Immunological detecting kit and preparation method and using method thereof

An immunological detection and kit technology, which is applied in the field of detection kits for lipoprotein-associated phospholipase A2 protein in human plasma, can solve the problems of increasing detection sensitivity, reducing accuracy, and many steps, so as to improve accuracy and accuracy Improvement, False Positive and False Negative Reduction Effects

Inactive Publication Date: 2011-07-13
TIANJIN KANGERKE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biggest disadvantage of the ELISA method is that there are many steps, time-consuming, and due to the use of cascade amplification, while increasing the detection sensitivity, it also brings the problem of reduced accuracy.

Method used

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  • Immunological detecting kit and preparation method and using method thereof
  • Immunological detecting kit and preparation method and using method thereof
  • Immunological detecting kit and preparation method and using method thereof

Examples

Experimental program
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preparation example Construction

[0032] In the preparation method of the kit of the present invention, the directly detectable label is preferably a quantum dot label or a gold nanometer label. In a preferred embodiment, the method for preparing the antibody is a method for preparing monoclonal antibodies using hybridoma technology. In a further preferred embodiment, it also includes the step of preparing another monoclonal antibody that recognizes an antigenic determinant different from the epitope recognized by the monoclonal antibody. This monoclonal antibody can also be prepared by hybridoma technology, for example, by fermentation Hybridomas were generated by tank culture or intraperitoneal injection in mice.

[0033] The kit of the present invention may also include the step of preparing recombinant Lp-PLA2 protein as a standard by fermentation. This standard can be used as a positive control or to create a standard curve.

[0034] According to the components of the kit, the method of using the kit of...

Embodiment 1

[0038] 1. Preparation of monoclonal antibody

[0039] 1) PANWNSPLRPGEKYC; 2) SFGQTKIPRGNGPYC; 3) PSQDNDRLDTLWIPC; 4) CDHGKPVKNALDLKF; 5) QHIMLQNSSGIEKYN immunized mice to prepare monoclonal antibody hybridomas, and selected two hybridomas for fermentation The monoclonal antibodies A and B of Lp-PLA2 protein were produced by tank culture or intraperitoneal injection of mice. The specific operation can be found in (Kohler et al., Nature 256:495, 1975; Kohler et al., Eur.J.Immunol.6 :511,1976; Kohler et al., Eur.J.Immunol.6:292,1976; Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981)

[0040] Purify and concentrate monoclonal antibodies A and B, and carry out formulation, concentration adjustment and sterilized subpackaging respectively. After the monoclonal antibody A is sterilized and subpackaged, it is the detection solution A agent.

[0041] Take monoclonal antibody B and mark it with quantum dots. The following are two methods that can...

Embodiment 2

[0051] The detection of the same process as Example 1, except that the protein to be tested is fibrinogen, or C-reactive protein (C-Reactive Protein, CRP), or thrombosis precursor protein (Thrombus Precursor Protein, TpP), or creatine kinase Creatine Kinase-MB (CK-MB) and other human serum proteins or all other proteins in the detection of cardiovascular and cerebrovascular diseases.

[0052] result

[0053] The detection box reagent of Example 1 was selected to detect the Lp-PLA2 protein concentration of 28 patients diagnosed with coronary heart disease and 21 normal control groups of the same age, and simultaneously compared with the results obtained by using the ELISA method. control. (see Figure 5A and Figure 5B )

[0054] The results showed that two of the 28 cases of the disease group showed false negatives, and one of the 21 normal control groups showed false positives when detected by the ELISA method. However, in the detection results of the kit of the present ...

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Abstract

The invention provides an immunological detecting kit, a preparation method and a using method thereof. The kit comprises a protein antibody which is to be detected and is marked by a marker capable of directly detecting, wherein the protein to be detected is the protein related to heart cerebrovascular disease examination. The protein to be detected comprises one or more of fibrinogen, C-reactive protein, thrombus precussor protein, creatine kinase and human body lipoprotein related phospholipase A2. The abundance or the concentration of the protein to be detected in the sample to be detected can be detected in one step by using the kit, so the step and the time are saved; and compared with the traditional method requiring signal cascade amplification such as enzyme-linked immuno sorbent assay (ELISA) and the like, the accuracy is improved.

Description

technical field [0001] The invention relates to an immunological detection kit used for related examinations of cardiovascular and cerebrovascular diseases, and methods for its preparation and use. In particular, it relates to a detection kit for lipoprotein-associated phospholipase A2 (Lipoprotein-associated Phospholipase A2, abbreviated as Lp-PLA2) protein in human plasma and a preparation and use method thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases have become the number one "killer" that endangers human health in the world. The detection, prevention and treatment of cardiovascular and cerebrovascular diseases, especially the detection, prevention and treatment of coronary artery sclerosis and embolism have always been the focus of medical research all over the world. relentless goal. At present, although three-dimensional angiography can accurately understand the degree of coronary artery blockage and provide key information for vasc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577
Inventor 李惠仇思东钟建
Owner TIANJIN KANGERKE BIOSCI
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