Laboratory diagnosis and research progress of Lyme disease / 中华检验医学杂志

Lyme disease is a natural zoonotic disease transmitted by the bite of tick. The clinical manifestations of Lyme disease are complex and varied, and it is easy to be misdiagnosed. Based on the review of the background knowledge of Lyme disease, this paper reviews the current situation of its diagnosis and the research progress of detection.

Laboratory diagnosis and research progress of Lyme disease / 中华检验医学杂志

Lyme disease is a natural zoonotic disease transmitted by the bite of tick. The clinical manifestations of Lyme disease are complex and varied, and it is easy to be misdiagnosed. Based on the review of the background knowledge of Lyme disease, this paper reviews the current situation of its diagnosis and the research progress of detection.

Immuno-PCR Assay on Detection of HIV-1 p24 Antigen:A Primary Evaluation / 中华医院感染学杂志

OBJECTIVE To evaluate primarily the detection limit,specificity and reproducibility of immuno-PCR assay on HIV-1 p24 antigen.METHODS We p24 antigen were detected by established immuno-PCR system,and then the detection limit,specificity and reproducibility were discussed.We quantitatively analyzed the nonspecific amplified bands with fluorescence intensity(FI),and a preliminary determination of the lower specific amplification limit was made.RESULTS We taken(x-+3s)FI of nonspecific amplification as the lower limit of specific amplification signal.The detection limit of immuno-PCR assay was 0.1 ng/L.CONCLUSIONS The detection limit,specificity and reproducibility can meet the needs of HIV-1 p24 antigen detection.

Detection of Serum IgE Specific to Mite Allergens by Immuno-PCR

BACKGROUND: Although a skin test is the primary option for detecting allergen-specific IgE in clinics, the serum IgE immunoassay is also important because it allows for the diagnosis of allergy without any accompanying adverse effect on the patient. However, the low detection limit of IgE levels by immunoassay may restrict the use of the method in some occasions, and improving its sensitivity would thus have a significant implication in allergy-immunology clinics. METHODS: In this study, we attempted to detect specific serum IgE by using immuno-polymerase chain reaction (IPCR) which combines the antigen-antibody specificity of enzyme-linked immunosorbent assays (ELISAs) with the amplification power of PCR. RESULTS: Our results demonstrated that Blo t5-specific serum IgE can be detected by IPCR with a 100-fold higher sensitivity than ELISA, and cross-reactivity of serum IgE to other mite allergens is able to be analyzed by using only 0.3 microliter of serum sample. Use of real-time IPCR seemed to permit more convenient determination of specific serum IgE as well. CONCLUSION: We believe that IPCR can serve as a valuable tool in determining specific serum IgE, especially when the amount of serum sample is limited.

Detection of HIV-1 p24 by Immuno-PCR Assay Using Gold-magnetic Particles as Carriers / 中华医院感染学杂志

OBJECTIVE To establishan immuno-PCR assay with the carriers of gold-magnetic particles for detection of HIV-1 p24. METHODS The feasibility of using gold-magnetic particles as the carriers was verified. The gold-magnetic particles were coated with mouse anti-p24 monoclonal antibody as the capture antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound through streptavidin to biotinylated polyclonal antibody as the detection antibody. HIV-1 p24 sandwiched by two antibodies was detected by amplifying the reporter DNA using PCR. RESULTS The efficiency of gold-magnetic particles coated with mouse anti-p24 monoclonal antibody could reach up to 95%. Furthermore, the amount of antibodies immobilization was consistent among different batches of gold-magnetic particles and there was nearly without nonspecific adsorption. The detection limit of immuno-PCR assay was 0.1 ng/L, an approximately 1.5?104-fold higher compared with an enzyme-linked immunosorbent assay. The linear range of p24 concentration was 0.1-100 ng/L. CONCLUSIONS Gold-magnetic particle is one of the ideal immuno-PCR reaction carriers. The immuno-PCR for detection of HIV-1 p24 reported in this article is indicated to be a promising detection method.

Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen / 대한진단검사의학회지

BACKGROUND: Immuno-PCR has been known as a highly sensitive and specific method, yet no standardized protocol is available. We analyzed each step of immuno-PCR to develop a reliable standardized method. METHODS: We made a protocol modified from several methods reported previously, and performed immuno-PCR, but false positive reactions were noted. To reduce the false positivity, we investigated the buffer reagents and biotin-labelled oligo-nucleotide probe. Using a finally determined protocol, we compared the detection-limits of the immuno-PCR and ELISA methods. RESULTS: Streptavidin was identified as a main reagent causing a non-specific binding, thus it was replaced by neutravidin. The employment of CAS block as a dilution buffer for the biotin-labelled oligo-nucleotide probe and Casein block as a buffer for the detection antibodies resulted in a dramatic reduction in the false positive reactions. The standardized immuno-PCR detected angiogenin antigen at a concentration as low as 5 fg/mL, while an ELISA method detected 5 pg/mL. CONCLUSIONS: The immuno-PCR procedure newly described in this study was ultra-sensitive with no false positivity. This method can be utilized as an epochal tool for detection of a small amount of antigen which would not be discovered by ELISA method.

STUDY ON THE DETECTION OF TOXOPLASMACIRCULATING ANTIGENS USING IMMUNO-PCR / 中国人兽共患病学报

Aim to explore an assay for the early diagnosis of Toxoplasma infection. Methods Free streptavidin was used to attach a biotinylated DNA to the biotinylated second antibody, through amplication of the DNA label, antigen which bound with antibodies was detected, thus Toxoplasma circulating antigens Immuno-PCR assay was estabished. detecting the CAg in serial dilutions of serum and the dynamic variation of serum CAg of Toxoplasma in experimental infected mice using Immuno-PCR and ELISA parallely to compare the sensitivity Result The detection limit of the immuno-PCR was at about 1/1000 dilution, it was at 1/5 dilution for ELISA. CAg of Toxoplasma could be detected as early as the third day after infection using Immuno - pcr, Wheareas the ELISA on the same samples, revealed no detectable CAg elevations until fifth day after infection. Conclusion In comparison with CAgdetecting by ELISA parallelly performed, this method is more sensitive than ELISA, The detection limit of the immuno-PCR was an 200 fold improvement compared with a conventional ELISA. the positive result could be detected earlier than that of ELISA,It provide scientific basis for early diagnosis of Toxoplasmosis.

Detection of human recombinant HIV-p24 antigen by Immuno-polymerase chain reaction / 中国输血杂志

Objective To describe a highly sensitive immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human recombinant HIV-p24 antigen. Methods We used gold-magnetic particles as the carriers,mouse anti-p24 monoclonal antibody as the capture antibody and biotinylated goat anti-p24 polyclonal antibody as the detection antibody. The reporter DNA was initially generated by PCR amplification using a biotinylated primer,and was bound with streptavidin to biotinylated polyclonal antibody. Human recombinant p24 antigen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. The optimal concentration of sreptavidin and DNA label were determined using square titration. The electrophoresis gels were imaged and analyzed by Quantity One software. Results The optimal concentration of sreptavidin and DNA label were determined to be 0.1 mg/ L and 10 ng/ L,respectively. The detection limit of the immuno-PCR assay was 0.1 ng/L,higher than that of the conventional ELISA. Conclusion A highly sensitive immuno-PCR for human recombinant HIV-p24 antigen was indicated to be an available method for early screening of HIV infectors in blood donors.

Detection of growth hormone(GH) in urine with immuno-PCR / 中国免疫学杂志

Objective:To establish a highly sensitive method by immuno PCR for measurement of GH in Urine.Methods:Sandwich ELISA was operated in PCR tubes which treated with glutarakdehyde,and PCR was tested in the same PCR tube.The PCR production were electrophoresis in Agarose gel and Scan for gel.Results:The results show that ELISA could be correctly reacted in PCR tubes which treated with glutarakdehyde,more than 0.1 pg/ml GH could be detected.Results of Scan showed that the GH values in urine overnight have significantly deference between 30 normal children and 10 GHD patients which were diagnosed by GH provocative test.Conclusion:The sensitivity of Immuno PCR is 10 3～10 4 times higher than that of ELISA,Immuno PCR could be used in detection of GH in urine of children.