CN110501492A - Fat arabian mannan detects sensitizer and combinations thereof and kit - Google Patents
Fat arabian mannan detects sensitizer and combinations thereof and kit Download PDFInfo
- Publication number
- CN110501492A CN110501492A CN201910720315.6A CN201910720315A CN110501492A CN 110501492 A CN110501492 A CN 110501492A CN 201910720315 A CN201910720315 A CN 201910720315A CN 110501492 A CN110501492 A CN 110501492A
- Authority
- CN
- China
- Prior art keywords
- mycobacterium tuberculosis
- seq
- polypeptide shown
- lipoarabinomannan
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
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- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of Mycobacterium tuberculosis LAM antibody test sensitizer, it is polypeptide shown in SEQ ID No 1, or polypeptide shown in polypeptide shown in SEQ ID No 2 and SEQ ID No 3 forms composition with polypeptide shown in SEQ ID No 1 respectively, it is coated with altogether with Mycobacterium tuberculosis LAM antigen again, realizes the detection of mycobacterium tuberculosis.The present invention is total to coated mode using fat arabian mannan antigen and polypeptide (or composition), realizes the specific detection to mycobacterium tuberculosis antibody, and sensitivity with higher, is suitable for vitro detection.
Description
Technical Field
The invention relates to a polypeptide auxiliary agent, in particular to a sensitizer for improving detection sensitivity and specificity, which is used for detecting lipoarabinomannan, a detection composition and a detection kit.
Background
Tuberculosis is an infectious disease that afflicts humans for thousands of years, and it is estimated that the adult population of europe 1/4 of the nineteenth century dies of tuberculosis. By the time of the fifties of the twentieth century tuberculosis has almost been eradicated by industrialized countries due to improvements in standards of living in the twentieth century and the discovery of antibiotics. However, tuberculosis remains rampant as a recurrent infectious disease. Currently, it is estimated that one of three people in the world population is infected with mycobacterium tuberculosis (latent tuberculosis). The total number of new-onset tuberculosis patients in the world is said to be nine hundred and forty-one million, and two million of them are estimated to die of the disease. Currently, approximately 95% of active tuberculosis (active tuberculosis) patients live in developing countries, with 99% of the people who die from tuberculosis concentrating in developing countries.
The LAM antigen tubercle bacillus survives and breeds in the genus host through a unique cell wall structure. The cell wall of tubercle bacillus contains various components, such as: porin (Porin), polysaccharides, Lipids (Lipids), mycolic acid (mycolic acid), phosphatidylinositol mannosides (PIM), Lipomannans (LM), Lipoarabinomannans (LAM). Wherein LAM is the major component of the cell wall of mycobacteria, up to 15 mg/g total weight of bacteria. LAM broadly affects the immune system of the infected host. It has been reported that LAM isolated from mycobacterium tuberculosis and leprosy causes inhibition of T cell proliferation, interference with interferon-mediated macrophage activation, elimination of toxic free oxygen radicals, inhibition of protein kinase activity and gene-encoded Interleukins (IL) -2 and IL-5 and granulocyte synthesis/macrophage colony stimulation of human T cell factor, induction of macrophage immediate early gene expression, promotion of monocyte tumor necrosis factor production and complement activation. Research results show that the LAM serving as a genus specific antigen has stronger immunogenicity and immunoregulation function, participates in a plurality of processes of tubercle bacillus infection, and is beneficial to timely detecting tuberculosis morbidity (tuberculosis) and other mycobacterial infections.
Enzyme-Linked ImmunoSorbent Assay (ELISA) is the most widely used technique in Enzyme immunoassay. The basic method is to adsorb known antigen or antibody on the surface of solid phase carrier (polystyrene micro reaction plate), to make enzyme-labeled antigen-antibody reaction proceed on the solid phase surface, to wash away the free component in the liquid phase by washing method, to detect the absorbance by enzyme-linked immunoassay detector, the method can not only determine the nature, but also determine the quantity of the antigen and antibody of various pathogens, the technique has wide application in medical inspection, scientific research, environmental monitoring and food safety.
The reagent is usually provided in the form of a complete kit, and is matched with a special enzyme-linked immunosorbent assay instrument, the result is judged according to the detected absorbance value, the enzyme used in the reagent is generally horseradish peroxidase (HRP), the specific activity of the enzyme is high and stable, the molecular weight is small, and pure enzyme is easy to prepare, so that the reagent has the advantages of high specific activity, stability and small molecular weightMost commonly used. HRP can be used as a substrate for peroxidase by H2O2And a luminol system, which fluoresces under the action of HRP (horse radish peroxidase). Therefore, the method is most widely applied to clinical diagnosis.
The performance indexes of such kits generally include: assay sensitivity, clinical sensitivity, specificity, precision, accuracy, linear range, stability, and the like. Only if the comprehensive requirements of the indexes are met at the same time, the selling license can be obtained through registering the application on the market.
An enzyme linked immunoassay kit typically comprises the following components: enzyme-linked immunosorbent assay plate, coating antigen (antibody), enzyme-labeled antibody and luminescent substrate. The preparation process comprises the following steps: coating the coated antigen (antibody) on an enzyme-linked immunoassay plate; the luminescence enzyme used is labeled to the antibody.
In the preparation process of the kit, the operation steps of coating the antigen or antibody on the ELISA plate have an important influence on the sensitivity of the kit, and usually, to increase the sensitivity of the kit, an engineer will choose two main solutions, one is to increase the coating concentration, and the other is to increase the marker concentration. In most cases, the two solutions can effectively improve the sensitivity of the test strip. However, in some cases, it is difficult to achieve the goal of increased sensitivity by both of these solutions. In order to meet the performance index requirements of the kit, other technical means are required to improve the sensitivity, so that the development goal can be realized.
