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Kit for detecting biomarker as well as preparation method and application of kit

A technology of biomarkers and kits, applied in the field of biomedicine, can solve the problem of lack of detection, and achieve the effect of good specificity

Active Publication Date: 2022-07-08
深圳粒影生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It has been reported that MBTPS2 (membrane bound transcription factor peptidase, site 2) may be used as a diagnostic marker for osteoporosis (Lindert, U., Cabral, W., Ausavarat, S. et al. MBTPS2 mutations cause defective regulated intramembrane proteolysis in X-linked osteogenesis imperfecta.Nat Commun 7,11920(2016).), but there is no kit for detecting MBTPS2 on the market at present, therefore, it is necessary to develop a kit for detecting MBTPS2 for the diagnosis of osteoporosis detection

Method used

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  • Kit for detecting biomarker as well as preparation method and application of kit
  • Kit for detecting biomarker as well as preparation method and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Design of MBTPS2 mutein antigen polypeptide and preparation of MBTPS2 recombinant protein

[0020] The MBTPS2 protein was selected from NCBI (NCBI Reference Sequence: NP_056968.1) and analyzed by ( figure 1 and Table 1), the protein transmembrane many times, and the extracellular region is short, through analysis and existing literature reports, when aa459 N is mutated to S, aa505 L is mutated to F, the detection is closely related to osteoporosis. related. Considering the protein transmembrane structure, mutation position and secondary structure, two antigenic polypeptides were designed, specifically: antigenic polypeptides (aa456~aa462): AIV S AVP; antigenic polypeptide 2 (aa502~aa508): SVL F AAN. Since the antigenic polypeptide is short, it is a hapten and has low immunogenicity. Therefore, the two antigenic polypeptides are respectively coupled with BSA to form a complete antigen for use.

[0021] Table 1 MBTPS2 protein transmembrane analysis

[0022...

Embodiment 2

[0024] Example 2: Preparation and testing of monoclonal antibodies that recognize antigenic polypeptides

[0025] The two antigen polypeptides designed and synthesized in Example 1 were immunized to mice respectively, and monoclonal antibodies were prepared according to conventional hybridoma screening (see Chinese Patent for Invention CN 106226512 B). After multiple rounds of repeated immunization and screening, a hybridoma cell was screened for each antigenic polypeptide and a batch of monoclonal antibodies was prepared. The numbers are hybridoma cells 1 and 2, monoclonal antibodies 1 and 2, corresponding to antigenic polypeptide 1 and 2. The titers of the two monoclonal antibodies were detected by conventional ELISA. After testing, the titer of monoclonal antibody 1 (10 6 ) compared to the titer of monoclonal antibodies (10 5 ) is higher, because the position of the two monoclonal antibodies binding to the MBTPS2 recombinant protein is relatively close, and there is steri...

Embodiment 3

[0028] Example 3 Detection of MBTPS2 mutant protein

[0029] 3.1 Werstern blot detection The protein extracted from urine was used as a sample for SDS-PAGE gel electrophoresis, and then transferred to the membrane, blocked after transfer, and blocked overnight at 4 °C, and the blocking solution was 5% nonfat milk powder; the prepared monoclonal antibody 1 ( The original concentration of 1 mg / ml) was diluted 5000 times and incubated as primary antibody for 1 hour at room temperature; after washing, 5000 times diluted HRP-labeled goat anti-mouse IgG secondary antibody was added, and incubated at room temperature for 1 hour; after washing, ECL was used to develop the results. display (such as figure 2 ), the positive sample has a specific band at 57KD, and the negative sample has no band at 57KD, indicating that the monoclonal antibody has good specificity.

[0030] 3.2 Double antibody sandwich ELISA detection

[0031]3.2.1 Detection steps and determination method MBTPS2 prote...

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Abstract

The invention discloses a kit for detecting a biomarker. The biomarker is an MBTPS2 protein; the kit comprises an effective amount of MBTPS2 mutant protein and an effective amount of a monoclonal antibody for specifically recognizing the MBTPS2 mutant protein, and the MBTPS2 mutant protein is characterized in that N at the aa459 site is mutated into S, and L at the aa505 site is mutated into F; the nucleotide sequence of the MBTPS2 mutant protein after codon optimization is as shown in SEQ ID NO. 1; the effective dose of the monoclonal antibody capable of specifically recognizing the MBTPS2 mutant protein is a monoclonal antibody 1, and the sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are as shown in SEQ ID NO.2 and SEQ ID NO.3. The invention further discloses a preparation method of the MBTPS2 mutant protein capable of specifically recognizing the MBTPS2 mutant protein. The prepared kit (such as werstern blot and double antibody sandwich ELISA) for detecting the MBTPS2 mutant protein has good specificity, sensitivity and accuracy, and is suitable for large-scale accurate detection of the MBTPS2 mutant protein.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a kit for detecting biological markers and a preparation method and application thereof. Background technique [0002] Osteoporosis is a systemic bone disease in which bone density and bone quality are decreased due to various reasons, and the microstructure of bone is destroyed, resulting in increased bone fragility, which is prone to fractures. Osteoporosis is divided into two categories: primary and secondary. Primary osteoporosis is further divided into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II), and idiopathic osteoporosis (including juvenile osteoporosis). Postmenopausal osteoporosis generally occurs in women within 5 to 10 years after menopause; senile osteoporosis generally refers to osteoporosis in the elderly after the age of 70; and idiopathic osteoporosis mainly occurs in adolescents, and the cause is unknown. . ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573C12N9/48C12N15/57C07K16/40
CPCG01N33/577G01N33/573C12N9/48C07K16/40C07K2317/56C07K2317/34G01N2333/948G01N2800/108
Inventor 赵正严陈永洪郑志东
Owner 深圳粒影生物科技有限公司