Laboratory Study Of HIV-1 P24 By Immuno-PCR

Objectives:To establish a sandwich immuno-PCR assay using golden-magnetic particles as the carrier and streptavidin-biotin system as the bridge system to laboratory detection of HIV-1 p24 antigen. Possible factors affecting immuno-PCR sensitivity and specificity will be optimized. We also discuss the sensitivity, specificity, reproducibility and how to process non-specificity signals in p24 detection. Finally we compare the sensitivity between immuno-PCR and ELISA and explore the possibility of detecting clinic samples.Methods:1. The marker DNA is a fragment of cinnamoyl coenzyme A reucing enzyme gene of Brassica napus L., and the cloning vector is plasmid pMD18-T. The plasmid was transferred into Escherichia coli DH5αto preserve. Plasmid DNA of recombinant bacteria, which is the amplification template, was extracted by thermal cracking method using TE Buffer (pH8.0).2. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and the PCR reaction conditions was optimized for high efficiency and specificity.3. Coating golden-magnetic particles with mouse anti-p24 monoclonal antibody, which is the capture antibody. The reporter DNA was bound through streptavidin to biotinylated goat anti-p24 polyclonal antibody, which is the detection antibody. HIV-1 p24 antigen sandwiched by two antibodies was detected by amplifying the reporter DNA using PCR.4. Optimizing the detection conditions of HIV-1 p24 system such as streptavidin concentration, reporter DNA concentration, sealant ingredients, washing buffer ingredients and washing times.5. The sensitivity, detection limit, specificity and reproducibility of immuno-PCR detecting HIV-1 p24 were discussed. We quantified the amplification bands through fluorescence intensity and determined the assessment standard of lower limit of specificity amplification. We also compared the difference of detection sensitivity between immuno-PCR and traditional ELISA method. Results:1. We have established sandwich immuno-PCR technique for HIV-1 p24 detection, which using golden-magnetic particles as carrier, streptavidin-biotin system as bridge system and gel electrophoresis as detection method for reporter DNA.2. The final concentrations of reagents in PCR reactions of reporter DNA preparing are as follows: primers: P1 0.8μmol/L, P2 0.8μmol/L, MgCl2: 2.5mmol/L, dNTPs(each): 0.25mmol/L, Taq DNA polymerase: 2.5×104U/L, template: 1/25 of total volume per PCR reaction. And we start the PCR cycles according to the following schemes: denaturation - 94℃, annealing - 55℃, extension - 72℃, repeat cycles 35 times.3. The efficiency of golden-magnetic particles coated with mouse anti-p24 monoclonal antibody can reach up to 95%. Furthermore, the amount of antibodies immobilization is consistent among different batches of golden-magnetic particles and there is nearly without nonspecific adsorption. And we can make the separation and washing steps simpler and more thoroughly.4. The capture antibody concentration is chosen as 1μg/ml and detection antibody concentration is 0.8μg/ml. The optimized detection conditions of immuno-PCR are as follows: streptavidin concentration is 0.1mg/L, reporter DNA concentration is 10ng/L, washing buffer PBST contained 150mmol/L NaCl is better and washing times should be more than 8 times. The established immuno-PCR system can detect low concentration of HIV-1 p24.5. We recommend the fluorescence intensity higher than 17741 be taken as the lower limit of specific amplification (positive signal). The HIV-1 p24 detection limit of immuno-PCR is 0.1ng/L, while the detection limit of ELISA is 1.5μg/L. The sensitivity of immuno-PCR is 1.5×104 times higher than that of ELISA. And the immuno-PCR detection system has good specificity and reproducibility.Conclusions:1. We have established a simple sandwich immuno-PCR detection system with golden-magnetic particle carrier, biotin-streptavidin bridge system, biotinylated dsDNA reporter, and gel electrophoresis detection system, and the immuno-PCR system can be applied in standardized laboratory.2. The method of using TE Buffer to extract recombinant bacteria plasmid DNA is quick and simple, and the DNA template can be kept in frozen for longer time. The optimized PCR conditions have improved amplification efficiency. And the conditions are not only the preparation conditions of reporter DNA but also the reporter DNA amplification conditions in HIV-1 p24 antigen detecting in immuno-PCR.3. Golden-magnetic particle has high immunological sensitivity and faster reaction rate. The amount of antibodies immobilization is consistent among different batches of golden-magnetic particles. And it can make the separation and washing steps simpler and more thoroughly. The golden-magnetic particle is one of the ideal immuno-PCR reaction carriers.4. In immuno-PCR detection, many factors can affect detection sensitivity, specificity and reproducibility, such as streptavidin concentration, reporter DNA concentration, sealant ingredients, washing buffer ingredients and washing times. We should investigate the best reaction conditions for certain target protein.5. We have realized detecting low concentration of HIV-1 p24 through established immuno-PCR system. It has high sensitivity and has good specificity and reproducibility. It can initially satisfy HIV p24 antigen detection demand and it is promising to be an available detection method for HIV p24 in serum.