Letters in Applied Microbiology 1987, 4, 85-89 MS/043

Production of monoclonal antibodies to Shigella. Enzyme-linked immunosorbent assay for screening hybridoma antibodies with intact bacteria

M . S . I S L A M & W . H . STIMSON Immunology Division, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G4 ONR, UK

Received 22 January 1987 and accepted 23 January 1987

I S L A M , M.S. & STIMSON, W . H . 1987. Production of monoclonal antibodies to Shigella. Enzyme-linked immunosorbent assay for screening hybridoma antibodies with intact bacteria. Letters in Applied Microbiology 4, 85-89.

A simple and rapid enzyme-linked immunosorbent assay (EL1SA)-type assay has been developed to screen hybridoma supernatant fluids with whole viable or killed bacteria as the antigen. The optimum concentration of acetone-killed and dried cell antigen for coating was 25100 pg/ml. Screening of hybridoma supernatant fluids against whole cells, both with and without fixation, was assessed and both were equally sensitive. The data indicate that bacterial fixation is detrimental in ELISA probably because of loss of antigenic structure. A highly specific monoclonal anti- body (laM3) was produced against ShigellafIexneri la and was employed to opti- mize the assay procedure.

Somatic cell hybridization to produce mono- clonal antibodies (Kohler & Milstein 1975) has become a valuable microbiological technique. A basic requirement of this procedure is to estab- lish a suitable screening assay to identify specific hybridomas that secrete antibodies with the appropriate reactivity.

Different assay methods have been developed to screen hybridoma-secreted antibodies, includ- ing the enzyme-linked immunosorbent assay (ELISA) (Engvall & Perlman 1972), ELTSA with whole mammalian cells (Handley et al. 1982), complement-dependent cytotoxicity (Ritz et al. 1980), radioimmunoassays (Stocker & Heusser 1979) and complement-dependent lysis of eryth- rocytes (Kohler & Milstein 1976). Enzyme- linked immunosorbent assay has been used in several circumstances and has already been proven to have advantages over the other immunoassays (Carlsson & Lindberg 1978; Suter et al. 1980).

Enzyme-linked immunosorbent assay has been described to detect antibodies against the lipopolysaccharide (LPS) fraction of Shigellu jlexneri (Keren 1979) and Sh. dysenteriae type 1 (Lindberg et al. 1984). Glycosylation,

acetylation or other modification of the 0- polysaccharide chain, which results in the expression of a new O-antigenic specificity and sometimes in the loss of an existing O-antigenic specificity after lysogenic conversion by bac- teriophage, has been reported by Lindberg (1977). This result also suggested that changes in the structure and conformation of the immuno- genic complex, during the extraction process, cannot be overlooked. The use of LPS, there- fore, to screen hybridomas produced after immunization with whole bacteria could fail to detect antibodies of interest.

We describe an ELISA method for the detec- tion of hybridoma antibodies specific against Shigella by using whole viable and killed cells as antigen; this method could also be used to detect antibodies against other bacteria.

Materials and Methods

FIXATION

Glutaraldehyde (0.1% v/v, Sigma), 50% (v/v) ethanol, 50% (v/v) acetone and 2% (v/v)

86 M . S . Islam and W . H . Stimson formalin, all as aqueous solutions, were used as fixatives. Phosphate-buffered saline (PBS), 20 mmol/l at pH 7.2, was used throughout. Poly-L-lysine (mol. wt 32 000-70 OOO), bovine serum albumin (fraction V) and Tween 20 were obtained from Sigma.

BACTERIA A N D P R E P A R A T I O N OF

A N T I G E N S

Shigella flexneri la, 2a, 2b and Sh. dysenteriae type 1 were obtained from the National Collec- tion of Type Cultures (Central Public Health Laboratory, London, UK). Escherichia coli (NCTC no. 8623) and Salmonella typhimurium (NCTC no. 5711) were obtained from the Divi- sion of Applied Microbiology, University of Strathclyde.

All the bacterial strains were grown on a large scale in Nutrient broth No. 2 (Oxoid) with maximum aeration. When the cells were in the late exponential phase of growth, purity was confirmed and the cells harvested by centrifug- ation at 3000 g for 15 rnin at 4°C. Thereafter, the cells were killed with ice-cold acetone and rapidly dried in vacuo according to Morgan (1937). Acetone-killed and dried (AKD) cells were used for both immunization and ELISA.

I M M U N I Z A T I O N A N D HYBRIDOMA

P R O D U C T I O N

Acetone-killed and dried cells of Sh. jexneri la, in saline (4 mg/ml), were emulsified with an equal volume of Freund's adjuvant (Gibcok complete for primary and incomplete for sec- ondary immunization. Female Balb-c/NZB F1 hybrid mice, 8-10 weeks old, were immunized with 0.5 ml emulsified AKD cells by two intra- peritoneal injections at a 9 d interval. The final injection of antigen, 1.5-12 weeks after second- ary immunization, did not contain Freund's adjuvant and was carried out, 3 d before fusion, by the intraperitoneal administration of 0.5 ml AKD cells in saline.

