CN110596382A - Tuberculosis diagnostic kit and preparation method thereof - Google Patents
Tuberculosis diagnostic kit and preparation method thereof Download PDFInfo
- Publication number
- CN110596382A CN110596382A CN201910872009.4A CN201910872009A CN110596382A CN 110596382 A CN110596382 A CN 110596382A CN 201910872009 A CN201910872009 A CN 201910872009A CN 110596382 A CN110596382 A CN 110596382A
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- solution
- tuberculosis
- streptavidin
- diagnostic kit
- solid phase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
Abstract
The invention provides a tuberculosis diagnostic kit and a preparation method thereof, belonging to the technical field of medical detection, wherein the kit comprises a streptavidin-coated solid phase carrier, a biotin-labeled antigen, a luminescent substance-labeled antigen, tuberculosis patient positive control serum, normal person control serum, a concentrated washing solution and a substrate solution, wherein the solid phase carrier is superparamagnetic beads, and a binding bacillus antigen is recombinant antigen protein. The kit adopts the solid phase carrier coated by the streptavidin, the streptavidin and the biotin molecule have stronger affinity, and more luminescent substances can be combined, which is beneficial to amplifying detection signals and improving the sensitivity of analysis.
Description
Technical Field
The invention belongs to the technical field of medical detection, and particularly relates to a tuberculosis diagnostic kit and a preparation method thereof.
Background
Tuberculosis is one of the most main infectious diseases in the world, about one third of the population in the world is infected with combined mycobacteria, about 900-.
At present, the diagnosis of tuberculosis is mainly based on clinical symptoms, chest X-ray films and bacteriological examination, for example, the smear examination of tubercle bacillus is simple and easy, but has low positive rate, and the result of the tubercle bacillus culture detection has high reliability but takes long time and can not meet the clinical requirements. The key for reducing the mortality rate of tuberculosis is early diagnosis, and an effective early diagnosis and detection means is helpful for timely and accurately making a treatment scheme. With the rapid development of molecular biology technology, the immunodiagnosis technology for detecting tuberculosis antibodies in serum has the advantages of simplicity, rapidness, no need of precise instruments, easiness in popularization and the like, but some kits have the problems of high false positive, low sensitivity and the like.
Therefore, a tuberculosis diagnostic kit capable of solving the existing problems and a preparation method thereof are urgently needed.
Disclosure of Invention
The invention aims to provide a tuberculosis diagnosis kit and a preparation method thereof aiming at the defects of the prior art, the kit is simple in preparation method, the kit adopts a solid phase carrier coated by streptavidin, the streptavidin and biotin molecules have stronger affinity, more luminescent substances can be combined, the amplification of detection signals is facilitated, and the analysis sensitivity is improved.
The invention provides the following technical scheme:
a tuberculosis diagnostic kit and a preparation method thereof are provided, the kit comprises a streptavidin-coated solid phase carrier, a biotin-labeled antigen, a luminescent substance-labeled antigen, a positive control serum of a tuberculosis patient, a control serum of a normal person, a concentrated washing solution and a substrate solution, wherein the solid phase carrier is superparamagnetic beads, and a binding bacillus antigen is recombinant antigen protein.
Preferably, the luminescent material is a material which can react with a substrate to generate chemiluminescence, and the luminescent material comprises azetidine ester, acridine sulfonamide, alkaline phosphatase, N- (4-aminobutyl) -N-ethyl isoluminol or luminol.
Preferably, the recombinant antigenic protein is the following fusion protein: ESAT-6-connecting peptide-CPF-10 or ESAT-6-connecting peptide-38 KD, in which the connecting peptide is a short peptide which does not affect the immunogenicity of ESAT-6 and CPF-10 or a short peptide which does not affect the immunogenicity of ESAT-6 and 38 KD.
Preferably, the kit is used for detection through enzyme-linked immunosorbent assay.
A method for preparing a tuberculosis diagnostic kit comprises the following steps: coating streptavidin on a solid phase carrier; preparing positive control serum of tuberculosis patients and control serum of normal people; preparing recombinant antigen protein, and marking the recombinant antigen protein with biotin; labeling the recombinant antigen protein with a luminescent substance; preparing a concentrated washing solution and a substrate solution; subpackaging the samples and assembling into finished products.
