Production and characteristics of monoclonal antibodies against staphylococcal enterotoxin A

Thesis/Dissertation · Tue Jan 01 04:00:00 EST 1985

OSTI ID:6990511

Edwin, C

Four hybridomas (designated B/sub 2/I, B/sub 2/II, CV/sub 3/ and C/sub 5/) secreting monoclonal antibodies (McAb) to staphylococcal enterotoxin A (SEA) were produced by fusion of NS-1 myeloma cells with spleen cells of BALB/c mice hyperimmunized against SEA. Immunization consisted of a intraperitoneal injection of 50 ..mu..g SEA in phosphate-buffered saline (PBS, pH 7.2) and complete Freund adjuvant, followed four weeks later with 150 ..mu..g SEA in PBS and incomplete Freund adjuvant. Three days prior to fusion an intravenous injection of 150 ..mu..g SEA was given per day. Monoclonal antibodies from all clones (tissue culture supernatant) showed high end-point titers (>10/sup -7/) in an indirect enzyme-linked immunosorbent assay (ELISA) with 1 ..mu..g SEA as the coating antigen in microtiter plate wells. Even nanogram levels of purified SEA yielded high absorbance with purified IgG/sub 1/ from clone B/sub 2/I. Saturation analysis with competitive ELISA indicated the McAb of all clones to be of similar and high affinity to SEA. Cross-reactivity with SEB, SEC/sub 1/, SED, and SEE was observed with McAb from all the clones. Specific reactivity of all McAb with each enterotoxin was noted in autoradiographs of electroimmunoblots (of SDS-PAG) of crude SEA, SEB, SEC/sub 1/, SED and SEE from growth of enterotoxigenic Staphylococcus aureus strains (265, 243, 293, 494 and ET-10 respectively) even when present in nanogram levels. Autoradiographs of crude SEB showed a 30,000 dalton SE band and an additional larger molecular weight band. Both polyclonal and monocloal antibodies demonstrated this second band with radiolabelled (/sup 125/I) Protein A and sheep anti-mouse F(ab/sup 1/)/sub 2/ probes.

• Research Organization: Minnesota Univ., Minneapolis (USA)
• OSTI ID: 6990511
• Country of Publication: United States
• Language: English

Related Subjects

550701* -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
ANTIBODIES
ANTIGENS
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME IMMUNOASSAY
HYBRIDOMAS
IMMUNOASSAY
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
MAMMALS
MATERIALS
MICE
MICROORGANISMS
MONOCLONAL ANTIBODIES
NUCLEI
ODD-EVEN NUCLEI
RADIOISOTOPES
RODENTS
SOMATIC CELLS
SPLEEN CELLS
STAPHYLOCOCCUS
TOXIC MATERIALS
TOXINS
TUMOR CELLS
VERTEBRATES