Spleen cells from mice immunized as described above were fused with cells from the X63.Ag8.6.5.3 murine myeloma line, in exponen- tial growth, in a ratio of 4 : 1 by the addition of 1 ml 46% (w/v) polyethylene glycol 1550 (Serva) in RPMI 1640 with gentle mixing for 3 min at

37°C. After standing for 2 rnin at room tem- perature, the mixture was slowly diluted by the drop-wise addition of 20 ml RPMI 1640 over 5 min, followed by standing at room tem- perature for 10 min. After washing twice with RPMI 1640, the cells were incubated for 2 h at 37°C in bicarbonate-buffered RPMI 1640, sup- plemented with 10% (v/v) fetal calf serum, 2 mmol/l L-glutamine, 50 IU/ml penicillin and 50 pg/ml streptomycin (Flow) and containing 1 x molfi hypoxanthine and 1.6 x lo-' molfi thymidine (HT medium). The cell suspen- sions (100 pl) were then dispensed into 96-well tissue culture plates (Costar) at three different concentrations (2.5, 1.25 and 6 x lo6 cells/ml). Finally, 200 p1 HT medium containing 4 x lo-' mol/l aminopterin (HAT medium) was added to each well, The plates were incubated at 37°C in a humid atmosphere of 5% CO, in air. Hybridoma cells were initially grown in HAT medium but this was slowly eliminated by step-wise replacement with HT medium after 10 d. Supernatant liquids were screened for spe- cific antibody by indirect non-competitive ELISA 14-18 d post-fusion. Specific hybri- domas were subsequently expanded into flasks and cloned three times or until f00% cloning efficiency was obtained. This procedure was carried out by limiting dilution in 96-well tissue culture plates containing a feeder layer of spleen cells (2 x lo5 cells/well) from non-immunized Balb-c/NZB F1 hybrid mice. Cell lines of inter- est were maintained in uitro in culture medium and were frozen, at a concentration of 5 x lo6 cells/ml, in RPMI 1640 cotitaining 30% bovine serum and 15% dimethyl sulphoxide (Sigma) and stored in liquid nitrogen.

ELISA P R O C E D U R E

Flat- and round-bottomed 96-well microtitre plates (Dynatech) were employed for ELISA and each well activated with 100 pl poly-~- lysine in PBS, at a concentration of 5 pg/ml, for 45 min at 37°C. The poly-~-lysine was removed and 100 p1 of AKD cells (50 pdml in PBS) adsorbed onto each well by centrifugation (500 g for I0 min) followed by incubation at 4°C for 18 h. Without additional fixation these plates could be stored dry for up to 8 weeks at 4°C. In some experiments cells were fixed after the centrifugation stage with 100 p1 fixative per well for 5 min at 18°C; both fixed and unfixed

Monoclonal antibodies to Shigella 87 plates were then washed three times with PBS. Thereafter all washing was achieved with PBS containing 0.05% (v/v) Tween 20.

Bovine serum albumin (BSA), in 100 p1 1% (w/v) in 0.02 mol/l Tris/HCI buffer, pH 9.0, was added to each well, incubated for 45 min at 37°C and then the unbound BSA was removed by washing five times. These treated plates could then be stored under dry conditions or used immediately for ELISA.

Neat or diluted tissue culture supernatant fluids (100 pl) were added to each well and incu- bated for 1 h at 37°C. Thereafter, following washing three times, 100 p1 sheep anti-mouse y- globulin-horseradish peroxidase conjugate (Scottish Antibody Production Unit, Carluke, Scotland), diluted 1 : 2000 in 0.15 mol/l NaCl containing 25% (v/v) normal sheep serum, were added; unbound conjugate was removed by washing five times after 1 h incubation at 37°C. Enzyme activity was measured with 200 pl tetramethylbenzidine substrate at pH 5-5 (Bos et al. 1981); the reaction was stopped after 30 min with 50 pl HzSO4 (10% v/v) and the A450 mea- sured.

The concentration of monoclonal antibodies in the tissue culture supernatant fluids was esti- mated using a sandwich-type enzyme immuno- assay (Stimson & Sinclair 1974). Briefly, neat or diluted tissue culture supernatant fluids and G 100 pg/ml purified mouse IgG were allowed to bind with sheep anti-mouse IgG (Scottish Anti- body Production Unit) coated plates; the rest of the procedure was as described above.