Preferably, the specific method for coating the solid phase carrier with streptavidin is as follows:
s1, uniformly mixing the PBS buffer solution and streptavidin to prepare coating solution;
s2, taking a magnetic bead stock solution, taking a reaction buffer solution to wash the magnetic beads, then suspending in a coating solution, and reacting overnight at 4-10 ℃;
and S3, washing the reaction product for three times by using a magnetic bead washing solution after the reaction is finished, and finally suspending the reaction product in a magnetic bead storage solution.
The invention has the beneficial effects that:
the streptavidin exists in a form of a homotetramer, has strong affinity with biotin molecules, can be combined with more luminescent substances, is beneficial to amplifying detection signals, and improves the sensitivity of analysis; the invention adopts recombinant antigen protein, such as ESAT-6-connecting peptide-CPF-10 or ESAT-6-connecting peptide-38 KD, ESAT-6, CPF-10 and 38KD, and has the advantages of high sensitivity and high specificity for the serum diagnosis of tuberculosis.
Detailed Description
Example 1
Coating a superparamagnetic magnetic ball with streptavidin; preparing positive control serum of tuberculosis patients and control serum of normal people; preparing recombinant antigen protein, and marking the recombinant antigen protein with biotin; labeling the recombinant antigen protein with alkaline phosphatase; preparing a concentrated washing solution and a substrate solution; subpackaging the samples and assembling into finished products.
Wherein, the recombinant antigen protein is ESAT-6-connecting peptide-38 KD, and the connecting peptide is short peptide which does not influence the immunogenicity of ESAT-6 and 38 KD.
The specific method for coating the superparamagnetic magnetic ball with streptavidin comprises the following steps:
s1, uniformly mixing the PBS buffer solution and streptavidin to prepare coating solution;
s2, taking a magnetic bead stock solution, taking a reaction buffer solution to wash the magnetic beads, then suspending in a coating solution, and reacting overnight at 5 ℃;
and S3, washing the reaction product for three times by using a magnetic bead washing solution after the reaction is finished, and finally suspending the reaction product in a magnetic bead storage solution.
Example 2
Coating a superparamagnetic magnetic ball with streptavidin; preparing positive control serum of tuberculosis patients and control serum of normal people; preparing recombinant antigen protein, and marking the recombinant antigen protein with biotin; labeling a recombinant antigenic protein with an azin ester; preparing a concentrated washing solution and a substrate solution; subpackaging the samples and assembling into finished products.
Wherein, the recombinant antigen protein is ESAT-6-connecting peptide-CPF-10, and the connecting peptide is short peptide which does not influence the immunogenicity of ESAT-6 and CPF-10.
The specific method for coating the superparamagnetic magnetic ball with streptavidin comprises the following steps:
s1, uniformly mixing the PBS buffer solution and streptavidin to prepare coating solution;
s2, taking a magnetic bead stock solution, taking a reaction buffer solution to wash the magnetic beads, then suspending in a coating solution, and reacting overnight at 5 ℃;
and S3, washing the reaction product for three times by using a magnetic bead washing solution after the reaction is finished, and finally suspending the reaction product in a magnetic bead storage solution.
The blood samples of the examples 1 and 2 and the conventional ELISA kit are detected, the antigen used by the conventional ELISA kit is single antigen ESAT-6, the detection samples comprise 150 clinically diagnosed active tuberculosis patient sera, 150 nontuberculous disease patient sera and 150 normal human control sera, and the specific detection results are shown in Table 1.
TABLE 1 comparison of test results
The detection data show that the kit has higher positive detection rate on active tuberculosis patients and obviously lower positive detection rate on non-tuberculous disease patients than the conventional ELISA kit, so that the kit is more suitable for tuberculosis detection and has more accurate detection effect compared with the conventional ELISA kit.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A tuberculosis diagnostic kit is characterized by comprising a streptavidin-coated solid phase carrier, a biotin-labeled antigen, a luminescent substance-labeled antigen, positive control serum of tuberculosis patients, normal control serum, a concentrated washing solution and a substrate solution, wherein the solid phase carrier is superparamagnetic magnetic beads, and a binding bacillus antigen is recombinant antigen protein.
