An analysis of β‐lactam‐derived antigens on spleen cell and serum proteins by ELISA and Western blotting

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Abstract
Penicillins and related β‐lactam antibiotics are known to conjugate to proteins to generate potentially antigenic (haptenic) determinants. In the present study, we used a rabbit polyclonal antibody raised against benzylpenicillin (BP) to investigate the capacity of six penicillins and one cephalosporin to generate haptenic groups in vitro on cultured mouse spleen cells and on serum proteins in the culture medium. All of the drugs tested, namely, BP, amoxicillin (AMX), ampicillin (AMP), cephalothin (CEP), cloxacillin (CLX), flucloxacillin (FLX), and phenoxymethylpenicillin (PMP) generated antigens in a concentration‐dependent manner on cell and serum proteins, which could be detected by ELISA, although antigens generated by BP, CEP, FLX, or PMP in either cell‐ or serum‐conjugated form were more readily detected than those generated by AMX, AMP, or CLX. Western blot analysis revealed that BP‐derived antigens were generated relatively slowly on cell proteins (maximum binding was not yet reached after 8 h), compared to serum proteins (maximum binding within 1 h). BP, CEP, and PMP all generated similar distinctive patterns of immunostaining of electrophoresed cell or serum proteins which did not reflect the relative abundance of different proteins as revealed by Coomassie brilliant blue staining. FLX, CLX, AMP, and AMX did not generate antigens that could be detected on Western blots. In conclusion, we have shown that various β‐lactam antibiotics generate antigens on cell and serum proteins that can be detected and characterized immunochemically with polyclonal antiserum. Further application of these methods may offer potential for further identification of immunologically relevant cellular and serum antigens generated by these drugs.