Use of an automatic cell harvester in a cellular radioimmunoassay

Journal o f l m m u n o l o g i c a l M e t h o d s ,

75 (1984) 159-166

159

Elsevier JIM03301

Use of an Automatic Cell Harvester in a Cellular Radioimmunoassay Denis Gerlier 1 and Th6r6se Avice INSERM

U. 218, Centre Lbon Bbrard, 28 rue LaOnne~, 69373 Lyon C e d e x 2, France

(Received 14 May 1984, accepted 3 September 1984)

An automatic cell harvester was used in the final step of a cellular radioimmunoassay to collect cell b o u n d anti-rat IgG 125l-F(ab')2. Studies on the reliability of this collection method were performed with antibodies directed against cell surface antigens induced by the Gross murine leukemia virus and produced by immunization of W / F u rats with the syngeneic (C58NT)D lymphoma. Glutaraldehyde-fixed as well as untreated Gross virus induced lymphoma cells could be used. Similar and specific antibody binding curves were obtained when the cells were incubated with the anti-(C58NT)D serum and anti-rat IgG 125I-F(ab')2 in the presence of 0.1% N a N 3. Background levels of non-specific binding of anti-rat 125I-F(ab')2 to mouse l y m p h o m a cells or rat thymocytes were only a few cpm above the background of the gamma-counter. This allowed detection of surface immunoglobulin positive lymphocytes among as few as 30,000 rat splenocytes. In addition, this cellular radioimmunoassay was found to be suitable for the measurement of solubilized cell surface antigen by its capacity to inhibit the binding of the specific antibodies to the target cells. Key words: cellular radioimmunoassay - cell surface antigens - automatic cell harvester - Gross murine leukemia virus - lymphoma - soluble antigen

Introduction

Numerous assays have been developed to detect cell surface antigens or their related antibodies with a specific 125I-labeled ligand such as anti-immunoglobulin antibodies or protein A used in a cellular radioimmunoassay. The need to be able to handle a large number of antibody samples has led to the development of microassays that use multiwell microplates (Longenecker et al., 1978) instead of tubes (Rockoff et al., 1979). Despite the initial proposal of Longenecker et al. (1978) to use an automatic cell harvester to collect the specific radioactivity bound to chicken red blood cells, most investigators have used other approaches in the final step of the assay. Collection of 125I-labeled ligand bound to cell surface was performed either by manual harvesting of the cell suspension from each microwell (Brown et al., 1979) or by elution with a detergent solution followed by absorption to a supernatant 1 D.G. is Charg6 de Recherches au C.N.R.S. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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collection system (Titertek device; Nilsson et al., 1982). Alternatively, the cells were initially fixed to the plastic surface of each well either as adherent monolayers (Brown et al., 1979) or by treatment with glutaraldehyde (Levy et al., 1981). AI Sheikly et al. (1980) proposed sealing the final cell pellet with Nobecutane aerosol spray. 125I-labeled ligand binding levels have been determined either semiquantitatively by autoradiography (Brown et al., 1979) or by counting in a gamma-counter each microwell, previously cut out (A1 Sheikly et al., 1980; Levy et al., 1981). However, each of these commonly used methods has some disadvantages. Manual harvesting is time consuming, glutaraldehyde fixation of the cell surface may affect antibody recognition of some antigens and simultaneous collection of the 125I-labeled ligand non-specifically bound to the plastic microwells may drastically increase the non-specific background (Nilsson and Sj6gren, 1984). Results presented here show that, in a quantitative cellular radioimmunoassay, the use of an automatic cell harvester, as rediscovered by one of us, is a very attractive method which combines very low backgrounds with swiftness and versatility. With anti-rat IgG 125I-labeled goat F(ab')2, cell surface immunoglobulin on rat spleen cells and rat antibodies against cell surface antigens induced by the Gross leukemia virus on rat and mouse lymphoma cells are detected without chemical fixation of the target cells. In addition, this assay is suitable to measure the amount of soluble antigen in cellular extracts by inhibition of the antibody cell binding.

