Protocol for measuring anti-fentanyl antibodies in mouse serum by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) offers an effective, inexpensive, and reliable approach for the analysis of humoral immune responses. Here, we describe a protocol for measuring anti-fentanyl antibodies generated by the immunization of mice with novel opioid vaccine candidates. We describe steps for coating BSA-fentanyl antigen and standard wells. We then detail procedures for preparing buffers and performing assays. This protocol is adaptable for use with either BALB/c or C57BL/6J mice strains and can measure antibody isotypes to elucidate class switching. For complete details on the use and execution of this protocol, please refer to Stone et al.1 [Display omitted] •This protocol can determine antibody class switching using the indirect ELISA method•This protocol detects mouse anti-fentanyl antibodies using a fentanyl-protein conjugate•This protocol can test up to 40 mouse samples in duplicate per plate and isotype•The protocol has been optimized to allow for the use of either BALB/c or C57BL/6 mice Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Enzyme-linked immunosorbent assay (ELISA) offers an effective, inexpensive, and reliable approach for the analysis of humoral immune responses. Here, we describe a protocol for measuring anti-fentanyl antibodies generated by the immunization of mice with novel opioid vaccine candidates. We describe steps for coating BSA-fentanyl antigen and standard wells. We then detail procedures for preparing buffers and performing assays. This protocol is adaptable for use with either BALB/c or C57BL/6 mice strains and can measure antibody isotypes to elucidate class switching.