Treponema pallidum specific antibody chemical light emitting detection kit and preparation method thereof

A technology for chemiluminescence detection and treponema pallidum, which is applied in the direction of chemiluminescence/bioluminescence, analysis by making materials undergo chemical reactions, and measurement devices, can solve the problems of inability to culture Treponema pallidum in vitro, high false positive rate of cardiolipin antibodies, inspection The positive rate is not high, and the detection time is shortened, the sensitivity is improved, and the precision is improved.

Inactive Publication Date: 2015-01-28

ZHONGSHAN HOSPITAL XIAMEN UNIV +1

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AI-Extracted Technical Summary

Problems solved by technology

[0003] Treponema pallidum cannot be cultured in vitro, and the positive rate of direct etiological dark-field microscopy is not high. Serological te...

Abstract

The invention discloses a treponema pallidum specific antibody chemical light emitting detection kit and a preparation method thereof, and relates to treponema pallidum. The kit comprises an outer package box, an alkaline phosphatase marked recombinant antigen bottle, a light emitting substrate bottle, a treponema pallidum specific antibody negative control bottle, a treponema pallidum specific antibody positive control bottle, a washing liquid bottle and a recombinant antigen coated micropore plate. The preparation method comprises the following steps: firstly, preparing reponema pallidum specific recombinant antigen and the recombinant antigen coated micropore plate, marking alkaline phosphatase of the recombinant antigen, further preparing a light emitting substrate, a washing liquid and a control group, and finally assembling the treponema pallidum specific antibody chemical light emitting detection kit. The treponema pallidum specific antibody chemical light emitting detection kit can be used for detecting syphilis specificity specific antibodies in clinical specimens, specific solid phase is adopted, immunity and light emitting reaction can be rapidly completed within a short time, the light emitting signals are greatly intensified, the sensitivity is improved, the detection time can be shortened, and the precision is improved.

Application Domain

Chemiluminescene/bioluminescence

Technology Topic

Solid phasesNegative control +9

Image

Examples

Experimental program(3)
Effect test(1)

Example Embodiment

[0028] Example 1

[0029] See figure 1 The Treponema pallidum specific antibody chemiluminescence detection kit is provided with an outer packaging box 1, an alkaline phosphatase labeled recombinant antigen bottle 2, a luminescent substrate bottle 3, a Treponema pallidum specific antibody negative control bottle 4, and a Treponema pallidum specific antibody Positive control bottle 5, washing solution bottle 6 and recombinant antigen-coated microplate 7; alkaline phosphatase labeled recombinant antigen bottle 2, luminescent substrate bottle 3, Treponema pallidum specific antibody negative control bottle 4, Treponema pallidum The specific positive control bottle 5, the washing solution bottle 6 and the recombinant antigen-coated microplate 7 are arranged in the outer packaging box 1. The alkaline phosphatase labeled recombinant antigen bottle 2 contains alkaline phosphatase labeled recombinant antigen and luminescent substrate Bottle 3 contains the luminescent substrate, Treponema pallidum specific antibody negative control substance. Bottle 4 contains Treponema pallidum specific antibody negative control substance and Treponema pallidum specific positive control substance. Bottle 5 contains Treponema pallidum specific positive control substance and washing solution. The bottle 6 contains washing liquid.

[0030] The Treponema pallidum specific antibody chemiluminescence detection kit and its preparation include the following steps:

[0031] (1) Preparation of Treponema pallidum specific recombinant antigen:

[0032] Using gene cloning technology, PCR amplifies the DNA encoding the Treponema pallidum antigen, and inserts it into E. coli to express it to obtain TPN17 and TPN47 specific antigens for Treponema pallidum.

