Abstract
Antigen Used for Immunization
Recombinant Escherichia coli O157:H7 Stx1B subunit with pGEX prokaryotic expression system.
Method of Immunization
Six-week-old BALB/c mice were immunized subcutaneously with 50 μg purified Stx1B protein in Freund's complete adjuvant and boosted weekly three times with an equivalent dose in Freund's incomplete adjuvant. Finally mice were injected intravenously and intraperitoneally with 10 μg of Stx1B in PBS before preparation of spleen lymphocytes.
Parental Cell Line Used for Fusion
SP2/0 myeloma cell line.
Selection and Cloning Procedure
After immunization, spleen lymphocytes were fused with SP2/0 myeloma cells using 50% polyethylene glycol. Hybridoma cells were cultured in 1640 medium containing 10% newborn bovine serum (NBS, Excel Bio, Beijing, China), hypoxanthine, aminopterin, and thymidine and cloned twice by limiting dilution.
Heavy and Light Chains of Immunoglobulin
The immunoglobulin isotype was determined as IgG2a by a mouse MAb isotyping kit.
Specificity
With ELISA, no reactivity was found with lysate of Salmonella enteritis, non-Stx1-producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae, and Staphylococcus aureus. The only positive reaction was with expressed EHEC O157:H7 Stx1B protein.
Specific Antigen Identified
Prokaryotic expressed EHEC O157:H7 Stx1B protein.