Preparation method of cystatin c detection kit

A technology of cystatin and detection agent, applied in the fields of laboratory medicine and biology, can solve the problems to be improved, etc.

Inactive Publication Date: 2011-12-14
王贤俊
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the SPIAs method can be the first choice in the routine clinical application of determination of serum cystatin C. Tanaka conducted research on the SPIAs method, but the sensitiv...

Method used

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  • Preparation method of cystatin c detection kit
  • Preparation method of cystatin c detection kit
  • Preparation method of cystatin c detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] antigen modification

[0092] 1. Prepare the purified cystatin C antigen according to the above method, and store it at -20°C for future use.

[0093] 2. Coupling of carrier proteins. Take 100mg of purified bovine serum albumin, 2mg of carbodiimide, and 8mg of N-hydroxysuccinimide, dissolve in pH=6.0 phosphate buffer, shake and mix, rotate at 4°C for 4 hours, dialyze 3 times, dilute to 10ml, set aside.

[0094] 3. Take 20 mg of purified and freeze-dried Cys C antigen, dissolve it in carbonate buffer solution with pH = 9.0 to 20 ml, add 10 ml of the carrier activation solution collected in step A into the Cys C antigen solution dropwise, shake and mix, and rotate at 4°C Reacted for 24 hours, dialyzed 3 times, concentrated to 10ml, and stored at 4°C for future use.

[0095] 4. Take 10 mg of phenylalanine, dissolve it in 30 ml of pH=7.4 phosphate buffer, add 10 ml of the solution containing Cys C antigen collected in step B, add 4 mg of carbodiimide, 5 mg of N-hydroxysu...

Embodiment 2

[0098] Antibody preparation

[0099] 1. Select 10 healthy adult male goats aged 2 years old and weighing about 40kg, and move them to the inoculation room. After 1 week of adaptation, start immunization.

[0100] 2. Take rabbit blood before immunizing with the antigen, extract the serum and prepare it as a negative control serum for antibody detection, use Freund’s complete adjuvant (FCA) as the antigen emulsion for the first vaccination, and use Freund’s complete adjuvant (FCA) for the second vaccination. Complete adjuvant (FIA) is an antigen emulsion.

[0101] 3. The first inoculation is by subcutaneous injection on the inner side of the hind leg and multi-point injection in the retroperitoneal cavity, 3ml / head, 0.5ml per point. The second inoculation was multi-point injection on the back and front abdomen, 3ml / head, 0.5ml per point. The third and fourth inoculations were the same as the second inoculation, and the interval between each inoculation was 2 weeks.

[0102] 4...

Embodiment 3

[0105] Preparation of immune latex

[0106] 1. Activation of carboxylated polystyrene latex: take 0.4ml of 10% carboxylated polystyrene latex and place it in a centrifuge tube, wash with 1ml PBS buffer solution for 3 times, centrifuge at 14000r / min, and discard the supernatant. Add 0.5ml of water-soluble carbodiimide (EDC) and 0.5ml of PBS, stir at room temperature for 30min, wash with borate buffer three times, centrifuge at 14000r / min, discard the supernatant, and resuspend with 1ml of borate buffer for use.

[0107] 2. Add 0.2ml of Cys C antibody to the latex suspension, shake on a shaker at room temperature for 4 hours, add 100μl of glycine terminator, react for 30min, centrifuge at 14000r / min, discard the supernatant, and reconstitute with PBS buffer with pH 7.4. Suspended and stored at 4°C for later use.

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Abstract

The invention relates to a preparation method of cystatin C antibody and a preparation method of a detection kit. Cystatin C (Cys C) is currently one of the most sensitive diagnostic markers for clinical kidney disease. The antibody preparation of the present invention is extracted and purified from human normal serum, and then a highly sensitive antigen is obtained by artificial modification. Animals were immunized multiple times to obtain hyperimmune serum containing Cys C antibody, and high-purity Cys C antibody was obtained by affinity chromatography. During the preparation process, the loss of antibody activity is small, and it is easy to produce in batches, and the antibody has high affinity and high titer, and does not generate cross-immune reaction. After the antibody is coupled with polystyrene latex balls, the prepared detection kit has higher sensitivity and linear range, good repeatability, low detection limit, easy automatic analysis, fast and simple operation. Due to the high purity of the antibody, the content of heteroantibody is very small, so there is less interference and the matrix is ​​stable.

Description

[0001] Technical field The present invention relates to a preparation method of a cystatin C detection kit, which belongs to the field of laboratory medicine and biotechnology. Background technique [0002] Cystatin C (Cys C) is a low molecular weight protein that can be produced by all nucleated cells in the body with a constant yield. Cystatin C in human circulation is only cleared by glomerular filtration, which is an ideal endogenous marker to reflect changes in glomerular filtration rate, while glomerular filtration rate (glomerular filtration rate rate, GFR) is an important indicator for monitoring renal function, especially for kidney transplant patients, it is very important to quickly and accurately grasp the changes in glomerular filtration rate. In addition, because the molecular weight of cystatin C is greater than that of creatinine, and it is positively charged, it is more likely to reflect the early changes in the permeability of the glomerular filtration membra...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K16/18
Inventor 王贤俊李锐吴蓓蕾
Owner 王贤俊
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