[0033] (2) Preparation of recombinant antigen-coated microplate

[0034] Dilute the syphilis spiral recombinant antigens TPN17 and TPN47 in step (1) to 20μg/mL with coating buffer, add 0.1mL per well to the microtiter plate, and coat at 4°C for 24h; take out the antigen plate and air dry; use 0.01 1.0% skimmed milk powder prepared in mmol/L pH7.4 phosphate buffer, 0.2 mL per well, sealed at 4°C for 24 hours, taken out and washed 5 times with phosphate buffer, air-dried and sterilized at room temperature, to prepare a recombinant antigen-coated microplate , Seal for spare.

[0035] (3) Alkaline phosphatase labeling of recombinant antigen

[0036] Alkaline phosphatase is activated by sodium iodate, and is respectively coupled with -NH2 of the recombinant antigen molecule in step (1) to form alkaline phosphatase-labeled recombinant antigen.

[0037] (4) Preparation of luminescent substrate

[0038] Artificially synthesized amantadine luminescent substrate (AMPPD) and its enhancer are sterile filtered to prepare luminescent substrate working solution.

[0039] (5) Preparation of washing liquid

[0040] The washing solution is a phosphate buffer solution in which Tween-20 is dissolved, wherein the final concentration of Tween-20 is 0.01%.

[0041] (6) Reference substance

[0042] Treponema pallidum specific antibody negative control substance: prepared from the serum of healthy people without syphilis infection;

[0043] Treponema pallidum specific positive control: prepared from the specific antibody positive serum of syphilis patients;

[0044] (7) Preparation of high-throughput detection kit for Treponema pallidum antibody

[0045] Recombinant antigen-coated microplate, alkaline phosphatase-labeled recombinant antigen, luminescent substrate, washing solution, Treponema pallidum specific antibody negative control substance, Treponema pallidum specific antibody positive control substance and outer packaging box composed of Treponema pallidum-specific Sex antibody chemiluminescence detection kit.

[0046] In step (7), the alkaline phosphatase-labeled recombinant antigen, luminescent substrate, washing solution, Treponema pallidum specific antibody negative control substance, and Treponema pallidum specific antibody positive control substance are all packed in corresponding polyethylene bottles.

Example Embodiment

[0047] Example 2

[0048] The Treponema pallidum antibody high-throughput detection kit is used to detect Treponema pallidum specific antibodies in clinical specimens of patients:

[0049] 1. Specimen processing: Serum: 5 mL of venous blood, placed in a 37°C water bath for 30 minutes, centrifuged at 3000 g for 10 minutes, and the supernatant is used as a test sample for future use.

[0050] 2. Add sample: add 100μL of sample to the reaction plate, and make blank, negative and positive control wells at the same time. Incubate at 37°C for 1h.

[0051] 3. Washing: After reacting at 37°C for 30 minutes, the tested syphilis-specific antibody is combined with the syphilis-specific recombinant antigen coated on the microplate, and the unbound free components are washed and separated;

[0052] 4. Add alkaline phosphatase to label the recombinant antigen, and then add the luminescent substrate working solution. The alkaline phosphatase catalyzes the dephosphorylation of the substrate and emits 463nm visible light. Measure the relative light units (RLU) of each sample hole at 10-20 min. The RLU of the sample is positively correlated with the concentration of syphilis antibody to be tested.

Example Embodiment

[0053] Example 3

[0054] The performance verification of the Treponema pallidum antibody high-throughput detection kit is given below.

[0055] (1) Compliance rate of positive samples

[0056] 50 syphilis-specific antibody positive reference sera were tested to calculate the positive coincidence rate.

[0057] (2) Compliance rate of negative specimens

[0058] 50 syphilis-specific antibody negative reference sera were tested to calculate the negative coincidence rate.

[0059] (3) Differences within batches

[0060] The same batch of kits, with characteristic serum testing, requires CV ≤ 10%.

[0061] (4) Differences between batches

[0062] Different batches of kits, with characteristic serum testing, require CV≤12%.

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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

high sensitivity
shorten detection time

Treponema pallidum specific antibody chemical light emitting detection kit and preparation method thereof

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Image

Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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