Development of monoclonal antibodies and simplified sandwich ELISA for canine neutrophil gelatinase-associated lipocalin A detection

Abstract

Abstract Background Acute kidney injury (AKI) is one of the most common canine diseases with a high mortality rate. Neutrophil gelatinase-associated lipocalin (NGAL) is the novel biomarker for early diagnosis of renal injury. Only few sandwich ELISA kits are commercially available, all of which require the use of expensive enzyme-conjugated secondary or biotinylated antibody. The aims of this study were to develop high affinity monoclonal antibodies (mAbs) and simplified sandwich ELISA for canine NGAL detection. Results Recombinant canine NGAL was expressed in E. coli and purified to a high purity. Six hybridoma cell lines were generated by immunization of mice with the purified protein, all of which secreted high titers of specific mAbs. By screening 36 different antibody combinations, a pair of mAbs with high additivity and P/N ratio was selected as the capture and detection antibodies. By conjugation of the detection mAb with horse radish peroxidase, a simplified sandwich ELISA was developed with a correlation coefficient of 0.9939, detection limit of 8.28 ng/mL. The parallel test of 42 samples from healthy and AKI dogs showed the good agreement in NGAL concentrations detected by the simplified sandwich ELISA and commercial ELISA kit. Conclusions We developed canine NGAL-specific monoclonal antibodies and simplified sandwich ELISA. The simplified sandwich ELISA could replace the commercial ELISA kits for canine NGAL detection with the advantages of reduced cost and detection time.