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Micro-sphere double-antibody sandwich detection method and kit for detecting soluble FAM19A4 protein

A FAM19A4, 1. FAM19A4 technology, applied to the quantitative detection method and kit for detecting soluble FAM19A4 protein, FAM19A4 protein as a disease diagnostic marker field, can solve the problem that it is difficult to meet the detection requirements of a small amount of samples, and the sample size is large

Active Publication Date: 2019-03-05
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of actually exploring and establishing this detection method, the inventors found that the conventional double-antibody sandwich ELISA method requires a large sample size, which is difficult to meet the detection requirements of a small amount of samples

Method used

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  • Micro-sphere double-antibody sandwich detection method and kit for detecting soluble FAM19A4 protein
  • Micro-sphere double-antibody sandwich detection method and kit for detecting soluble FAM19A4 protein
  • Micro-sphere double-antibody sandwich detection method and kit for detecting soluble FAM19A4 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Materials and Methods

[0070] 1. Preparation of recombinant protein

[0071] The eukaryotic recombinant proteins FAM19A1-myc-his, FAM19A4-myc-his, FAM19A5-myc-his and LYG1-myc-his were prepared according to conventional eukaryotic recombinant protein preparation methods, and the concentration was determined by the BCA quantitative method to be not less than 1mg / mL.

[0072] 2. Antibodies

[0073]Rabbit anti-human FAM19A4 polyclonal antibody was obtained by immunizing rabbits with FAM19A4-myc-his eukaryotic recombinant protein. the migration and phagocytosis of macrophages, Cell.Mol.Immunol., 2015, Volume 12, pages 615-624). Mouse anti-human FAM19A4 monoclonal antibody was used to immunize BALB / c nude mice with FAM19A4-myc-his eukaryotic recombinant protein, and 12 hybridoma clones were obtained by ELISA screening. The affinity and specificity of the 12 antibody strains were identified by Western blot and flow cytometry analysis, and the best clone was s...

Embodiment 2

[0089] Example 2 Establishment of standard curve for detection of soluble FAM19A4 protein by microsphere double-antibody sandwich method

[0090] First, dilute FAM19A4-myc-his eukaryotic recombinant protein to 10ng / mL with detection dilution buffer, and then double dilution to obtain concentrations of 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL mL, 0.312ng / mL, 0.156ng / mL, 0.078ng / mL, and 0.039ng / mL standard gradient solutions. Test the dilution buffer as a blank control. The above-mentioned standard substance was detected according to the detection steps in Example 1, and the APC-A GMFI data was collected, and a standard curve was drawn using CurveExpert 1.4 software corresponding to the concentration of the standard substance. figure 2 Shown in Table 1 is a representative data, according to which the standard curve drawn is as image 3 shown.

[0091] Table 1

[0092]

[0093]

Embodiment 3

[0094] Example 3 Specificity, recovery and precision of detection of soluble FAM19A4 protein by microsphere double-antibody sandwich method

[0095] The method of the present invention verifies its specificity by detecting FAM19A1 and FAM19A5. Both belong to the TAFA family and share 74% and 68% sequence similarity with FAM19A4, respectively. Prepare 10 000 and 1 000 pg / mL FAM19A1 and FAM19A5 standard substances respectively, use the method of the present invention to detect, and the results are all lower than the detection limit (Table 2). This shows that there is no obvious cross-reaction between FAM19A4 and its analogues, and the method of the present invention is highly specific.

[0096] Add a certain concentration of FAM19A4 standard protein into different substrates to be tested, use the method of the present invention to detect, compare the detected concentration with the actual concentration, and calculate the recovery rate of the detection method. As shown in Tab...

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Abstract

As an FAM19A4 protein is possibly related to various physiological and pathological conditions and plays an important role in the physiological and pathological conditions, and quantitative detectionof the soluble FAM19A4 protein in specific samples such as blood, body fluid and cell culture supernatant has significance. The invention firstly relates to an application of the FAM19A4 protein serving as a diagnosis marker of a disease including an infectious disease and an autoimmune disease. The invention further relates to a method and kit for quantitatively detecting the soluble FAM19A4 protein. According to the method and kit, the soluble FAM19A4 protein is detected through a micro-sphere solid carrier and a flow cytometry technique by the aid of the double-antibody sandwich method principle, and the method and kit can be used for quantitatively detecting the FAM19A4 protein in body fluids such as serum, plasma, urine, pleuroperitoneal fluids, joint fluids, cerebrospinal fluids, amniotic fluids and follicular fluids of a clinic patient, basic research and various biological samples such as cell culture fluids and mouse blood in a mouse model.

Description

technical field [0001] The invention belongs to the field of immunology and biotechnology, and firstly relates to the use of a FAM19A4 protein as a disease diagnosis marker; the invention also relates to a quantitative detection method and kit for detecting soluble FAM19A4 protein. The method and kit can be used for the detection of FAM19A4 protein in human and mouse body fluid samples, the detection of FAM19A4 protein in various biological samples (such as cell culture supernatant, cell lysate) in basic research, and the detection of FAM19A4 protein derived from animal models. Detection of FAM19A4 protein in biological samples. technical background [0002] FAM19A4 (family with sequence similarity 19 (chemokine (C-C motif)-like), member A4; also known as TAFA4) is a cytokine of FAM19 / TAFA family with chemotactic function. Bioinformatics found that the FAM19A4 gene is located at 3p14.1, the full-length sequence is 2248bp, the full-length ORF is 423bp, encoding 140 amino aci...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6863G01N33/6893G01N2800/102G01N2800/104
Inventor 李婷韩文玲马大龙王文彦程迎迎朱凤雪李纾李荷楠张烜石爽
Owner PEKING UNIV
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