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Anti-flavin antibodies.

ABSTRACT: Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.

SUBMITTER: Barber MJ

PROVIDER: S-EPMC1147668 | biostudies-other | 1987 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundAnti-drug antibodies (ADAs) to bococizumab were detected in > 40% of subjects in the SPIRE lipid-lowering trials. The risk of cross-reactivity between anti-bococizumab antibodies and other approved anti-proprotein convertase subtilisin/kexin type-9 (PCSK9) monoclonal antibodies (mAbs) was investigated using a single-assay approach.MethodsBococizumab immunogenicity was assessed in SPIRE-HR, a 52-week study. The highest ADA titer sample from each ADA-positive subject (n = 155) was tested in vitro for cross-reactivity to alirocumab and evolocumab using a novel ADA assay approach. Additional specificity tiers within the bococizumab ADA assay against each drug were validated using recombinant PCSK9 as a surrogate cross-reactive positive control. If the highest ADA titer sample showed cross-reactivity, additional samples from that subject were analyzed. Cross-reactivity was determined by the ability of alirocumab or evolocumab to inhibit the sample signal greater than or equal to the cross-reactivity cut-points.ResultsADAs were detected in 44.0% (155/352) of bococizumab-treated subjects, and 27.0% also developed neutralizing antibodies (NAbs). Median ADA and NAb titers ranged from 276 to 526 and 8 to 12 over the course of the study, respectively. From 155 ADA-positive subjects tested for cross-reactivity, one (0.6%) subject showed weak cross-reactivity to both alirocumab and evolocumab. This cross-reactivity signal was transient (from Days 337 to 373) and undetectable at the last ADA-positive timepoint (Day 407).ConclusionA novel, single-assay approach was validated to assess the potential cross-reactivity of anti-bococizumab antibodies to alirocumab and evolocumab. In subjects who developed ADAs to bococizumab, the likelihood of clinically relevant cross-reactivity to marketed anti-PCSK9 mAbs is remote, based on the low frequency of cross-reactivity observed, which was weak in signal inhibition and transient in nature.Clinical trial registrationThe SPIRE-HR study is registered on ClinicalTrials.gov under the identifier NCT01968954.

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Anti-flavin antibodies.

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