Materials and Methods Animals and tumors

C 5 7 B L / 6 / J I c o mice and W / F u / I c o rats originally obtained from IFFA CREDO (France) were bred in our colonies. The mouse Gross leukemia virus E8 G 2 lymphoma (Old et al., 1965) and the rat Gross leukemia virus induced (C58NT)D lymphoma (Geering et al., 1966) were maintained by weekly passages in syngeneic C57BL/6 mice and W / F u rats respectively. Tumor cells and normal spleen cells were prepared as previously described (Gerlier et al., 1981). Antisera and crude soluble antigen extract

Anti-(C58NT)D sera were produced by immunization of syngeneic rats with viable (C58NT)D tumor cells (Gerlier et al., 1977). These antisera are mainly directed against the Gross cell surface antigen (GCSAa) which is expressed on E 8 G 2 and (C58NT)D lymphoma cells (Old et al,, 1965; Geering et al., 1966), as glycosylated precursors of the viral core proteins (Ledbetter and Nowinsky, 1977; Snyder et al., 1977). Anti-rat IgG goat (F(ab')2 without cross-reactivity toward mouse immunoglobulins (A1 Sheikly et al., 1980) was kindly provided by M.R. Price. After labeling with 125I according to the iodogen procedure (Salacinski et al., 1981), 125I-F(ab')2 was separated from free lzsI by gel chromatography on a Sephadex G-25 (Pharmacia, Sweden) microcolumn (Parkinson et al., 1981). Trichloroacetic acid precipitation showed that more than 95% of the iodine was linked to precipitable protein in the 12~I-F(ab')2 peak. The specific activity of the a25I-F(ab')2 was about

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107 cpm//~g protein and, as previously reported (A1 Sheikly et al., 1980), approximately 50% of the labeled F(ab')2 rebound to rat IgG covalently linked to Sepharose 4B. A crude soluble G C S A a extract was prepared from the cytosol of (C58NT)D lymphoma cells as previously described (Gerlier et al., 1983). Briefly, after cell disruption, the 100,000 × g supernatant was fractionated by 60% a m m o n i u m sulfate precipitation and the G C S A a was further purified by gel filtration through Sephadex G-200 (Pharmacia, Sweden) and concentrated to 25-35 mg p r o t e i n / m l on Amicon XM50 membranes (Amicon, Denvers, MA).

Cellular radioimmunoassay After 3 washes in phosphate-buffered saline p H 7.4 (PBS), the cells were resuspended in PBS supplemented with 1% bovine serum albumin (Sigma, St. Louis, MO) and 0.1% N a N 3 (BSA-PBS) usually at 8 × 1 0 6 cells/ml. In some experiments, the cells were previously fixed with 0.01% glutaraldehyde in PBS for 30 min at 4°C and extensively washed. Cells (25 ~1, 2 × 105) were added to each well of 96 round-bottomed wells microplate. Serial dilutions in triplicates of anti-(C58NT)D or normal rat serum (25 ~1) were added and the microplates were incubated for 30 min at room temperature. After addition of 150 ~1 of BSA-PBS to each well, the plates were centrifuged for 2 min at 400 x g. The supernatant of each well was then carefully aspirated with a vacuum source. To ensure nearly complete removal of the supernates without loss of cells, a simple device was used; namely the needle of an infusion set (Butterfly', Abbott Ireland, Ireland) cut 1 m m shorter than the well depth and connected to a vacuum source. The cells were carefully washed 3 times with 200/~1 of BSA-PBS. The last supernatant was discarded and 50/~1 of anti-rat I g G 125I-F(ab')2 (105 cpm) was added to each well and the plates were incubated for 30 min at room temperature. The cells were then carefully washed 4 times as described above and the final pellets were resuspended in 0.1% N a N 3 PBS. Cells were collected from individual wells on glass-fiber filter strips with an automatic cell harvester flushed with 0.9% NaC1 solution (MASH II, Microbiological Associates, Bethesda, MA). The filter discs were transfered to plastic tubes and counted in an automatic gamma-counter. In control microwells, tumor cells were incubated with the conjugate alone in order to determine the background levels. Standard deviations with triplicates were usually less than 10% of the mean value. The results were expressed after subtraction of background levels and antibody titers were defined as the antibody dilution giving 50% of the maximal azsI-F(ab')2 binding. Antigen dosage was determined by modifying the initial step as follows. Serial dilutions (in triplicate) of the crude G C S A a extract (20/~1) were incubated together with the anti-(C58NT)D serum diluted 1 : 500 or 1 : 1000 (20/~1) for 30 min at room temperature. 2 × 105 E 8 G2 tumor ceils were then added (10 ~1) and incubated for 30 min at room temperature, and the standard procedure was carried out. Results were expressed as percentages of 125I-F(ab')2 bound to E 8 G2 cells in the absence of crude G C S A a extract. The antigen titer was defined as the dilution of extract inhibiting 50% of the anti-(C58NT)D antibodies diluted 1 : 500 or 1 : 1000.

