Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof

A technology of monoclonal antibody and hemoglobin, applied in the direction of anti-animal/human immunoglobulin, microbial-based methods, biochemical equipment and methods, etc., can solve problems affecting scientific evaluation of drug toxicity, false negative results, etc., and achieve sensitivity High, scientific evaluation, good accuracy and precision

Active Publication Date: 2011-10-26
成都华西海圻医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection of canine fecal occult blood is realized by detecting the presence of canine hemoglobin in the sample to be tested, but there are structural differences between canine hemoglobin and human hemoglobin, resulting in false negative results in most tests, which seriously affects the scientific evaluation of drug toxicity

Method used

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  • Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof
  • Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof
  • Diagnostic kit containing anti-dog hemoglobin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The preparation of embodiment 1 monoclonal antibody

[0020] The flow chart of the preparation of anti-canine hemoglobin monoclonal antibody is as follows: figure 1 shown.

[0021] 1. Antigen preparation

[0022] Canine red blood cells were separated and lysed, and the cell debris was removed by centrifugation, and the supernatant was collected; the supernatant was separated and purified with a Q column, eluted with a salt concentration gradient, and different components were collected; identified by electrophoresis, the results were as follows figure 2 As indicated, the fraction of canine hemoglobin (canine Hb) with a purity greater than 85% detected by SDS-PAGE was collected as the antigen for immunization.

[0023] 2. Preparation of hybridoma cells

[0024] Immune animals: Take six Bal b / c mice (M1-M6 respectively), dilute the immunogen with phosphate buffer saline (PBS), and then mix it with the corresponding adjuvant 1:1, the antigen and adjuvant are completely...

Embodiment 2

[0103] Embodiment 2ELISA kit composition and detection steps

[0104] 1. Kit composition

[0105] (1) Anti-canine hemoglobin monoclonal antibody: prepared as in Example 1 of the present invention, with a concentration of 2.5 mg / mL, and stored at -18 to -24°C.

[0106] (2) HRP-labeled anti-canine hemoglobin polyclonal antibody: prepared as in Example 1 of the present invention, with a concentration of 2.5 mg / mL, and stored at -18 to -24°C.

[0107] (3) Coating solution (carbonate buffer): Na 2 CO 3 1.59g, NaHCO 3 2.93g is fixedly dissolved in 1000mL pure water, the pH is 9.6~9.8.

[0108] (4) PBS buffer: NaCl 8.0g, KCl 0.2g, NaCl 2 HPO 4 2.9g, KH 2 PO 4 0.2g must be dissolved in 1000mL pure water, the pH is 7.2~7.4.

[0109] (5) Washing solution: PBS buffer containing 0.1% Tween20, pH 7.2-7.4.

[0110] (6) Blocking solution: 5.00 g of skimmed milk powder was added into 100 mL of PBS buffer solution and fully dissolved.

[0111] (7) Standard substance diluent: add...

Embodiment 3

[0127] The characteristic of embodiment 3 kits of the present invention

[0128] 1. Standard curve and its linear range verification

[0129] Dilute canine hemoglobin standard with standard diluent (2% skimmed milk powder) to multiple non-zero concentration points, respectively 640ng / mL, 320ng / mL, 160ng / mL, 80ng / mL, 40ng / mL, 20ng / mL , 10ng / mL, 5ng / mL, using the kit and method provided in Example 2 to detect. Test results such as Figure 4 As shown, the correlation coefficient is greater than 0.99, and the linear relationship is good.

[0130] according to Figure 4 As shown in the standard curve, calculate the measured concentration of each group, and the test results are shown in Table 4:

[0131] Table 4 Linear range verification results

[0132] Marking concentration (ng / mL)

320

160

80

40

20

10

5

first day

367.440

138.721

85.807

40.242

19.192

9.878

5.380

second day

317.80...

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Abstract

A diagnostic kit containing an anti-dog hemoglobin monoclonal antibody and an application thereof. The invention relates to a monoclonal antibody, and particularly relates to an anti-dog hemoglobin monoclonal antibody. The invention provides a mouse hybridoma cell strain, which is a strain preserved in China general microbiological culture collection center with a preservation number of CGMCC No. 4619. The invention also provides a monoclonal antibody generated by the mouse hybridoma cell strain with a preservation number of CGMCC No. 4619, and a kit containing the monoclonal antibody. The kit provided by the invention has high sensitivity, strong specificity, good accuracy and precision, and good stability, can detect dog hemoglobin in a sample to be detected specifically and sensitively, can exactly evaluate the gastrointestinal toxicity, the hepatic toxicity, the hematopoietic system toxicity, and the like of a medicament, and provides more scientific and accurate evaluation of the nonclinical safety of a medicament.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to an anti-canine hemoglobin monoclonal antibody. Background technique [0002] Dogs are one of the most commonly used experimental animals in the non-clinical safety evaluation of drugs. The gastrointestinal toxicity and liver toxicity of drugs are routine indicators for non-clinical safety evaluation, and the detection of fecal occult blood in dogs is to determine the gastrointestinal toxicity and liver toxicity of drugs. Important method of toxicity. [0003] At present, there is no dog-specific detection reagent for the detection of fecal occult blood in dogs, and only human-derived reagents can be used. The detection of canine fecal occult blood is realized by detecting the presence of canine hemoglobin in the sample to be tested, but there are structural differences between canine hemoglobin and human hemoglobin, resulting in false negative results in most tests, which seriously affec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577G01N33/543C12R1/91
Inventor 岑小波王莉刘斌
Owner 成都华西海圻医药科技有限公司
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