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Myelin basic protein antibody detection kit and preparation method thereof

A myelin basic protein and antibody detection technology, applied in the fields of peptide chemistry and immunology, can solve the problems of inability to activate, unclear pathogenic mechanism, lack of auxiliary functions, etc., and achieve good stability, stable results, and anti-interference ability strong effect

Pending Publication Date: 2019-11-19
安徽恩禾生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the exact pathogenic mechanism of MBP-Ab disease is still unclear. Most studies believe that specific B cells that recognize MBP antigens may exist in peripheral blood, but due to the lack of expression of MBP antigens in the bone marrow, MBP-specific immature B cells The cells are in an anergic state, and at the same time, due to the lack of corresponding helper T cells (Th cells), the specific B cells that recognize MBP cannot be activated, but only proliferate in the peripheral blood

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The composition of a kind of myelin basic protein antibody detection kit:

[0024] 1) MBP-coated plate: a microwell plate coated with MBP. 48-well / 96-well plate

[0025] 2) 1 set of MBP calibrator A-F: derived from human serum, concentration 0, 0.5, 1, 2, 5, 10g / L.

[0026] 1×0.5ml

[0027] 3) MBP-binding antigen: biotin-labeled human myelin basic protein (MBP).

[0028] 1×2.5ml / 1×5.0ml

[0029] 4) MBP-Ab enzyme conjugate: streptomycin-labeled horseradish peroxidase.

[0030] 1×5.0ml / 2×5.0ml

[0031] 5) MBP quality control: derived from human serum, the quality control range is 7μg / L±1.05μg / L. 1×0.5ml

[0032] 6) Color developer A: the main component is urea peroxide. 1×5.0ml

[0033] 7) Chromogenic agent B: the main ingredient is 3,3,5,5,-tetramethylbenzidine hydrochloride (TMB·2HCl). 1×5.0ml

[0034] 8) Concentrated washing solution (25×): Concentrated phosphate buffer solution, diluted 1:24 before use.

[0035] 1×20.0ml

[0036] 9) Termination solution: ...

Embodiment 2

[0040] A preparation method of a myelin basic protein antibody detection kit:

[0041] 1) Prepare MBP-coated antigen and prepare MBP-bound antigen:

[0042] The X segment and the Y segment were intercepted from the human MBP antigen epitope peptide, and the X segment and the Y segment were respectively transformed into Escherichia coli for cultivation. The bacteria were collected and centrifuged to obtain the recombinant precipitated protein, and the recombinant protein was purified separately. The target proteins were obtained, named X protein and Y protein respectively, X protein is the MBP coating antigen, Y protein is the MBP binding antigen.

[0043] 2) Preparation of MBP binding antigen-HRP marker:

[0044] The coupling of MBP-labeled antigen and HRP specifically adopts the NaIO4 oxidation method, including the following steps:

[0045] 2.1) dialyze the MBP-labeled antigen obtained in step S1 in PBS solution overnight at 4°C;

[0046] 2.2) UV spectrophotometer is used...

Embodiment 3

[0092] A detection method of a myelin basic protein antibody detection kit, comprising the following steps:

[0093] 1) Preparation of washing solution: 25× concentrated washing solution stored at 2-8°C may form crystals at the bottom of the bottle. Use a 500ml container to fully dissolve the entire bottle of concentrated washing solution in 480ml of distilled or deionized water.

[0094] 2) After the kit is equilibrated to room temperature (20°C-30°C), take out the required microwell strips and fix them on the plate rack, and compile the sequence;

[0095] 3) Adding samples: first add 50ul of the sample to be tested / MBP calibrator / MBP quality control, then add 50ul of MBP-bound antigen to the corresponding well, pat and mix well, and set a blank well;

[0096] 4) Incubation: seal the strips with parafilm (to prevent contamination), and incubate at 37°C for 30 minutes;

[0097] 5) Plate washing: take out the reaction plate, shake off all the liquid in the plate, fill each wel...

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Abstract

The invention belongs to the technical field of polypeptide chemistry and immunology and discloses a myelin basic protein antibody detection kit. According to the kit, a double-antigen sandwich methodis adopted to detect the content of a human MBP antibody; a coated plate, a calibration product, an MBP conjugated antigen, an enzyme conjugate, a quality control product, a color developing agent A,a color developing agent B, a stop solution and a concentrated washing solution are arranged in the kit; the coated plate is coated with an MBP coating antigen; an X fragment in the epitope peptide of the human MBP antigen is coupled with a carrier protein, so that the MBP coating antigen can be prepared; a Y fragment in the epitope peptide of the human MBP antigen is coupled with a carrier protein, so that the MBP conjugated antigen can be prepared; and the MBP conjugated antigen is labeled with biotins. With the method adopted, defects in the prior art are eliminated; the content of the human MBP antibody is quantitatively detected through the double-antigen sandwich method. The myelin basic protein antibody detection kit has the advantages of simple, convenient and rapid operation, high detection accuracy, high precision, high specificity, high sensitivity, and high stability.

Description

technical field [0001] The invention relates to the technical field of polypeptide chemistry and immunology, in particular to a detection kit for myelin basic protein antibody. Background technique [0002] At present, the exact pathogenic mechanism of MBP-Ab disease is still unclear. Most studies believe that specific B cells that recognize MBP antigens may exist in peripheral blood, but due to the lack of expression of MBP antigens in the bone marrow, MBP-specific immature B cells The cells are in an anergic state, and at the same time, due to the lack of corresponding helper T cells (Th cells), the specific B cells that recognize MBP cannot be activated, but only proliferate in the peripheral blood. When the neurotropic virus infects the body, the blood-brain barrier is destroyed, MBP antigen leaks into the periphery, activates CD4+ T cells, increases the recruitment and activation of MBP-specific B cells, and produces a large amount of MBP-IgG; at the same time, pro-infl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/6893G01N33/535
Inventor 余旭亮
Owner 安徽恩禾生物技术有限公司
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