Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting human toxoplasma gondii antibody and preparation method of indirect ELISA kit

A technology for antibody detection and Toxoplasma gondii, applied in the biological field, can solve the problems of low specificity, unstable results, high false negative rate, etc., and achieve obvious specificity and sensitivity, easy promotion, and good repeatability

Inactive Publication Date: 2018-11-02
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] The present invention provides a kind of indirect ELISA kit and its preparation method that use recombinant protein GRA15 as antigen to detect human toxoplasma gondii antibody. and other shortcomings and deficiencies, used for the qualitative detection of human Toxoplasma gondii antibody

Method used

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  • Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting human toxoplasma gondii antibody and preparation method of indirect ELISA kit
  • Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting human toxoplasma gondii antibody and preparation method of indirect ELISA kit
  • Indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting human toxoplasma gondii antibody and preparation method of indirect ELISA kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the preparation of the indirect ELISA kit that human toxoplasma gondii antibody detects

[0051] 1. Prepare various solutions:

[0052] ①Coating solution: 0.75g sodium carbonate, 1.46g sodium bicarbonate, add deionized water to 500mL, store at 4°C.

[0053] ②Sample diluent: 0.2g BSA plus prepared 0.02mol / L phosphate buffer solution (0.2g potassium dihydrogen phosphate, 2.90g disodium hydrogen phosphate, 8g sodium chloride, add deionized water to 1000mL) for dissolution and quantification to 100mL.

[0054] ③Blocking solution: 2.0gBSA was added to the prepared 0.05mol / L carbonate buffer solution to dissolve and quantify to 100mL.

[0055] ④Washing solution: Dissolve 50μL Tween-20 in 100mL 0.02mol / L phosphate buffer, shake and mix well.

[0056] ⑤ Chromogenic solution: Liquid A: Weigh 20 mg of TMB and dissolve it in 10 mL of absolute ethanol. After it is completely dissolved, add double distilled water to 100 mL. Liquid B: weigh Na 2 HPO 4 12H 2 O 14....

Embodiment 2

[0076] Embodiment 2: the operating steps of the indirect ELISA kit that human toxoplasma gondii antibody detects

[0077] (1) After diluting human serum with sample diluent at 1:1000, add to the wells of GRA15 pre-coated microtiter plate, 100 μL per well, and incubate at 37°C for 1 hour;

[0078] (2) Wash with washing liquid 5 times, 5 minutes each time;

[0079] (3) Add HRP-labeled goat anti-human IgG (working concentration), 100 μL per well, and incubate at 37°C for 1 hour;

[0080] (4) Wash with washing liquid 5 times, 5 minutes each time;

[0081] (5) Add chromogenic solution, 50 μL per well, and develop color for 5 minutes in the dark;

[0082] (6) Add stop solution, 50 μL per well;

[0083] (7) Utilize a microplate reader to measure the optical absorption value (OD) value at 450nm;

[0084] (8) Result judgment: After measuring the OD450 value, judge the sample value according to the critical value.

Embodiment 3

[0085] Embodiment 3: the determination of the indirect ELISA kit performance of human toxoplasma gondii antibody detection

[0086] (1) Sensitivity test: The ELISA plate coated with Toxoplasma gondii GRA15 protein was used to detect positive sera with different dilutions, and the sensitivity was 1:25600, as shown in Table 1.

[0087] Table 1 Sensitivity detection of the indirect ELISA kit for human Toxoplasma gondii antibody detection

[0088]

[0089] (2) Specificity experiment: The established indirect ELISA method was used to detect the positive sera of human cytomegalovirus infection and human rubella virus infection. The results showed that the method had no cross-reaction with the two standard positive sera. Better, as shown in Table 2.

[0090] Table 2 Specific detection of indirect ELISA kits for human Toxoplasma gondii antibody detection

[0091]

[0092] (3) Repeatability experiment: select 10 parts of Toxoplasma gondii positive serum to apply established ind...

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Abstract

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) kit for detecting a human toxoplasma gondii antibody and a preparation method of the indirect ELISA kit, and belongs to the field of biotechnology. The kit comprises a recombinant antigen GRA15 pre-coated plate, a sample diluent, a positive control, a negative control, an enzyme conjugate, a blocking solution, a washingsolution, a color developing solution and a terminating solution, wherein the amino acid sequence of a recombinant antigen GRA15 is shown in SEQ ID NO: 1. Compared with the existing detection technology, the indirect ELISA detection method has the advantages of being simple to operate, good in repeatability, economic and suitable for large-scale sample detection. Toxoplasma gondii Chineses1 strain GRA15 protein isolated in China is used as the coating antigen of the indirect ELISA method, and the kit has higher specificity for detection of toxoplasma gondii infection of Chinese people.

Description

technical field [0001] The invention discloses an indirect ELISA kit for detecting human toxoplasma gondii antibody and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Toxoplasma gondii is a parasitic protozoan that can infect almost all warm-blooded animals. Human infection with Toxoplasma gondii can lead to miscarriage, stillbirth, deformity, stunted fetal growth and development, and even death in pregnant women. It will also cause diseases such as toxoplasma eye disease and toxoplasma encephalopathy to immunocompromised people, AIDS infected persons and organ transplant recipients. Moreover, there is no effective vaccine for Toxoplasma gondii, which poses a serious threat to human health. Therefore, the detection of human Toxoplasma gondii infection is of great significance for the prevention and control of diseases. Establishing an effective technical means and method for detecting Toxoplasma gondii infection is part...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/54306G01N33/6893G01N2333/45
Inventor 蔡亦红陈守斌罗庆礼沈继龙
Owner ANHUI MEDICAL UNIV
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