Screening Of High Affinity Monoclonal Antibodies By Bio-layer Interferometry And Its Application On Immunochromatographic Colloidal-gold Test Strips

Legionella pneumophila(Lp)is an important respiratory pathogen that can infect humans.At present,the traditional detection methods for pathogenic have the characteristics of long operation time,delayed results,strong professional operation,and no bedside diagnosis,which are greatly limited in practical applications.In this study,a pair of Hybridoma cell lines that efficiently secreted monoclonal antibodies against pathogen-specific protein PAL were screened rapidly and accurately by Bio-layer Interferometry(BLI).Based on these antibodies,a immunochromatographic colloidal-gold test strip assay was successfully established for rapid,sensitive and accurate detection of Legionella pneumophila antigens.In this study,the gene encoding the out membrane protein PAL was cloned,expressed in E.coli.and the recombinant protein was purified.The 6-8 week old BALB/c female mices were immunized multiple times by the purified artificial antigen(Lp-PAL),then cell fusion was performed by chemical methods and the hybridoma cells secreting specific antibodies were screened by BLI technology and indirect ELISA.The MAbs were then prepared from mouse ascites after inoculating the hybridoma cells.The spot crossing tests were used to verify the effectiveness of these antibodies.The purified monoclonal antibodies were analyzed for its specificity and sensitivity by indirect ELISA,Western blot and BLI.The results of spot crossing tests showed that three antibodies were positive by BLI,and only three of the eleven antibodies screened by indirect ELISA showed positive reaction,indicating that BLI screening method is more accurate and persuasive than traditional indirect ELISA.Two cell lines(1G10B2G1 and 1G10B2F5)screened by BLI technology were used to prepare monoclonal antibodies(Lp-7 and Lp-26).The titer of antibodies detected by indirect ELISA reached 1:409600 at less.It is verified that the paired antibodies recognize different antigenic epitopes of the same antigen,using the in-Tandem assay and Classical sandwich assay in the BLI technology platform,which means,there is no competitive effect between the two antibodies,and there is no spatial binding steric effect.According to the screening results of BLI technology,the antibody(Lp-26)with fast binding rate was preferentially selected as capture antibody,on a immunochromatographic colloidal-gold test strip platform,wherein the antibody Lp-26was used as the capture antibody and antibody Lp-7 was used as the gold-labeled antibody for the test strips,respectively.The minimum detection limit of the test strips prepared was 1×10~7 CFU/mL.The strip made of this pair of antibodies specifically recognized Legionella pneumophila and did not react with ten of other common respiratory pathogens such as Streptococcus pneumoniae and Moraxella catarrhali et.al.Compared with the traditional ELISA detection method,BLI technology is more beneficial to the rapid screening of antibodies with strong binding force for the preparation of immunochromatographic colloidal-gold test strips,which is conducive to the rapid detection of pathogenic bacteria infections.