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Bird's-nest enzyme linked immunosorbent assay kit

An enzyme-linked immunosorbent reagent and detection method technology, which is applied in the field of bird's nest enzyme-linked immunosorbent immunoassay kit and its detection, can solve the problems of low content of bird's nest, cumbersome detection operations, low sensitivity, etc., and achieve good anti-matrix interference effect and detection The effect of simple operation and low detection cost

Inactive Publication Date: 2014-09-17
SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) PCR method: using specific gene fragments in bird’s nest for PCR detection, this method has good specificity; however, it cannot achieve quantitative detection, and the finished products of bird’s nest often require thermal processing, which may easily lead to DNA degradation in the sample, thus making the test results A false negative occurs;
[0005] (2) Electrophoresis method: analysis is based on the protein map of bird’s nest, but the ability to resist matrix interference is poor and the sensitivity is low. It is limited to the detection of bird’s nest and cannot be used for the detection of bird’s nest products;
[0006] (3) Specific component analysis method: use chromatography, spectroscopy and other means to identify the authenticity of various chemical components in bird’s nest, such as specific elements, amino acids, sialic acid, sugars, etc., but its specificity is not high , easily disturbed by artificial additives
[0007] Therefore, the existing detection technology has low specificity, is easily interfered by artificial additives, cannot carry out quantitative analysis, and has a narrow scope of application. For effective detection, the detection operation is cumbersome, the detection time is long, it needs to be equipped with large analytical instruments and requires professional operators, etc.

Method used

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  • Bird's-nest enzyme linked immunosorbent assay kit
  • Bird's-nest enzyme linked immunosorbent assay kit
  • Bird's-nest enzyme linked immunosorbent assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Separation and purification of target antigen.

[0026] The target antigen is bird's nest sialoglycoprotein, which consists of two proteins with the same antigenic determinant but different degrees of glycosylation modification, with molecular weights of 106 kDa and 128 kDa, respectively, and the isoelectric point ≤ 3.0.

[0027] The bird's nest was ultrasonically extracted with 7M urea for 10 minutes, centrifuged to take the supernatant, and separated and purified using a liquid phase focuser. The target protein was precipitated at the positive electrode and washed with deionized water to obtain the target protein. The target protein was verified by SDS-PAGE and 2-D PAGE, the results are as follows figure 1 As shown, no other foreign protein bands were seen, and the purity met the immune requirements.

Embodiment 2

[0028] Example 2 Preparation and purification of anti-sialoglycoprotein antibody.

[0029] Female balb / c mice were immunized with bird's nest sialoglycoprotein for 6 weeks, 3 in each group. For the first immunization injection, 100 μL of 100 μg / mL immune antigen was fully emulsified with the same amount of complete Freund’s adjuvant, and injected directly into the intraperitoneal cavity. After an interval of two weeks, take the sampled antigen, emulsify it with 100 μL of incomplete adjuvant, and inject it in the same way.

[0030] One day before cell fusion or on the same day, the Kunming rats were killed by pulling the neck, soaked in 70% alcohol, and disinfected the body surface; the Kunming rats were fixed on the wax board with pins, the abdomen was cut open on the ultra-clean workbench, and the peritoneum was picked up with small tweezers. Inject 5 mL of RPMI-1640 complete culture solution (obtained by adding 15% fetal bovine serum to GIBICO RPMI-1640 basic culture solu...

Embodiment 3

[0037] Example 3 Construction of bird's nest ELISA kit.

[0038] Coating of microtiter plate: add 0.1mL of 1mg / L target antigen (bird’s nest sialoglycoprotein) to high adsorption microtiter plate, coat overnight, seal with 5% skimmed milk powder for 2h, and dry in vacuum.

[0039] The anti-sialoglycoprotein antibody was diluted 1:10000 with PBS to obtain the antibody working solution.

[0040] The enzyme-labeled secondary antibody was diluted 1:10000 with PBS to obtain the enzyme-labeled secondary antibody working solution.

[0041] Substrate chromogenic solution: obtained by mixing equal volumes of chromogenic agent A and chromogenic agent B; weigh 17.2 mg tetramethylbenzidine, add 1 mL DMSO to dissolve, then add 0.1 M pH5.5 acetic acid-sodium acetate Add 66 mL of buffer solution to obtain chromogenic agent A; take 100 mL of double distilled water and add 17 μL of 30% hydrogen peroxide to obtain chromogenic agent B.

[0042] The bird's nest ELISA kit provided by the prese...

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Abstract

The invention provides a bird's nest ELISA kit, which comprises sialoglycoprotein standard solution, anti-sialoglycoprotein antibody, enzyme-labeled secondary antibody, enzyme-labeled plate and substrate chromogenic solution. The invention can perform quantitative detection on bird's nest and related products, has wide application range, good specificity, high sensitivity, good anti-matrix interference effect, good detection result accuracy and good repeatability, and can conveniently carry out on-site detection and a large number of samples. Screening and detection costs are low.

Description

technical field [0001] The invention relates to the detection of bird's nest and its related products, in particular to a bird's nest ELISA kit and a detection method thereof. Background technique [0002] Bird's nest is a nest formed by the condensation of saliva or down feathers of swiftlets of the Swiftlet family Swiftlet and similar species. Its origin is distributed in Southeast Asia, such as China, Indonesia, Thailand, Vietnam and Malaysia. Bird's nest is a kind of precious traditional Chinese medicine, which has the functions of nourishing yin and moistening dryness, nourishing the middle and nourishing qi, nourishing the stomach and spleen, resolving phlegm and relieving cough; bird's nest is rich in active glycoprotein, calcium, iron, phosphorus, iodine and vitamins and other natural nutrients And minerals, it also has the effect of preventing influenza virus, promoting cell growth and strengthening the function of the immune system. Due to the high nutritional val...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 张世伟杨国武赖心田刘小青李芸陈血剑严琼英陈薇陈国培陈雁叶秀玲
Owner SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
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