Improved double-antigen sandwiching immunity detection method
A double-antigen sandwich and immunoassay technology, applied in the field of immunoassay, can solve the problems of high false positive rate, reduced sensitivity, high background, etc., and achieve the effect of improving specificity, reducing background and false positive rate
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Embodiment 1
[0029] Example 1 HCV double antigen sandwich method gold-labeled test strip with anti-human IgG polyclonal antibody added to the sample pad
[0030] 1.1 Preparation of HCV coating antigen
[0031]PCR amplifies the NS3 part of the hepatitis C virus gene as the DNA segment corresponding to SEQ ID NO.1 (part of the NS3 protein), and its upstream primer is as shown in SEQ ID NO.2 with a BamHI site, and downstream primers As shown in SEQ ID NO. 3, with HindIII site. After the PCR fragment was recovered, it was digested with BamHI and HindIII, and then ligated into the PQE30 vector (Qiagen) digested with BamHI and HindIII to obtain the positive recombinant plasmid PQE30-NS3. The plasmid PQE30-NS3 was transformed into ER2566 bacteria (New England Biolabs, USA), and cultured with 500ml LB medium containing 100ug / ml ampicillin sodium (Shanghai Shenggong Bioengineering Technology Service Co., Ltd., catalog number A0339) at 37°C with shaking to OD600= About 1.0, IPTG (Shanghai Shenggong Biol...
Embodiment 2
[0058] Example 2 HCV double antigen sandwich method gold-labeled test strip with anti-human IgG polyclonal antibody added to the marker
[0059] Refer to Examples 1.1, 1.2, 1.3, and 1.4 to prepare HCV coated antigen, HCV labeled antigen, colloidal gold, and labeled antigen-colloidal gold complex.
[0060] 2.5 Assembly of test strips
[0061] Dilute the mouse anti-human IgG polyclonal antibody with PBS buffer to 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0 mg / ml, and soak glass fibers (Watman), -50 Store at 4℃ after freeze-drying in vacuum for 6h. PBS without mouse anti-human IgG polyclonal antibody was used as a control.
[0062] The labeled antigen-colloidal gold complex was diluted 10 times with the colloidal gold diluent and then 0.45μl / mm 2 Add it to the glass fiber treated with mouse anti-human IgG polyclonal antibody, freeze-dry it in vacuum at -50°C for 5 hours, and store it at 4°C to make a marker pad.
[0063] Dilute the HCV coating antigen PQE-NS3 and the contro...
Embodiment 3
[0077] Example 3 HCV double antigen sandwich method gold-labeled test strip with anti-human IgG monoclonal antibody added to the sample pad or the marker pad
[0078] Refer to Examples 1 and 2 to prepare HCV double-antigen sandwich gold-labeled test strips with sample pads or marker pads with anti-human IgG monoclonal antibodies. Finally, similar results to Examples 1.7 and 2.7 were obtained, which shows that , Adding anti-human IgG monoclonal antibody or polyclonal antibody to the sample pad or marker pad can improve the specificity of gold-labeled test strip detection.
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