Enzyme linked immunosorbent assay kit for detecting human epididymis secretory protein 4

A technology for epididymis secreted protein and human detection, which is applied in the field of enzyme-linked immunosorbent assay kits, can solve the problems of high false positive rate, and achieve the effects of high detection sensitivity and accuracy, convenient detection method, and strong reliability

Inactive Publication Date: 2015-06-24
北京神州细胞生物技术集团股份公司
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  • Abstract
  • Description
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Problems solved by technology

However, CA125 is still insufficient in the detection of ovarian cancer: First, the false positive rate is high, and it can also appear in other cancers, endometriosis, pelvic inflammatory disease and other benign gynecological diseases and physiological conditions (such as menstruation, pregnancy) positive expression; secondly, only 50%-60% of patients with stage I ovarian cancer have elevated CA125 values, and the diagnostic positive rate of CA125 alone is <10%. Combined with other detection methods such as ultrasound, the positive diagnostic rate can only be increased to 20%, therefore, a more sensitive and specific marker is needed to improve the diagnosis of ovarian cancer

Method used

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  • Enzyme linked immunosorbent assay kit for detecting human epididymis secretory protein 4
  • Enzyme linked immunosorbent assay kit for detecting human epididymis secretory protein 4
  • Enzyme linked immunosorbent assay kit for detecting human epididymis secretory protein 4

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Component preparation of embodiment 1 ELISA kit

[0015] 1. Preparation of human HE4 protein mouse monoclonal antibody:

[0016] 1) Animal immunity

[0017] Balb / c mice were used as immunized animals, and the biologically active recombinant human HE4 protein produced by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. was used as the immunogen, and the immunization dose was 50 μg / mouse. Complete Freund's adjuvant to make emulsifier, abdominal subcutaneous injection in multiple points, take the same dose of immunogen and equal amount of incomplete Freund's adjuvant to make emulsifier at intervals of 2-3 weeks, boost immunization twice, and measure serum after three times of immunization For titer, mice with high serum titer were taken for intraperitoneal booster immunization once, and splenocytes were taken for fusion after 4 days.

[0018] 2) Cell fusion and cloning

[0019] Splenocytes from immunized mice were mixed with SP2 / 0 myeloma cells at a ratio of 4:1, and fus...

Embodiment 2

[0033] Example 2 The formation of the ELISA kit for detecting human HE4

[0034] Set up an enzyme-linked immunosorbent assay kit for detecting human HE4, including the following reagents:

[0035] 1) Mouse anti-human HE4 monoclonal coating antibody;

[0036] 2) HRP-labeled rabbit polyclonal antibody;

[0037] 3) Recombinant human HE4 protein standard;

[0038] 4) The coating buffer is pH7.4, 0.1mol / L phosphate buffer;

[0039] 5) The blocking solution is a phosphate buffer containing 2% bovine serum albumin;

[0040] 6) The sample diluent is a phosphate buffer containing 0.1% bovine serum albumin;

[0041] 7) The concentrated washing solution is a Tris buffer containing 2% Tween or a phosphate buffer containing 2% Tween washing solution;

[0042] 8) Color developing solution A and color developing solution B are composed, and color developing solution A is hydrogen peroxide or carbamide peroxide, and color developing solution B is tetramethylbiphenyl;

[0043] 9) The sto...

Embodiment 3

[0044] Example 3 Preparation of ELISA Kit for Detecting Human HE4

[0045] 1. Explore the optimal antibody concentration of the enzyme-linked immunosorbent immunoassay kit by using orthogonal experiments

[0046]According to the ultraviolet spectrophotometer method, the concentration of antibody and antigen was determined. Using the orthogonal test method, the anti-HE4 monoclonal antibody was diluted to a concentration of 4 μg / ml, 2 μg / ml, and 1 μg / ml, and the recombinant HE4 protein concentration was diluted to 1000 pg / ml, 100 pg / ml, and 0 pg / ml. HE4 rabbit polyclonal antibody was diluted to 2μg / ml, 1μg / ml, 0.5μg / ml, 0.25μg / ml. Considering the background and the light absorption value of the recombinant protein, the coating concentration of the selected antibody was 2 μg / ml, and the working concentration of the HRP-labeled antibody was 0.5 μg / ml.

[0047] 2. Batch preparation of kits

[0048] 1) Coating microtiter plate: use phosphate coating buffer (weigh 8g of sodium chl...

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Abstract

The invention discloses a kit for detecting human epididymis secretory protein 4 (HE4) by a double antibody sandwich method, which comprises a HE4 resistant mice monoclonal coated antibody, an enzyme-labeled HE4 rabbit polyclonal antibody and a recombinant HE4 albumen standard substance. The detection kit provided by the invention can be used for qualitatively or quantitatively detecting the HE4 level in sample and has the advantages that the detection method is convenient and practicable, the detection sensitivity and accuracy are high, the specificity is strong, and the detection kit can simultaneously rapidly detect bulk samples. Through quantitative determination of a tumor marker HE4, the kit can provides effective methods for early stage diagnosis and disease monitoring of ovarian cancer, and can play a vital role in clinical research.

Description

technical field [0001] The invention relates to an ELISA kit for detecting human epididymis secretory protein 4 in the technical field of ELISA. Background technique [0002] Ovarian cancer is a common malignant tumor of female reproductive organs. Its incidence rate ranks second only to cervical cancer and endometrial cancer. Due to its hidden onset, it is difficult to be detected early. the first. The current screening methods mainly include gynecological examination, tumor markers and imaging examination. Serological diagnostic testing based on tumor markers is simple and non-invasive, and compared with imaging, it can significantly advance the diagnostic window period of ovarian cancer. It has a very broad application prospect in tumor screening, diagnosis, guiding treatment, and estimating prognosis. . [0003] As far as ovarian cancer is concerned, only the detection of cancer antigen 125 (CA125) is widely used in clinical diagnosis of ovarian cancer. A large numbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/57449G01N2800/7028
Inventor 张杰王加兰黄序
Owner 北京神州细胞生物技术集团股份公司
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