The Research Of Screening Of Aptamers To IgG Antibodies Purified From TB Patient Serum And Assessing Aptamers In TB Serological Diagnosis

Objective To screen tuberculosis （TB） IgG antibodies specific aptamers bySELEX (systematic evolution of ligands by exponential enrichment) and to evalue thecombinations of aptamers or aptamers binding to antigens in TB serologicaldiagnosis,which would be used in clinical TB serological diagnosis and to explore thepotential laboratory diagnosis value of this method.Methods IgG antibodies from TB patient serum were purified as targets of thescreening. A random DNA library was subjected to rounds of selection by SELEXmethod. Available ampters were cloned and sequenced. The affinity of the aptamers tothe antibodies was examined by biotin-streptavidin-horseradish peroxidase system.DNAMAN package was employed to analyze their sequences and second structures ofthe aptamers. Moreover, both aptamers and combining aptamers were tested in TBpatients (n=48) and health controls (n=40) by ELISA. Aptamer combined with antigenin different ratios were also tested in the same serum above. ELISA systems based onthe aptamer（s） and/or antigen were used to evaluate the potential diagnosis value ofthese methods by detecting clinical serum samples.Results IgG antibodies with the concentration1.8mg per ml were obtained.After12rounds of selection,4aptamers from12random selected aptamers showed highabsorbency to TB IgG antibodies (A>1.0). Pocket and stem-loops were the basicalstructure of aptamers binding to the antibodies by the analysis of structures. Fouraptamers were tested in88cases of clinical serum samples from TB and healthy controland the result showed that there were cross and complemental effects. Four aptamerscombined with each other and antigens both were tested in the same serumsamples.Detection systems based on N1and N6aptamer combinations had the bestcomplemental effect and the highest ratio of average value of TB group to control group.Aptamer and16KD antigen combinations in different ratios were also tested inthe same serum samples and the best ratio was half to half. Aptamer (N6) and/orantigen (16KD)were evaluated by88cases of clinical serum samples from TB patients(n=48), health controls (n=40). The differences of TB group and healthy group werestatistically significant (P <0.05) in all these three detection methods. The specificitieswere all100%,and the sensitivities were47.92%(based on N6aptamers),56.25%(based on16KD antigen),64.58%(based on the combination of aptamer and antigen),respectively.The χ2tests of the three detection methods all showed no statisticallysignificance (P>0.05). And aptamers (N1+N6) and/or antigen (16KD) which werepreliminarily evaluated by221cases of clinical serum samples from TB patients (n=48),healthy controls (n=40) and other non-TB patients (n=46) showed the same result.Conclusion A set of aptamers highly affinitive to TB IgG antibodies wassuccessfully selected and had considerably serological diagnosis value.Althoughaptamer and antigen had cross and complemental effects and the sensitivity ofcombining detection was higher, which meaned the method of combining aptamer andantigen was feasible, the differences among the three detection methods weren’tstatistically significant.