Preparation and application of tumor marker calreticulin detection kit

A calreticulin and kit technology, applied in the fields of genetic engineering and clinical diagnosis

Inactive Publication Date: 2013-05-08
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the mechanism of tumor development has not been elucidat

Method used

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  • Preparation and application of tumor marker calreticulin detection kit
  • Preparation and application of tumor marker calreticulin detection kit
  • Preparation and application of tumor marker calreticulin detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, preparation and identification of CRT monoclonal antibody

[0045] 1. Preparation of monoclonal antibody

[0046] Before immunizing Balb / c mice, about 100 μl of serum was obtained and stored at -20°C, and then 50 μg of purified CRT antigen was mixed with Freund’s complete adjuvant to make an emulsion, and then immunized by multi-point injection in the back of the skin. One month later, 25 μg of the antigen plus Freund's incomplete adjuvant was injected intradermally for booster immunization three times with an interval of 2 weeks each time. After the titer test reaches the standard, the mouse splenocytes obtained by immunization are fused with SP2 / 0 myeloma cells, the feeder layer cells are mouse peritoneal macrophages, and the fusion reagent is 50% PEG4000, splenocytes and myeloma cells The ratio of cells was 5:1, placed in 5% CO 2 Cultivate in a 37°C incubator with saturated humidity. On the 3rd, 6th, 9th, and 10th day, change into the complete 1640 ...

Embodiment 2

[0059] Example 2, Preparation and Identification of CRT Polyclonal Antibody

[0060] 1. Preparation of multiple antibodies

[0061] First obtain about 5ml serum of New Zealand white rabbits before antigen injection, and then mix 500μg CRT antigen with Freund's complete adjuvant to make emulsion, and inject it into the back of the skin at multiple points for immunization. One month later, 250 μg of the antigen plus Freund's incomplete adjuvant was injected intradermally for 3 booster immunizations, with an interval of 2 weeks each time. After the titer was determined, blood was collected from the carotid artery and centrifuged to collect serum, purified by saturated ammonium sulfate precipitation, and then added with an equal volume of glycerol to store at -20°C.

[0062] 2. Determination of antibody titer by ELISA method

[0063] Coat CRT antigen (concentration: 1 μg / ml, 100 μl per well) with carbonate buffer solution for 16 hours at 4°C;

[0064] Wash the ELISA plate onc...

Embodiment 3

[0073] Embodiment 3, establishment of double-antibody sandwich ELISA detection method

[0074] 1. Basic steps of double-antibody sandwich ELISA assay

[0075] With carbonate buffer (0.05MNa 2 CO 3 / NaHCO 3 , pH9.6)) was coated with 1:800 CRT monoclonal antibody, 100 μl per well was coated at 4°C for 16 hours, and washed once with PBST;

[0076] Add 200 μl blocking solution (1% BSA and 1% goat serum PBS) to block at 37°C for 1 hour, and wash once;

[0077] Add 100 μl of CRT antigen diluted with 1% BSA (0.5 μg / ml, 100 μl / well) at 37°C for 1 hour, and wash 3 times with PBST;

[0078] Add 100 μl CRT polyclonal antibody (4500×), incubate at 37°C for 1 hour, then wash with PBST 3 times;

[0079] Add 100 μl of HRP-labeled goat anti-rabbit antibody (3000×) and incubate at 37°C for 1 hour, wash with PBST 3 times;

[0080] 100μl / well A+B color developing solution was developed for 15 minutes at room temperature in the dark;

[0081] Add 50μl / well of 2mol / LH 2 SO 4 The reactio...

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Abstract

The invention discloses preparation and application of a tumor marker calreticulin detection kit, and belongs to the fields of genetic engineering and clinical diagnosis. A monoclonal antibody and a polyclonal antibody of the CRT (calreticulin) are prepared by recombinant CRT genetic immune BALB/c mice and new Zealand white rabbits; and an ELISA (enzyme-linked immunosorbent assay) kit with high specificity and sensitivity of which the lowest CRT content is 0.3125ng/ml and the detection limit is 40-0.3125ng/ml is built. The collected clinical sample is detected through the prepared ELISA kit; the result shows that CRT in blood serum of a patient with lung cancer is obviously higher than that in normal blood serum; significant statistic difference exists (P is smaller than 0.01); and an experimental method is provided for diagnosis of the clinical patients with lung cancer.

Description

technical field [0001] The invention belongs to the field of genetic engineering and clinical diagnosis, and relates to the preparation and application of a tumor marker calreticulin detection kit. Background technique [0002] Calreticulin (CRT) is a 46KD endoplasmic reticulum molecular chaperone protein with a variety of biological functions. It is not only distributed in the endoplasmic reticulum, but also expressed in the nucleus, nuclear membrane, cell membrane surface and extracellular matrix. Play a variety of biochemical and physiological effects. When tumors occur, secretory CRT has a rising trend in the serum of liver cancer patients and the urine of bladder cancer patients. In addition, studies on gynecological tumors such as breast cancer and cervical cancer have also shown that compared with healthy controls, CRT The expression of CRT has increased; when gastric cancer is studied by immunohistochemical staining, it is found that CRT-positive patients are often ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 刘朝奇吴薇杨建林汪龚泽
Owner CHINA THREE GORGES UNIV
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