2. A tuberculosis diagnostic kit according to claim 1, wherein the luminescent material is a material which can react with a substrate to generate chemiluminescence, and the luminescent material comprises acridine ester, acridine sulfonamide, alkaline phosphatase, N- (4-aminobutyl) -N-ethyl isoluminol, or luminol.
3. A tuberculosis diagnostic kit according to claim 1, characterized in that, the recombinant antigen protein is the following fusion protein: ESAT-6-connecting peptide-CPF-10 or ESAT-6-connecting peptide-38 KD, in which the connecting peptide is a short peptide which does not affect the immunogenicity of ESAT-6 and CPF-10 or a short peptide which does not affect the immunogenicity of ESAT-6 and 38 KD.
4. A tuberculosis diagnostic kit according to claim 1, wherein the kit is for detection by enzyme linked immunosorbent assay.
5. A preparation method of a tuberculosis diagnostic kit is characterized by comprising the following steps: coating streptavidin on a solid phase carrier; preparing positive control serum of tuberculosis patients and control serum of normal people; preparing recombinant antigen protein, and marking the recombinant antigen protein with biotin; labeling the recombinant antigen protein with a luminescent substance; preparing a concentrated washing solution and a substrate solution; subpackaging the samples and assembling into finished products.
6. The method for preparing a tuberculosis diagnostic kit according to claim 5, characterized in that the specific method for coating streptavidin on the solid phase carrier comprises:
s1, uniformly mixing the PBS buffer solution and streptavidin to prepare coating solution;
s2, taking a magnetic bead stock solution, taking a reaction buffer solution to wash the magnetic beads, then suspending in a coating solution, and reacting overnight at 4-10 ℃;
and S3, washing the reaction product for three times by using a magnetic bead washing solution after the reaction is finished, and finally suspending the reaction product in a magnetic bead storage solution.
Priority Applications (1)
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| CN201910872009.4A CN110596382A (en) | 2019-09-16 | 2019-09-16 | Tuberculosis diagnostic kit and preparation method thereof |
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| CN201910872009.4A CN110596382A (en) | 2019-09-16 | 2019-09-16 | Tuberculosis diagnostic kit and preparation method thereof |
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| CN110596382A true CN110596382A (en) | 2019-12-20 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388378A (en) * | 2002-06-17 | 2003-01-01 | 四川大学 | Tubercle mycobaterium detecting reagent |
| KR20040092299A (en) * | 2003-04-26 | 2004-11-03 | 주식회사 인투젠 | A newly discovered tuberculosis-specific antigen and diagnostic kit using these antigens |
| CN1810838A (en) * | 2005-01-28 | 2006-08-02 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
| CN101235090A (en) * | 2008-01-31 | 2008-08-06 | 复旦大学 | Specificity fusion protein applied to tuberculosis rapid diagnosis |
| CN101377494A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Chemiluminescence immune analytic reagent kit for detecting tuberculosis antibody |
| CN101382548A (en) * | 2008-10-10 | 2009-03-11 | 中国人民解放军总医院第二附属医院 | Tuberculosis antibody multi-antigen ELISA detecting kit and making method |
-
2019
- 2019-09-16 CN CN201910872009.4A patent/CN110596382A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1388378A (en) * | 2002-06-17 | 2003-01-01 | 四川大学 | Tubercle mycobaterium detecting reagent |
| KR20040092299A (en) * | 2003-04-26 | 2004-11-03 | 주식회사 인투젠 | A newly discovered tuberculosis-specific antigen and diagnostic kit using these antigens |
| CN1810838A (en) * | 2005-01-28 | 2006-08-02 | 中国农业科学院哈尔滨兽医研究所 | Recombinant antigen protein for diagnosing ox tuberculosis and its prepn process |
| CN101235090A (en) * | 2008-01-31 | 2008-08-06 | 复旦大学 | Specificity fusion protein applied to tuberculosis rapid diagnosis |
| CN101377494A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Chemiluminescence immune analytic reagent kit for detecting tuberculosis antibody |
| CN101382548A (en) * | 2008-10-10 | 2009-03-11 | 中国人民解放军总医院第二附属医院 | Tuberculosis antibody multi-antigen ELISA detecting kit and making method |
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