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Results

Reactivity of anti(C58NT)D serum on Gross virus induced lymphoma cells Normal rat serum and syngeneic anti-(C58NT)D serum were tested on 2 × 105 glutaraldehyde fixed normal C 5 7 B L / 6 mouse spleen cells, or Gross virus induced mouse E ~ G2 and rat (C58NT)D lymphoma cells. The results are shown in Fig. 1. Only the anti-(C58NT)D serum bound the anti-rat IgG 125I-F(ab')2 to the Gross virus infected E ~ G2 and (C58NT)D target cells. The maximum 125I-F(ab')2 binding was however higher on E ~ G2 cells than on (C58NT)D cells, which may indicate somewhat lower expression of the surface antigens recognized by the anti-(C58NT)D serum, previously identified as mainly Gross cell surface antigen, G C S A a (Herberman, 1972). In addition, this result confirms the species specificity of the anti-rat I g G goat F(ab') 2 (A1 Sheikly et al., 1980). Application to unfixed target cells In preliminary experiments, glutaraldehyde fixed cells were used because such treatment results in almost indestructible particles (Price et al., 1981), likely to be retained on the glass fiber strip of the automatic cell harvester. Such fixation is considered highly suitable for cells kept for several days before their use (Levy et al., 1981). However, when glutaraldehyde fixed E 8 G2 lymphoma cells were kept 7 days before use, a significant decrease was found in their ability to bind anti-(C58NT)D antibodies and anti-rat IgG a25I-F(ab')2, as compared with freshly prepared target cells (Fig. 2). In order to avoid glutaraldehyde fixation which might result in the loss of antibody recognition of some cell surface antigen, the procedure was tentatively carried out with untreated target cells. N a N 3 (0.1%) was maintained in the buffer throughout the procedure, in order to avoid any cell processing of the antibody-antigen complexes. As shown in Fig. 2, the antibody binding curve was very similar to that obtained with freshly prepared glutaraldehyde treated E 8 G2 cells used as

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Fig. 1. Titration curve of anti-(C58NT)D rat serum ( ) or normal rat serum (. . . . . . ) tested by cellular radioimmunoassayon Gross virus induced mouse E ~ G2 (A, zx),rat (C58NT)D (11,r~) lymphoma cells or on normal mouse spleen cells (e, O). Target cells were used after 0.01% glutaraldehyde fixation.

163

targets. Collection on glass fiber strip was thus also suitable for unfixed target cells. All the following experiments were performed with unfixed cells.

Application to soluble antigen measurement A soluble GCSAa extract was tested for its ability to inhibit binding of anti(C58NT)D serum diluted 1:500 or 1:1000 to E 8 G2 target cells. Reproducible titration curves of antibody binding inhibition were obtained (Fig. 3), the 50% antigen titer being 959 and 1071 with the antiserum diluted 1:1000, and roughly half this (377) with 2-fold less dilution of the anti-(C58NT)D serum. Background level, sensitivity and reproducibility of the assay In a typical experiment where the maximum specific binding of anti-rat IgG

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Fig. 2. Titration curves of anti-(C58NT)D rat serum tested on mouse E ~ G2 lymphoma cells either untreated ([3. . . . n) or after 0.01% glutaraldehyde fixation (,7 xz). The third target cells (v I,) were 0.01% glutaraldehyde treated E ~ G2 cells kept 7 days at 4°C before their use in the assay.