Results and Discussion

An enzyme immunoassay was initially devel- oped for whole Sh. flexneri l a cells (live and AKD) employing an antiserum derived from mice immunized against the bacterium. Uniform adsorption of the bacteria onto the microtitre plate was observed microscopically, both with and without fixation, although it was apparent that the number of cells per well was higher after fixation. Serial dilutions of sera, from immunized mice, were tested against both fixed and unfixed cells and both systems showed equal reactivity thus confirming that both types of bacteria could be used to screen hybridoma supernatant fluids. However, Stya et al. (1984) have suggested that exposure of intracellular

antigens or alterations in the structural configu- ration of membrane antigens during extraction and fixation can result in the identification of hybridomas which secrete monoclonal anti- bodies that bind only to fixed cells and not to live bacteria. For this reason it was decided to confirm whether it was possible to use whole cells in ELISA without fixation and thus, during hybridoma screening, results from fixed and unfixed cells were compared.

Two weeks after cell fusion growth of hybri- domas was observed in more than 75% of the microtitre plate wells. Thereafter, culture super- natant fluids were tested by ELISA against glutaraldehyde- or ethanol-fixed cells and unfixed AKD cells of Sh. flexneri la. Of 294 culture wells, 79 (26.9%) showed positive reac- tions against Sh. Jlexneri l a when tested against both fixed and unfixed cells (group A). Inter- estingly, the antibodies from 17 wells (5.8%) showed highly positive reactions only with fixed cells (group B).

The positive cell lines were tested again 5 d after the first screening and all, except five from group A, produced a positive reaction. Loss of activity due to genetic instability is well known and also very slow growing specific cell lines could be overgrown by rapidly growing non- specific cell lines. However, 13 cell lines from group B did not give any positive reaction while, unexpectedly, the remaining four cell lines lost activity after a further 2 weeks. The reason for this is unknown but the result indicates that screening of false positive hybridomas could not have been overlooked because of fixation.

Positive cell lines were transferred from 96- to 24-well plates when they were 75% confluent and their specificity was checked against Sh. flexneri la, 2a, 2b, E . coli and Salm. typhimu- rium. Of the 79 cell lines produced, 48 (60.8%) showed cross-reactivity with all the bacteria tested. Twenty-one cell lines (26.6%) reacted with Sh.Jlexneri la and 2a only and seven cell lines (8.9%) reacted against all the Sh. Jlexneri assessed. Only three cell lines bound exclusively to Sh. flexneri l a ; these were expanded into flasks and their specificity confirmed against dif- ferent Shigella, Salmonella and E. coli. One cell line was found to be highly specific, reactive and stable and chosen for further expansion and cloning. One clone, named la3M, was finally chosen to optimize the assay procedure.

The relationship between absorbance values

88 M . S. Islam and W . H . Stimson Table 1. Effect of microtitre plate-coating conditions on the binding of monoclonal antibody laM3

(anti-Shi~ella.pexneri la), as assessed by enzyme-linked immunosorbent assay

Absorption (A,,,)* Microtitre plate-

coating conditions Cell fixation procedure

Coating 0.1 Yo 50% 50% 2% Pre-treatment bacteria? Unfixed glutaraldehyde acetone ethanol formalin

Untreated AKD 0.75 0.80 1.03 0.84 0.79 Live 1.31 1.28 1.50 1.21 1.21

Poly-L-lysine AKD 1.31 1.51 1-61 1 44 1.38 Live 1.89 1.53 1.70 1.51 1.29

~

* Values expressed as the mean of 10 determinations. t Fifty p g Sh.fiexneri la/ml employed for coating. AKD, Acetone-killed and dried.

and the concentration of coating antigen is indi- cated in Fig. 1. The decrease in absorbance observed at higher concentrations of coating has already been reported by other workers (Keren 1979). Assessment of the antibody binding at 37°C for different periods indicated that 90% of the binding potential was realized after 30 min; further increases in absorbance were not observed after 60 min. After quantitat- ing the total amount of antibody in the tissue culture supernatant fluids of laM3, serial dilu- tions were tested by ELISA to check its sensi- tivity in terms of the amount of antibody which could be detected; it was found that less than 1 ng/ml antibody could be estimated with both fixed and unfixed cells.

Table 1 shows the absorbance values

1.6r

O l l t ‘ I l l 1 I ] 5 10 25 50 100 200 5001000

Antigen Concentration (pg/rn!)

Fig. 1. Relationship between the absorbance at 480 nm and the concentration of acetone-killed and dried Shigellaflexneri la cells used to coat microtitre plates. Each point represents the mean f S.D. of eight determinations.

obtained for laM3 under different conditions of antigen coating-pre-coating of the plates with poly-L-lysine always gave higher values. With killed cells, fixation improved ELISA sensitivity, but with live cells sensitivity was decreased. Thus, the data show that in the assay described both fixed and unfixed bacteria can be employed.

The results presented indicate that live or killed bacteria can be attached to the microtitre plates with or without fixation. However, to screen hybridoma supernatant fluids we suggest the use of unfixed cells to avoid false positive values being obtained. This ELISA-type assay could also be used for diagnosis and for sub- class identification of bacteria by employing the appropriate antisera.

M.S. Islam is in receipt of an ORS award.

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