Mycobacterium tuberculosis ESAT-6 protein detection kit, as well as preparation method and use method

Abstract

The invention relates to a mycobacterium tuberculosis ESAT-6 protein detection kit based on the nano-immunomagnetic bead-near infrared fluorescence labeling method. In the detection kit, a monoclonal antibody targeting the ESAT-6 is labeled with a near infrared fluorescence dye; the surface of a nano-magnetic bead is coated with a polyclonal antibody targeting the ESAT-6. Based on the double-antibody sandwiching principle, combined substances and free substances are separated from each other magnetically; a portable high-sensitivity low-noise stimulating fluorescence detection instrument is adopted for detection of the fluorescence intensity of a magnetic combined substance, so as to detect the ESAT-6 protein content in a sample to be detected. The detection kit is suitable for ESAT-6 detection of a clinical pathological sample, and has the advantages of being instant, sensitive, stray fluorescence excitation-resistant, and long in emitted fluorescence saving time.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to a detection kit for mycobacterium tuberculosis ESAT-6 protein, a preparation method and a use method. technical background [0002] The invention establishes a nano-magnetic bead-near-infrared fluorescent label-detection kit for detecting the Mycobacterium tuberculosis-associated antigen ESAT-6. Including the near-infrared fluorescent dye labeling method of monoclonal antibodies, the polyclonal antibody coating method of nano magnetic beads and a series of related reagents. The invention uses a near-infrared fluorescent dye as a mark and adopts the principle of immunoassay to prepare a near-infrared fluorescent kit for detecting the ESAT-6 double-antibody sandwich method. The fluorescence intensity of the magnetic bead-conjugate was measured using a portable flow injection fluorescence detector. In quantitative detection, the concentration of the target protein in the test sa...

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Problems solved by technology

[0004] At present, the detection methods for Mycobacterium tuberculosis mainly include smear microscopy. This method requires the detection personnel to have skilled detection skills, and the detection sensitivity of the method is low. Generally, 1*10 3 CFU / ml bacteria have a positive rate of 10-15%, and the bacteria cannot be accurately identified; the culture method is mainly carried out in infectious disease hospitals, and the detection cycle is long, and it generally takes 6 weeks to issue a report Although the automatic microbial identification instrument that appeared recently has greatly shortened the inspection cycle, it usually takes about 2 weeks, this still does not fundamentally solve the problem of efficiency, and there is a defect of high cost. The simple reagent cost is 120 yuan / test, And it requires large-scale equipment; serological testing methods mainly focus on the detection of Mycobacterium tuberculosis-related antibodies, because patients with tuberculosis infection have low immunity, often do not express relevant antibodies, or because adults have a high chance of being infected with Mycobacterium tuberculosis, due to Immunological memory, many people who have been infected can also detect related antibodies, which makes it difficult to judge; molecular biology methods have been widely used in clinical detection recently, such as PCR method, probe detection method, etc. These methods have good specificity, However, special detection instruments are required, and there are problems in popularization and application. The ultra-high sensitivity also brings insurmountable problems-false positives; tuberculosis infection T cell spot test is used to detect gamma-interferon secreted by specific T cells after tuberculosis infection , and based on the results to determine whether you are infected with tuberculosis, this method is characterized by cumbersome, high cost, and is not suitable for promotion. However, it also has the disadvantages of cumbersomeness, high cost, and the need for sufficient experience in judging the results.

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