Production of Polyclonal Antibodies and Development of Competitive ELISA for Quantification of the Lantibiotic Paenibacillin

Abstract

The discovery and biotechnological application of new antimicrobial peptides are impeded by a lack of sensitive methods for peptide quantification. Paenibacillin is an emerging antimicrobial lantibiotic that was discovered in Paenibacillus polymyxa OSY-DF ATCC PTA-7852, isolated from the fermented vegetable Kimchee. This lantibiotic has potency against many foodborne pathogenic and spoilage bacteria. To advance the research and application of paenibacillin, a rapid, specific, and sensitive detection and quantification immunoassay was developed. After anti-paenibacillin polyclonal antibodies (pAbs) were generated and purified, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and optimized for paenibacillin quantification. The dynamic range of the cELISA was determined by using a three-parameter nonlinear regression model, achieving a correlation (R2) value of 0.95. The cELISA displayed high sensitivity, with the ability to detect paenibacillin at levels as low as 15.6 ng/mL, which is significantly lower than the limit of detection of the conventional antimicrobial assay (20 µg/mL paenibacillin). The cELISA successfully differentiated paenibacillin concentrations in cell-free crude supernatants of P. polymyxa wild type and its mutant strain when grown at 30 °C and 37 °C; higher paenibacillin levels were found in the mutant (0.248–0.276 µg/mL) than in the wild type (0.122–0.212 µg/mL) culture. These findings were validated by the transcriptional analysis of 11 paenibacillin biosynthetic genes, which were significantly upregulated (≥2-fold increase) in the mutant compared with the wild strain. Additionally, the cELISA exhibited high sensitivity by recovery of paenibacillin titers spiked at 2.5 and 10 µg/mL in de Man, Rogosa, and Sharpe (MRS) broth and diluted skim milk. These results suggest that the anti-paenibacillin pAbs and the developed cELISA could be valuable in quantifying paenibacillin in complex matrices and in aiding the discovery of paenibacillin-producing natural microbiota.