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Flow-type detection reagent for detecting InhibinB content and preparation method and application thereof

A technology of flow detection and content, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems that scientists cannot be sure, cannot detect the degree of sample acquisition, and the detection sensitivity is not high enough

Inactive Publication Date: 2017-08-08
ZHEJIANG CELLPRO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the laboratory normal reference values ​​of serum INH B reported in foreign literatures are different from each other, and they are concentrated in the range of 176.8-238ng / L, but they overlap with some pathological conditions, which may be related to the fluctuation of testicular volume, test sample size, and FSH. It is related to obesity, etc., which makes it impossible for scientists to determine a unified standard to give a definitive opinion on which samples are normal and which samples are beyond the normal range
In addition, INH B can be detected in normal adult male serum and seminal plasma, but not in urine, indicating that the detection sensitivity of the most common double-antibody sandwich ELISA method currently on the market is not high enough to detect some Patients with difficulty in obtaining samples
Since the currently used detection method is the double-antibody sandwich ELISA method, steps such as incubation of antibody antigens, elution, and absorbance measurement are required during the detection process, making the detection steps cumbersome, time-consuming (more than 2 hours), expensive, and unable to achieve high throughput of multiple samples detection

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  • Flow-type detection reagent for detecting InhibinB content and preparation method and application thereof
  • Flow-type detection reagent for detecting InhibinB content and preparation method and application thereof

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specific Embodiment 1

[0033]A flow detection reagent for detecting Inhibin B content, including Ab1 antibody microsphere conjugates and Ab2 antibody fluorescent molecular markers for detecting INH B content in serum or seminal plasma, wherein the Ab1 antibody microsphere conjugates General structure such as figure 1 As shown, the general structural formula of Ab2 antibody fluorescent molecular marker is as follows figure 2 As shown, M is the monodisperse microsphere made of polymer material, R is the functional bond, Ab1 is INHBα subunit monoclonal antibody Ab1, and Ab2 is INHBβ B Subunit monoclonal antibody Ab2, F is a fluorescent molecule.

[0034] The diameter of the above-mentioned monodisperse microspheres is 1-50 microns, and the material is polystyrene (PS), polymethyl methacrylate (PMMA), silicon dioxide (SiO2), polylactic acid (PLA) or polylactic acid glycolic acid polymerized (PLGA), the outer surface of the monodisperse microspheres is functionally modified with carboxyl, amino, hydro...

specific Embodiment

[0039] The above-mentioned specific embodiment one is used to detect the preparation method of the flow detection reagent of Inhibin B content, comprises the following steps:

[0040] (1) INHBα and β B Production of subunit antibodies

[0041] Obtain the coding sequence of the INHBα subunit and the amino acid sequence of the translated mature corresponding protein from the NCBI website, use protein-related software to analyze the stability of the amino acid sequence and epitope, and select some mature fragments (mature fragments are more stable and will not be The amino acid sequence SEQ-1 of degraded) was used as the antigen for preparing Ab1 antibody, and the base sequence encoding this antigen was cloned into the expression vector PET-28, PET-32 or PET-41, and the E.coli prokaryotic expression system expressed the INHBα subunit Unit antigen protein, purified INHBα subunit antigen protein; use INHBα subunit antigen protein as immunogen to synthesize mouse anti-human Inhibin...

specific Embodiment 3

[0049] The application of the flow detection reagent for detecting the content of Inhibin B described in the above specific embodiments one and two, the specific steps of using the flow detection reagent to detect the content of Inhibin B are as follows:

[0050] Add the serum or seminal plasma of the sample to be tested into the Ab1 antibody microsphere conjugate solution, then add a certain volume of Ab2 antibody fluorescent molecular marker solution, incubate at 37 degrees for 30 minutes, and the INHB in the sample is coupled with the Ab1 antibody microspheres respectively Ab2 antibody and fluorescent molecular markers combined to form a microsphere-Ab1-INHB-Ab2-fluorescent molecule complex, the fluorescence emitted by the fluorescent molecule can be detected by flow cytometry, and calculated according to the relationship between the fluorescence intensity and the concentration of INHB Inhibin B content in the sample, wherein the molar ratio of Ab1 antibody microsphere conju...

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Abstract

The invention discloses a flow-type detection reagent for detecting InhibinB content and a preparation method and application thereof. The flow-type detection reagent is characterized by comprising an Ab1 antibody microsphere conjugate and an Ab2 antibody fluorescent molecular marker which are used for detecting INH B content in serum or seminal plasma. The preparation method comprises: a step of preparing INHB alpha and beta B sub-unit antibodies; a step of coupling the INHB alpha sub-unit antibody Ab1 with mono-dispersion microspheres; a step of carrying out fluorescent molecular marking on the INHB beta B sub-unit antibody Ab2. By an antibody microsphere coupling technology and an antibody fluorescent marking technology and through adopting specific combination of antigen antibodies, microspheres and fluorescent molecules, which have a certain particle size, can be identified by a flow cytometry; fluorescent intensity directly reflects the INHB content in a sample; flow cytometry quantitative detection on the INHB in the sample is implemented; the flow-type detection reagent has the advantages of high sensitivity, high detection speed and high accuracy.

Description

technical field [0001] The invention relates to a detection technology for Inhibin B (inhibin B) content in serum or seminal plasma, in particular to a flow detection reagent for detecting Inhibin B content, a preparation method and application thereof. Background technique [0002] Inhibin B (INH B) is a member of the transforming growth factor β superfamily and belongs to glycoprotein hormones; it consists of α and β B The two subunits are connected by disulfide bonds to form a heterodimer (α-βB), and only the dimer has biological activity. Inhibin binding protein (INHBP) and transforming growth factor beta receptor type III (TGFβRIII) are considered INH B receptors. INH B mainly has the following functions: ① Regulating the physiological function of activin by antagonizing type II receptors. But only active in the case of activin response. ②Regulate and reflect Sertoli cell function, as one of the indicators for monitoring testicular function and evaluating spermatogen...

Claims

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Application Information

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IPC IPC(8): G01N33/74G01N33/533
CPCG01N33/74G01N33/533
Inventor 房海燕高攀陈继勇
Owner ZHEJIANG CELLPRO BIOTECH
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