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POCT fluorescence immunochromatography quantitative kit and application thereof

A technology of fluorescent immunochromatography and fluorescent microspheres, which is applied in measuring devices, analytical materials, biological tests, etc., can solve the problem that the screening and diagnosis results of cervical cancer and precancerous lesions are not accurate enough, and cannot accurately reflect the actual situation of HPV16 E7 protein. Concentration, cell number and other parameters are difficult to achieve the effect of low false negative, high positive detection rate and improved accuracy

Active Publication Date: 2018-08-03
河南省生物工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the collection of human cervical exfoliated cell samples is different from homogeneous samples such as blood and urine. Due to the problem of the collection operation method, it is difficult to quantitatively control the parameters such as the number of cells in the collected sample. The concentration of HPV16 type E7 protein in the lysate does not accurately reflect the actual concentration of HPV16 type E7 protein in the unit tissue or cell. The concentration of type E7 protein can cause inaccurate screening and diagnosis of cervical cancer and precancerous lesions
On the other hand, the POCT fluorescent quantitative detection kit disclosed in the above literature only detects a single indicator (the concentration of HPV16 E7 protein), and high-risk HPV16 and 18 persistent infection is the primary factor leading to cervical cancer. Only HPV16 Type E7 protein will miss some cervical cancer patients caused by HPV18 infection

Method used

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  • POCT fluorescence immunochromatography quantitative kit and application thereof
  • POCT fluorescence immunochromatography quantitative kit and application thereof
  • POCT fluorescence immunochromatography quantitative kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Preparation of immunochromatographic test strips coated with HPV16 type E6 antibody and HPV18 type E6 antibody

[0064] Prepare as follows:

[0065] HPV16 type E6 antibody, HPV18 type E6 antibody, mouse anti-β-Actin protein monoclonal antibody and goat anti-rabbit polyclonal antibody were coated on nitrocellulose membrane (NC membrane) using Gold Bio three-dimensional dot film gold spraying instrument , respectively used as two detection lines (T1 line, T2 line), internal reference line (A line) and quality control line (C line), dried in a 37 ℃ drying oven for 2 hours; at the same time, the fluorescent microspheres coupled with HPV16 type E6 antibody, HPV18 type E6 antibody, anti-β-Actin protein monoclonal antibody and goat anti-rabbit IgG were sprayed on the binding pad respectively, and dried in vacuum at 37°C for 2 hours; the NC membrane, binding pad, sample pad, water-absorbing pad and After the PVC sticky bottom plate is assembled, cut it into test str...

Embodiment 2

[0066] Embodiment 2: Preparation of human cervical exfoliated cell lysate

[0067] Prepare 1% Triton100 with 100mM Tris-HCL solution, and add protease inhibitor PMSF at a final concentration of 1mM before use.

Embodiment 3

[0068] Example 3: Preparation of POCT fluorescence immunochromatography quantitative detection kit for joint detection of HPV16 / 18 type E6 protein

[0069] The test kit contains:

[0070] ① The immunochromatographic test strip prepared in Example 1 coated with HPV16 E6 antibody, HPV18 E6 antibody, anti-β-Actin protein monoclonal antibody and goat anti-rabbit polyclonal antibody.

[0071] ② HPV16 type E6 protein calibrator 0, 1, 2, 3, 4, 5, 6, 7, 8: calibrator is freeze-dried powder, add 1mlddH20 to dissolve respectively, the linear concentration of calibrator solution is 0, 1, 2, 5, 20, 40, 80, 160, 200ng / ml;

[0072] HPV18 type E6 protein calibrator 0, 1, 2, 3, 4, 5, 6, 7, 8: calibrator is freeze-dried powder, add 1ml ddH20 to dissolve respectively, the linear concentration of calibrator solution is 0, 1, 2 respectively , 5, 20, 40, 80, 160, 200ng / ml;

[0073] β-Actin protein calibrator 0, 1, 2, 3, 4, 5, 6, 7: the calibrator is freeze-dried powder, add 1ml ddH20 to dissolv...

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Abstract

The invention relates to a POCT fluorescence immunochromatography quantitative test strip and kit. The test strip comprises a base plate, a sample addition area, a sample pad, a bonding pad and a water absorbing pad. A nitrocellulose membrane is arranged between the bonding pad and the water absorbing pad; the bonding pad is sprayed with a fluorescent microsphere labeled antibody. The test strip is characterized in that the nitrocellulose membrane is coated with a to-be-detected protein antibody, an anti-beta-Actin protein monoclonal antibody and a sheep anti rabbit polyclonal antibody. The concentration of a beta-Actin protein constantly expressed in cells is used as an internal reference to eliminate the influence of the number of cells in a sampling sample on the corresponding protein concentration in a lysate, so that the accuracy of the detection result is improved.

Description

technical field [0001] The invention belongs to the technical field of cancer diagnosis, and in particular relates to a POCT fluorescence immunochromatography quantitative kit, in particular to a POCT fluorescence immunochromatography quantitative kit combined with quantitative detection of HPV16 / 18 type E6 protein and an application thereof. Background technique [0002] Fluorescence immunochromatography technology combines the two advanced technologies of fluorescence immunology technology and immunochromatography technology. It is based on immune labeling technology. Detect the marker to reflect the situation of the antigen, so that the content of the tested antigen or antibody can be detected indirectly. Fluorescence immunochromatography technology supports the detection of whole blood, serum, plasma, urine, cells and other samples, and is widely used in cardiology, laboratory, emergency, ICU, oncology, nephrology, pediatrics, endocrinology, gynecology, geriatrics, respi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/574G01N33/533
CPCG01N33/533G01N33/57411G01N33/57484G01N33/577G01N33/6893
Inventor 王云龙石英杰王继创李玉林
Owner 河南省生物工程技术研究中心
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