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Chemiluminescence immune detection kit of anti-trophoblast cell membrane antibody and preparation method of chemiluminescence immune detection kit

A technology of trophoblast cell membrane and chemiluminescence immunity, which is applied in chemiluminescence/bioluminescence, biological testing, and analysis through chemical reactions of materials, etc., which can solve the problems of high detection cost, cumbersome reagent filling operation, and heavy burden on patients, etc. problem, to achieve the effect of high detection accuracy

Inactive Publication Date: 2016-09-07
SHENZHEN YHLO BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) Use 12×8 type, 6×8 type, 8×12 type or whole plate type 96-well special microwell plate as antigen coating equipment and reaction container, which can only be divided into 12 batches and 6 batches when used , 8 batches or the whole board can be used at one time, and independent and single-person testing cannot be carried out;
[0007] (2) There are many types of reagents used in quantitative determination, and each detection reagent must be contained in a reagent bottle, and each time a reagent is used, it is necessary to replace the suction nozzle to fill it into the microwells of the microwell plate , not only there are many types of reagent bottles, but also the operation of filling reagents is extremely cumbersome;
[0008] (3) There is a lack of corresponding labeling of the testing information. The production batch number and expiry date information of the testing reagent can only be known or known by checking the label on the outer packaging box of the kit, and the known information is not controlled during the testing process, which has great potential. large randomness;
[0009] (4) The detection reagents are in an open space during the detection process, which may easily cause cross-contamination among various reagents and affect the accuracy of the detection results;
[0010] (5) Manual operation is mostly used in the detection process, the addition of reagents or samples is not very precise, the operation process is extremely cumbersome and complicated, and operation errors are prone to occur, and the accuracy and precision of the detection results are poor;
[0011] (6) The quantity configuration and use of the complete set of reagents for the test items are the number of items × 48 / 96 persons. If 10 items need to be tested, the configuration and use of reagents must be 10×48 / 96 persons. If Only one sample needs to be tested for 10 different items, and reagents for 10×48 / 96 people need to be configured, which has the disadvantage of not being economical and reasonable
[0014] Enzymatic chemiluminescence mainly includes horseradish peroxidase (HRP) and alkaline phosphatase, but both have certain limitations. The main disadvantage of horseradish peroxidase is: In the presence of biomolecules, they will also be oxidized by H2O2 to emit light. The background is relatively high, which affects the signal-to-noise ratio. The reaction kinetics is complicated, and there are many influencing factors. easy
The main disadvantages of alkaline phosphatase are: it takes a long time for the substrate to reach the plateau, and the cost of the substrate is high, resulting in high detection cost and heavy burden on patients

Method used

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  • Chemiluminescence immune detection kit of anti-trophoblast cell membrane antibody and preparation method of chemiluminescence immune detection kit
  • Chemiluminescence immune detection kit of anti-trophoblast cell membrane antibody and preparation method of chemiluminescence immune detection kit
  • Chemiluminescence immune detection kit of anti-trophoblast cell membrane antibody and preparation method of chemiluminescence immune detection kit

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preparation example Construction

[0052] like figure 1 The shown preparation method of the anti-trophoblast cell membrane antibody chemiluminescence immunoassay kit includes the following steps:

[0053] Take the suspension of carboxylated magnetic particles, remove the supernatant by magnetic separation and resuspend with MES buffer, then add EDC aqueous solution to activate the surface carboxyl groups of the carboxylated magnetic particles, then add anti-trophoblast cell membrane antibody recombinant protein, at room temperature Suspended for 2h-10h, removed the supernatant by magnetic separation, and resuspended with Tris buffer to obtain carboxylated magnetic particles coated with recombinant protein of anti-trophoblast cell membrane antibody.

[0054] The concentration of MES (2-(N-morpholino)ethanesulfonic acid) buffer was 0.02 M and the pH was 5.5.

[0055] The concentration of Tris buffer is 0.1 M and contains 2% BSA, pH 8.0.

