| BackgroundDetection of anti-HIV specific antibodies, are common method of HIV diagnosis. But antibodies appear later than nucleic acid and HIV antigen after HIV infection. So HIV antibodies can not be detected on the earlyier stage of disease, while HIV-1p24antigen and nucleic acid are major biomarkers. Detection of HIV nucleic acid is great sensitive and specific, but also limited by the high variation, high quality of personnel instruments and technology. Detection of HIV-1core protein, p24antigen, shows advantagement to some degree and has been paid more attention. It has been used for early diagnosis of HIV-1infection, blood screening, monitor of disease process and evaluation of curative efficacy. HIV-1p24antigen is most detected by an enzyme-linked immunosorbent assay (ELISA), which is simple and mature to operate, high degree of automation, and has been widely used in HIV laboratory. But its low sensitivity is unable to meet the requirements of early diagnosis and trace detection.With the rapid development of nano-materials and technologies, Nanoparticle-based Bio-barcode Amplification (BCA) assay to detect trace protein with high sensitivity received widespread concern. BCA is based on two kinds of probes, one is the magnetic microsphere probes (MMPs), magnetic microspheres coated with a recognition element of the target protein. The other one is gold nanoparticle probes (GNPs), gold nanoparticles dually modified with another recognition element and signal identification oligonucleotides, bio-barcode DNA. Recognition elements on the two probes and the target protein form sandwich complexes,Then identification of bio-barcode DNA on the complexes reflects the level of the target protein. BCA combines DNA markers with the target protein, and amplifies signals during the protein identification process, and completes protein quantitation through the identification of the marker DNA. So it can build a new platform for high-sensitivity detection of biological markers and can be widely applied into biomolecular detection, surveillance of infectious diseases, early detection and monitoring of cancer.Here we tried building two types of BCA assay, based on magnetic microspheres and microplates, combined with PCR to detect HIV-1p24antigen, and reach a high sensitivity.Objective1. Monocolonal antibodies against HIV-1p24antigen were selected and screened for paired matching, which were used to construct a sandwich ELISA to detect HIV-1p24antigen and determine the detection sensitivity.2. Based on the screened paired antibodies, the initial establishment of nanoparticle-based BCA assay for detection of HIV-1p24antigen was performed, and PCR technique was introduced into the terminal detection for bio-barcode DNA, in order to significantly improve the sensitivity of HIV-1p24antigen detection.3. Try replacing the traditional magnetic microspheres with microplates to capture target p24antigen, establishing a new BCA assay to detect HIV-1p24antigen. And provide the technology platform for developing an effective way for early diagnosis of HIV infection.Methods1. Anti-HIV-1p24antibodies were screened according to the principle of paired monoclonal antibodies with sandwich ELISA, among which capture antibody and HRP modified detection antibody were determined by direct and indirect ELISA.2. Sandwich ELISA was built to detect HIV-1p24antigen based on the selected paired antibodies. Then the key technical conditions were explored and detection process was determined, and the sensitivity and detection range were ultimately evaluated.3. Bio-barcode DNA was a DNA fragment of47bp selected from the Arabidopsis genome, and capture DNA was disigned by modification with thiol group at5’end of the complement sequence of bio-barcode DNA. Primers were designed and optimal conditions for PCR and SYBR Green real-time PCR amplification of bio-barcide DNA were explored.4. Gold nanoparticles were conjugated with the detecting anti-HIV-1p24antibody, and double-stranded DNA formed by hybridization of bio-barcode DNA and capture DNA were connected to the surface of the gold nanoparticles through the thiol group, then the GNPs were made.5. Magnetic probe was made by magnetic microspheres modified with capturing anti-HIV-1p24antibody, which capture the target HPV-1p24antigen. Then the GNPs combined with them and formed sandwich complexes, on the surface of which the bio-carcode DNA were released by heating. The liberated bio-barcode DNA were determined by PCR and4%agarose gel electrophoresis analysis as the detection signal. Then sensitivity and detection range were determined and compared with ELISA.6. Capturing anti-HIV-1p24antibody1G12coated in the microplates seized target HIV-1p24antigen in PBS or viral culture supernatant, and combined with GNPs to form sandwich complexes, on which bio-barcode DNA were dissociated by heating as the detection signal. Then PCR and4%agarose gel electrophoresis analysis and SYBR Green real-time PCR were performed to determine the bio-barcode DNA, and the amounts of p24antigen were identified. Finally, comparison of sensitivity and the linear detection range were done between the new methods and ELISAResults1. Anti-HIV-1p24antibodies,1D4and1G12, were screened and used into BCA assay as detecting antibody and capturing antibody in detection of HIV-1p24antigen respectively.2. Sandwich ELISA was establised based on screened paired anti-HIV-1p24antibodies to detect HIV-1p24antigen. The limit of detection was1000pg/ml and the linear detection range was from3000to100,000pg/ml.3. Traditional BCA based on magnetic microspheres combined with PCR gel electrophoresis analysis was constructed to detect HIV-1p24antigen. There is good correlation between the intensity of PCR products and the concentration of target p24antigen. The DNA amounts of PCR products were weakening with the reducing quantity of HIV-1p24antigen, The limit of detection can be reached down to0.1pg/ml and exhibited a wide linear dose-dependent pattern within the detection range from0.1to1000pg/mL4. New BCA based on microplates combined with PCR gel electrophoresis analysis was constructed to detect HIV-1p24antigen. The limit of detection can be reached down to1pg/ml and exhibited a wide linear dose-dependent pattern within the detection range from1to10,000pg/mL. SYBR Green real-time PCR was used to terminal detection, CT value decreased with the concentration increasing of p24antigen to be tested, It showed a good linear relationship between the p24antigen concentration and CT value, ranging from0.1pg/ml to1000pg/ml.5. New BCA based on microplates can be used to detect HIV-1p24antigen in viral culture supernatant, and the sensitivity was7.9pg/ml. Negtive results were found for7HIV-1negtive serum by this new BCA assay. Conclusions1. Anti-HIV-1p24monoclonal antibodies,1G12and1D4, can be used in the BCA assay for detection of HIV-1p24antigen.2. Traditional BCA based on magnetic microspheres combined with PCR gel electrophoresis analysis can be used to detect HIV-1p24antigen. The sensitivity can be improved by4orders of magnitude than that of ELISA, and the linear detection range can be widen by2orders of magnitude than that of ELISA.3. The new BCA based on microplates combined with PCR gel electrophoresis analysis showed great advantage of simplicity and operability for detection of HIV-1p24antigen. The sensitivity and linear detection range can be improved by3and2orders of magnitude than those of ELISA.4. The new BCA assay based on microplates can be used to detect HIV-1viral culture supernatant, and it showed a good Analytical sensitivity and specificity,... |
PDF Full Text Request