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164

125I-F(ab')2 to 2 × 105 E 8 G2 cells in the presence of anti-(C58NT)D serum reached 4021 _+ 352 cpm, non-specific 125I-F(ab')2 binding to the same target cells was 124 _+ 32 cpm, which compares very favorably with the background for the gammacounter itself (87 _+ 4 cpm). The non-specific background level was thus only 0.037% of the initial 125I-F(ab)2 input, whereas maximum binding of anti-rat IgG 125I-F(ab')2 to 2 × 105 E 8 G2 cells coated with rat antibody reached 4-10% of the initial input. To determine further the sensitivity of the assay, direct determinations of the respective anti-rat Ig 125I-F(ab')2 binding ability of graded amounts of rat spleen and thymus cells were also made. Fig. 4 shows that, up to 10 6 thymocytes, the amount of anti-rat IgG 125I-F(ab')2 bound to these targets was as low as 30 cpm and that 3 × 10 6 thymocytes bound only 110 cpm. In contrast, good correlation was found between the number of rat spleen cells and the level of bound 125I-F(ab')2 (r = 0.988, 2~ < 0.001). The sensitivity of the assay allowed detection of Ig surface positive lymphocytes among as few as 30,000 rat spleen cells. It should be stressed that the assay is highly specific, since 2 × 105 mouse spleen cells did not significantly bind the anti-rat IgG 125I-F(ab')2 (see Fig. 1). The sensitivity of the cellular radioimmunoassay for measuring anti-(C58NT)D antibodies and GCSAa soluble antigen was assessed by comparison with the classical complement dependent cytotoxicity test. Seven anti-(C58NT)D sera were tested by both assays and a 6-7-fold increase in the 50% antibody titer was found with the cellular radioimmunoassay. Five soluble antigen extracts were also tested by

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Fig. 4. Direct binding of anti-rat IgG ~25I-F(ab')2 to rat thymocytes (C) . . . . O) and rat splenocytes (e e). Target cell numbers used in each well are plotted on the y-axis. Standard deviations within triplicates are indicated by vertical bars.

165

both assays and similarly a 10-12-fold increase in the 50% antigen titer was found for the radioimmunoassay. Moreover, antibody and antigen titers were highly reproducible from one experiment to another, with a standard deviation lower than 1 dilution.

Discussion These studies demonstrate that an automatic cell harvester can readily be used to collect the cell surface bound radioactivity in the final step of the cellular radioimmunoassay. This procedure proved useful for measuring antigen-antibody complexes bound to the cell surface and cell surface antigen in soluble form. The advantages of the cellular radioimmunoassay described are the following. (1) No pretreatment of the target cells such as glutaraldehyde fixation is required, which allows its use with aldehyde sensitive cell surface antigens. (2) Target cells are used in suspension form which eliminates time-consuming natural or artificial cell binding to the microweUs as previously proposed (Levy et al., 1981; Nilsson et al., 1982). (3) The use of the automatic cell harvester allows rapid collection of the 125I-F(ab')2 bound to the target cells and reduces non-specific binding almost to the background of the gamma-counter. This very low background is likely to be due both to the additional washes during flushing and the non-collection of the ~25I-labeled ligand non-specifically bound to the plastic wells. Such non-specific binding to the plastic may seriously hamper the sensitivity and the specificity of the cellular radioimmunoassay (Nilsson and SjOgren, 1984), since it is included when each microwell is cut out (A1 Sheikly et al., 1980; Levy et al., 1981) or when the ~25I-labeled ligand is solubilized from each well with a strong ionic detergent (Nilsson et al., 1982). (4) It is quantitative, sensitive and highly reproducible. In conclusion, this method should be very helpful in the serological detection of any cell surface antigen or for determining the dosage of a solubilized antigen. Monoclonal antibodies have been successfully tested in this way and we are currently using this cellular radioimmunoassay to follow purification of the GCSAa.

Acknowledgement We thank J.F. Dor6 for his helpful encouragement.

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