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Treponema (TP) antibody detection method and detection kit thereof

A technology of antibodies and antigens, applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve excellent detection specificity, improve sensitivity and specificity, and improve specificity

Inactive Publication Date: 2013-06-26
PERKINELMER MEDICAL DIAGNOSTICS PROD SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the detection of TP antibody in human serum using the double-antigen sandwich method using small molecules as indirect labels to detect TP antibodies at home and abroad.

Method used

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  • Treponema (TP) antibody detection method and detection kit thereof
  • Treponema (TP) antibody detection method and detection kit thereof
  • Treponema (TP) antibody detection method and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of biotin-labeled TP antigen

[0057] Proceed as follows:

[0058] (1) Dialyze the purchased TP recombinant antigens (N15, N17 and N47) against pH 7.4 100mMPBS at room temperature (20-25°C) for 24 hours, and change the medium twice, 2000mL each time. Adjust the concentration to about 1 mg / mL.

[0059] (2) Take the required amount of Biotin-cap-NHS, dissolve it in dimethylformamide (DMF) to 10 mg / mL, and immediately add it to the dialyzer according to the mass ratio of Biotin-cap-NHS to antigen of 1:2 After the TP recombinant antigen solution, mix quickly. Let stand at room temperature (20-25°C) for 1 hour.

[0060] (3) At room temperature (20-25°C) against pH 7.8, 50mM TSA [50mM Tris, containing 150mM NaCl, 1% (mass / volume) BSA, 16% (mass / volume) sucrose] fully stirred and dialyzed for 24 hours, replaced Liquid 2 times, 1000mL each time. Purified biotin-labeled TP antigen was obtained.

Embodiment 2E

[0061] Embodiment 2 Preparation of Eu-labeled streptavidin

[0062] Proceed as follows:

[0063] (1) Dissolve 1 mg of streptavidin in 2000 μL of pure water, and adjust pH 9.5 to 100 mM CBS (100 mM Na 2 CO 3 -NaHCO 3 Buffer (containing 150mM NaCl) was dialyzed for 24 hours, and the solution was changed twice, 2000mL each time.

[0064] (2) Take 1mg Eu-DTTA and dissolve it with 1000μL pH 9.5 100mM CBS. Add the completely dissolved Eu-DTTA into the dialyzed streptavidin solution (about 1 mg / mL), shake while adding, and let stand at room temperature (20-25°C) for 72 hours. The mass ratio of Eu-DTTA to streptavidin is about 1:1.

[0065] (3) Separate Eu-SA (Eu 3+ -labeled streptavidin) with free Eu-DTTA, eluted with pH 7.8 50mM TSA. The separation process was monitored by a nucleic acid and protein detector. to obtain purified Eu 3+ labeled streptavidin. Marking efficiency is an indicator for detecting the marking process, and the specific calculation method is as follows...

Embodiment 3

[0075] Example 3 Preparation of TP antigen-coated microwell plate

[0076] Proceed as follows:

[0077] (1) Pipette an appropriate amount of TP recombinant antigen into the required volume of coating buffer (50mM TSA buffer, pH 7.8), mix well to avoid the generation of air bubbles, and prepare TP antigen with a final concentration of 2-8μg / ml Coating solution;

[0078] (2) Add 100 μL of TP antigen coating solution to each well of the microwell plate, and let stand at room temperature (20-25°C) for 24 hours;

[0079] (3) Wash twice with 50mM pH7.8TSA buffer containing 0.1% Tween-20; inject blocking buffer [containing 2% (mass / volume) casein, 0.1% (volume / volume) Tween-20 50mM pH7.8TSA buffer solution] 300μL, stand overnight at room temperature (20-25℃) for about 16 hours;

[0080] (4) Blot up the blocking buffer, put the plate into a spin dryer and dry it for 5 minutes; turn on the freeze dryer to make the vacuum degree <50Pa, keep it for 3 hours, stop the machine, put the d...

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Abstract

The invention discloses a treponema (TP) antibody detection method and a detection kit of the TP antibody detection method. The detection method comprises the following steps of: (a) marking a first TP antigen by a small molecular substance; (b) coating a solid phase material by a second TP antigen; (c) marking an anti-small-molecular substance antibody or other substances capable of being combined with a small molecule by a signal generating substance, and then preparing a signal reporter molecule; (d) carrying out immune reaction between the second TP antigen on the surface of the solid phase material, the first TP antigen marked by the small molecular substance and the TP antibody in a sample, and then carrying out conjugation reaction between the signal reporter molecule and the small molecular substance, thus forming a detachable signal generation substance for detecting the content of the TP antibodies in the sample. The detection kit comprises (a) the first TP antigen marked by the small molecular substance, (b) the solid phase material coated with the second TP antigen, and (c) the signal reporter molecule. By adopting the TP antibody detection method and the detection kit of the TP antibody detection method, the sensitivity and specificity in the detection of the TP antibody can be effectively improved.

Description

technical field [0001] The invention relates to a Treponema pallidum (TP) antibody detection kit and a detection method thereof, in particular to a method for detecting Treponema pallidum antibody by a small-molecule indirect label-based double-antigen sandwich method and a corresponding detection kit. Background technique [0002] Syphilis, tuberculosis, and leprosy are listed as the three major chronic infectious diseases in the world. They are mainly infected through sexual behavior, and may also be infected through blood transfusion. When a woman is infected with syphilis during pregnancy, it will be transmitted to the fetus through the placenta, resulting in congenital syphilis. Syphilis is caused by the pathogen Treponema pallidum. It is not easy to prove that you are infected with syphilis, because there are no clinical symptoms, and the incubation period is 10 to 90 days. You can only rely on the detection of syphilis serum to help diagnose. At present, the markers ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/571
Inventor 张小寒杨挥
Owner PERKINELMER MEDICAL DIAGNOSTICS PROD SHANGHAI
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