[0056] The concentration of EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbod...

Embodiment 1

[0072] Example 1: Preparation of Anti-trophoblast Cell Membrane Antibody Chemiluminescence Immunodetection Kit

[0073] (1) Preparation of carboxylated magnetic particles coated with recombinant protein of anti-trophoblast cell membrane antibody:

[0074] Take the suspension containing 50 mg of carboxylated magnetic particles (MagnaBind21353) with a particle size of 0.05 μm to 1 μm, magnetically separate the supernatant, resuspend it with 0.02 M MES buffer with a pH of 5.5, and add 1 mL of freshly prepared 10 mg / mL EDC aqueous solution, activate the carboxyl groups on the surface of the magnetic beads, add 4 mg of recombinant protein anti-trophoblast cell membrane antibody (biorbyt, catalog number orb48780), suspend at room temperature for 6 hours, magnetically separate, remove the supernatant, and use 0.1 M containing 2% BSA, pH 8.0 Resuspend to 1 mg / mL in Tris buffer to obtain carboxylated magnetic microparticles coated with anti-trophoblast cell membrane antibody recombinan...

Embodiment 2

[0079] Example 2: Chemiluminescence immunodetection method of anti-trophoblast cell membrane antibody

[0080] The automatic chemiluminescence immunoassay analyzer (YHLO, product number iFlash3000) was used as the detection tool, and the method was indirect immunoassay, that is, the instrument added 50 μL of sample and 50 μL of anti-trophoblast cell membrane antibody recombinant protein-coated carboxylation in sequence. magnetic particles and 50 μL of anti-trophoblast cell membrane antibody treatment solution. After 20 min of reaction, add 50 μL of anti-human immunoglobulin acridinium ester. After 20 min of reaction, magnetic separation was performed, and the instrument sent the reaction mixture into a dark room. , followed by adding luminescent substrate A solution (H 2 O 2 ) and B solution (NaOH) for luminescence reaction, and finally record the luminescence value.

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Abstract

The invention discloses a chemiluminescence immune detection kit of an anti-trophoblast cell membrane antibody and a preparation method of the chemiluminescence immune detection kit. The chemiluminescence immune detection kit of the anti-trophoblast cell membrane antibody comprises carboxylated magnetic micro particles coated with anti-trophoblast cell membrane antibody recombinant protein and an immunoglobulin-marked chemiluminescence marker. By the chemiluminescence immune detection kit of the anti-trophoblast cell membrane antibody, the anti-trophoblast cell membrane antibody is detected by a full-automatic chemiluminescence immunity analyzer as a detection tool. Through an experiment, the detection sensitivity of the chemiluminescence immune detection kit of the anti-trophoblast cell membrane antibody reaches 1U / L; compared with a traditional detection method of the anti-trophoblast cell membrane antibody, the sensitivity is improved by 10 times at least; and the chemiluminescence immune detection kit of the anti-trophoblast cell membrane antibody is relatively high in detection accuracy.

Description

technical field [0001] The invention relates to the field of in vitro detection, in particular to an anti-trophoblast cell membrane antibody chemiluminescence immunodetection kit and a preparation method thereof. Background technique [0002] Infertility is a hot spot in current reproductive medicine research. In recent years, with the development of reproductive immunology research, it has been recognized that immune factors are one of the important factors that cause infertility. Immune infertility accounts for the majority of female infertility. About 10%, the production of autoantibodies is an important reason. Anti-trophoblastic antibody (ATA) is one of the autoantibodies affecting infertility. The production of anti-trophoblast cell membrane antibodies can affect the development and function of the ovary and follicles. Therefore, many infertility diseases are caused by anti-trophoblastic cell membrane antibodies, so the determination of anti-trophoblastic cell membra...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/68
CPCG01N21/76G01N33/6854
Inventor 邓爱凤代洪飞夏福臻钱纯亘王刚
Owner SHENZHEN YHLO BIOTECH