Preparation Of Fluorescent Antibodies Against Lipoarabinomannan From Mycobacterium Tuberculosis

Objective To prepare the fluorescent antibody against lipoarabinomannan(LAM)from Mycobacterium tuberculosis(Mtb)and discuss its application value in sputum smear microscopy.Methods After subcutaneous immunization of rabbits with LAM,an enzyme-linked immunosorbent assay(ELISA)was established to detect antibody titers.Rabbit blood were collected from the heart to obtain sufficient high titer rabbit serum.The rabbit serum was sequentially extracted by salting-out method,and the antibody was purified by G protein affinity chromategraphy.The purification effect was identified by SDS-PAGE.After dialysis and desalting,the concentration was determined.Then coupling the antibody by fluorescein isothiocyanate(FITC),and calculate the F/P ratio of the conjugate.Results Successful 6 subcutaneous immunizations of rabbits with LAM,and constructed an ELISA method to detect the antibody titer.The antibody titer reached 1:51200.Blood was successfully collected from the heart and about 100 mL of rabbit serum containing anti-LAM antibodies was obtained.The salting-out method was successfully completed,antibodies were purified by G protein affinity chromatography,and identified by SDS-PAGE,and high-purity polyclonal IgG was obtained.The antibody was concentrated after desalting,and the antibody concentration was 7.26 mg/mL by BCA method.After FITC coupling,a fluorescent antibody was prepared,and the F/P value was measured at 2.7.Conclusion Fluorescent antibody was successfully prepared,which laid a foundation for the establishment of direct immunofluorescence method to detect Mycobacterium tuberculosis in sputum.