Abstract
Antigen Used for Immunization
Staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) were obtained from Sigma (S-9399 and S-4881, Munich, Germany). SEA and SEB was used for immunizations and ELISA assays. Staphylococcal enterotoxins cause syndromes like toxic shock, arthritis, allergic reactions, and autoimmune diseases.(1,2) In this study a multiple immunization method was applied in order to obtain monoclonal antibodies with high specificity against SEA.
Method of Immunization
Two immunization methods were used. Two mice were injected with a mixture of two antigens (2 μg SEA+ 2 μg SEB); the other mice were immunized with 2 μg SEA antigen. A solution containing these antigens was prepared in phosphate-buffered saline (PBS, 10 mM K2HPO4, 10 mM KH2PO4, 0.15 M/L NaCl [pH 7.2]) and mixed with equal volumes of Freund's complete adjuvant (Sigma). 0.2 mL of these antigens were given by intraperitonal injection to four 8-week-old BALB/c mice. Booster injections were performed in three weekly intervals using Freund's incomplete adjuvant (Sigma).
The indirect enzyme linked immunosorbent assay was used for the screening of hybridoma supernatant and serum of the mice.(3) 96-well polystrene plates (Nunc immunoplates) were coated with 50 ng SEA in 100 μL PBS. The coating of the plates was carried out at 4°C overnight. The plates were washed three times with washing buffer (0.005% Tween-20 [Merck, Darmstadt, Germany] in PBS). Then, 0.1% casein in 200 μL PBS was added to the wells and the plates were incubated for 1 h at 37°C. After washing, the culture supernatant of hybridoma or mice sera was added and incubated for 1 h at 37°C. Anti-SEA antibody-binding reaction was detected by using an alkaline phosphatase-conjugated goat anti-mouse polyvalent (IgA, IgM, IgG) antibody (A0162, Sigma, St. Louis, MO) as secondary antibody. Primary-secondary antibody binding reaction was visualized by p-nitrophenyl phosphate (PNPP, Merck) hydrolysis reaction. After 45 min, the optical density was read at 405nm by using a microplate reader (Elia Reader, Bio-Rad, Tokyo, Japan).
Parental Cell Line Used for Fusion
Lymphocytes from spleen and all lymph nodes of the BALB/c mouse immunized with SEA and mouse myeloma cells (F0; ATTC CRL 1646) were used for fusion. The myeloma cells were cultivated in DMEM medium (Gibco, Scotland) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany) under humidified atmosphere of 5% CO2.
Selection and Cloning Procedure
Monoclonal antibodies were produced by a modified method of Köhler and Milstein.(4) Mice that developed the IgG response against SEA were selected for fusion studies. Four days before fusion, a booster immunization was done both intraperitoneally and to four footpads of the selected mouse with 1 μg of SEA dissolved in 100 PBS. One day prior to fusion, the peritoneal macrophages of the normal mice were prepared and seeded into 96-well plates used as feeder cells. For hybridoma preparation, the spleen cells and all lymph nodes of the BALB/c mouse immunized with SEA and mouse myeloma cells (F0; ATTC CRL 1646) were used in fusion in the presence of 50% polyethylene glycol (PEG) 4000.(5–7) PEG was added slowly over a 1 min period while gently stirring and kept without any stirring for another min. The fusion suspension was then diluted adding 4 mL of DMEM medium (Gibco) over a period of 2 min, followed by 40 mL at a rate of 20 mL per min. Fusion product, resuspended in DMEM containing 20% fetal calf serum (FCS, Biochrom) and antibiotic were distributed into the 96-well culture plates and incubated overnight at 37°C, 5% CO2, 95% humidity. On day 15, the wells were screened for choice of the desired antibody by indrect ELISA. The hybridoma cells positive for SEA were subjected to three rounds of cloning using limited dilution method.(8) At each stage of growth, aliquots of hybrid cultures (3–5.106 cells) were frozen in liquid nitrogen in 80% DMEM, 20% FCS, and 10% dimethylsulphoxide (Sigma). Three fusions were performed with SEA-immunized mice. The results of the fusions are summarized in Table 1. From the third fusion, only one (1D8) anti-SEA antibody producing hybridoma cells were obtained.
Heavy and Light Chains of Immunoglobulin
One SEA specific monoclonal antibody was obtained. Monoclonal antibody was identified as the IgG class, which was IgG1 subtype using a hybridoma subisotyping kit (Behring Diagnostics, La Jolla, CA).
Specificity
All monoclonal antibodies were tested by indirect ELISA with different proteins such as Staphylococcal enterotoxin B, bovine serum albumin (BSA), and milk. MAM 1D8 did not elicit any cross-reaction with the BSA and milk, only SEB recognized by half (Table 2).
Specific Antigen Identified
The enzyme-linked immunosorbent assay showed that the antibody MAM 1D8 reacts with SEA.