.-The optimal conditions established for the micro-RIA for different tumors are given in the next section. All systems involved are illustrated in text-figure 1. RESULTS Fixation

Dried MC-D cultures were treated with Kahle's fixative (21), absolute methanol, 10% buffered formaldehyde (pH 7.0), or 2.5% glutaraldehyde in PBS for 15 minutes at room temperature. After fixation, the wells were precoated with PBSA and incubated with rabbit antiserum to MC-D or normal rabbit serum (diluted to 1: 10 in PBSA), and subsequently with 125 1 sheep antibody to rabbit IgG (1 :200 dilution containing approximately 0.25 ftg iodinated IgG/ml) as outlined in text-figure 1. The reactivity of rabbit antiserum to MC-D with cells






1 Add

0.1 ml of medium 2 A'pirate containing 10'cell, and - culture medium culture 24 -48 h,..

~ \


3 Air-dry at roam temperature. Fix with 109f, neutralized formalin. 15 min. ot room

Reduction of Background



., ,

In fixation experiments with Po and PW13 tumors, methanol and formaldehyde were equally effective agents. Hence in all subsequent experiments, the cells were fixed with either methanol or formaldehyde. Fixed cells retained their antigenicity at 4° C for at least 1 year, the period of investigation up to the time of the preparation of this article.



.. Wa,h thrice with PBS.

5 Shake out



6 Add 0.2 ml

7 Add ,erial dilution,

of PBSA to each well and incubate at 37"c for 2 h". Shake out PBSA.


of anti-tumor antibody. Incubate for I ~ h,..at 37"

A microradioimmunoassay for antibodies to tumor-associated antigens.

A versatile microradioimmunoassay for the detection of antibodies to tumor-associated and other tissue antigens was described. The method involved: a)...
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