| Background and Objectives:AIDS has become a major public health and social problem.Antiviral therapy can inhibit viral replication and improve immune function.However,viral load and CD4+T lymphocytes should be detected regularly to evaluate the therapeutic effect.Traditional detection methods are based on different detection platforms,time-consuming and costly,while immunochromatography technology has the advantages of simple,rapid,easy to operate,and low cost,and widely used.This paper intends to develop a new method for the combined detection of HIV P24 antigen and CD4+T cell number in peripheral blood based on lateral immunochromatography technology,so as to provide a simple and affordable POCT detection method for the assessment of virus content changes and immune status in HIV infected persons.Methods:Based on the previous research results of the research group,this study was completed in two steps.First,the principle of double antibody sandwich was adopted,europium fluorescent microspheres were used as labeled probes,and relevant technological conditions were optimized to establish fluorescent immunochromatographic strips for quantitative detection of HIV P24 antigen,and the analytical performance was evaluated.Then,based on the dual-function detection principle of"sandwich" and"competition",EuNPs-anti-P24 polyclonal antibody signal probe and QDNPs-anti-CD4 monoclonal antibody were used as signal probes,The anti-P24 monoclonal antibody was coated on NC membrane as detection line 1,sheep anti-mouse IgG was coated on NC membrane as detection line 2,and chicken IgY was used as control line.Based on the research of the first part,the relevant experimental conditions were optimized.By detecting different concentrations of standard solution,standard curves were prepared to realize quantitative detection of P24Ag and CD4+T cell counts.This method was used to detect the amount of CD4+T cells in clinical whole blood samples,and the results were compared with flow cytometry,and the correlation and consistency of the two methods were analyzed.Results:1.The immunochromatographic test strips for quantitative detection of HIV P24 antigen had a good linear relationship in the range of 0-40 ng/mL(R2=0.997),the limit of blank was 1.5 ng/mL,the CV in the batch was less than 15%,and there was no cross reaction with TP、HBV、EB、HSV-2.Jaundice and hemolyzed samples had no significant interference with the results.Compared with CLIA method,the sensitivity of clinical serum sample results was 100%,the specificity was 87.5%,the total coincidence rate was 92.3%,and the consistency was good(Kappa=0.843).2.The immunochromatographic test strip for HIV P24 and CD4+T cells combined detection was developed and used for the detection of HIV P24 antigen standard solution,which had a good linear relationship in the range of 0-100 n g/mL(R2=0.9907),and the limit of blank was 8.7 ng/mL.When used for the detection of human CD4+T cell culture medium,it had a good linear relationship in the range of 0-700 cells/μL(R2=0.9839),and the limit of detection was 160 cells/μL.Eleven clinical whole blood samples were detected by immunochromatographic test strips,and the results showed a high correlation with FCM analysis(R2=0.9734,P<0.0001).Bland-Altman consistency analysis showed that the evaluation bias of the two methods was+29.06 cells/μL.The 95%confidence interval was-48.52 to+106.6 cells/μL.Conclusions:In this study,a new method for the detection of HIV P24 antigen and CD4+T cells counts was successfully established based on lateral immunochromatography technology.With excellent performance,it can meet clinical needs,and may become a quick,convenient and affordable POCT detection tool for dynamic monitoring of blood virus content changes and evaluating immune status of HIV infected persons during the antiviral therapy. |
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