Disclosure of Invention
The invention aims to provide a mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer.
Another objective of the invention is to provide a mycobacterium tuberculosis lipoarabinomannan antibody detection composition.
Still another object of the present invention is to provide a kit for detecting antibodies against mycobacterium tuberculosis lipoarabinomannan.
Still another object of the present invention is to provide a pretreatment method for detecting a mycobacterium tuberculosis lipoarabinomannan antibody, in which a lipoarabinomannan antigen is coated in combination with a polypeptide composition by sample pretreatment, for high sensitivity and high specificity in detection of mycobacterium tuberculosis.
In the present invention, the terms "lipoarabinomannan", "lipoarabinomannan antigen" or "antigen of the present invention" are used interchangeably and refer to a glycan derived from mycobacterium tuberculosis that constitutes the cell wall. The lipoarabinomannan antigen can be prepared, isolated and purified by conventional methods.
A mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer is a polypeptide shown in SEQ ID No1, and the polypeptide and a lipoarabinomannan antigen are coated together for improving the sensitivity and specificity of lipoarabinomannan antibody detection.
The mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer further comprises a polypeptide shown in SEQ ID No. 2 or a polypeptide shown in SEQ ID No. 3, and the polypeptide shown in SEQ ID No. 1 forms a composition, wherein the molar ratio of the polypeptide shown in SEQ ID No. 1 to the polypeptide shown in SEQ ID No. 2 or the polypeptide shown in SEQ ID No. 3 is 2-5: 1.
On the detection reaction plate, the coating amount of the mycobacterium tuberculosis lipoarabinomannan antigen is 0.5 mu g/hole to 2 mu g/hole, and the preferable examples are that: 1 μ g/well.
The amount of polypeptide coated on the assay plate is 0.5. mu.g/well to 1. mu.g/well. Such as: the coating amount of the polypeptide shown in SEQ ID No1 is 0.5 mu g/pore-1 mu g/pore, and the coating amount is as follows: the coating amount of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No 2 is 0.5 mu g/hole to 1 mu g/hole; the following steps are repeated: the coating amount of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No 3 is 0.5 mu g/hole to 1 mu g/hole
A method for pretreating a sample to be tested, wherein a sample treatment solution is an acidic solution, such as: perchloric acid, hydrochloric acid, dilute nitric acid, dilute sulfuric acid, trifluoroacetic acid and the like, treating the sample to be detected, and controlling the final pH value of the sample to be detected to be 6.0-8.0.
A kit for detecting a mycobacterium tuberculosis lipoarabinomannan antibody comprises a sample treatment solution, a mycobacterium tuberculosis lipoarabinomannan antigen, a sensitizer and a sample diluent, wherein the mycobacterium tuberculosis lipoarabinomannan antigen and the sensitizer are coated on a detection plate.
Blocking agents are added to the sample dilution, such as: the HIER-E-014, HIER-R-001, HIER-M-004, HIER-M-005 and HIER-E-001 are purchased from Fengcong biological products Ltd, the concentration is preferably 10 mu g/ml to 100 mu g/ml, and the HIER-E-001 is preferably selected.
The kit also comprises a coating plate, an enzyme conjugate working solution, a substrate color development solution A, a substrate color development solution B, a washing solution, a negative and positive control and a stop solution.
Enzyme conjugate working solution: na (Na)2HPO4.12H2O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH2PO40.2g/L, 2.0g/L of horseradish peroxidase protective agent, 500ml/L of calf serum, 500ml/L of goat serum, 1g/L of thimerosal and a proper amount of anti-TB-HRP.
Positive control: na (Na)2HPO4.12H2O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH2PO40.2g/L, 150ml/L ethylene glycol, 250ml/L negative serum, 1g/L merthiolate and positive serum.
Negative control: na (Na)2HPO4.12H2O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH2PO40.2g/L and 150ml/L of ethylene glycol; thimerosal 1g/L and negative serum 250 ml/L.
Washing liquid: NaCl 160 g/L; na (Na)2HPO4.12H2O 58g/L；KCl 4g/L；KH2PO44g/L and Tween-2010 ml/L.
Substrate color developing solution A: 8g/L of citric acid, 0.004g/L of 8-hydroxyquinoline, 2.4ml/L of glacial acetic acid and 0.7g/L of carbamide peroxide.
Substrate color developing solution B: 2.1g/L of citric acid, 0.38g/L of ethylene diamine tetraacetic acid and 0.4g/L of TMB.
Stopping liquid: concentrated sulfuric acid 110 ml/L.
Sample diluent: tris Base 2.4g/L, NaCl 17.8.8 g/L, casein sodium 5g/L, bovine serum albumin 2g/L, Tween-201 ml/L, Triton X-1002 ml/L, goat serum 50ml/L, thimerosal 0.4g/L and blocking agent.
According to the scheme, the lipoarabinomannan antigen is verified to be an antigen which is specific to mycobacterium tuberculosis and is suitable for a combined enzyme immunization method after pretreatment.
The scheme of the invention adopts a mode of coating the lipoarabinomannan antigen and the polypeptide (or the composition) together, realizes the specificity detection of the tubercle bacillus antibody, has higher sensitivity, and is suitable for in vitro detection.
The scheme of the invention also applies the pretreatment method of the serum sample to the enzyme-linked immunosorbent assay detection method for combined coating of the lipoarabinomannan antigen and the polypeptide (or the composition), skillfully utilizes the chemical characteristics of the lipopolysaccharide of the LAM antigen, and successfully detects the antibody of the lipoarabinomannan antigen in the sample by the pretreatment of the serum sample and the use of the polypeptide (or the composition). Compared with the existing mycobacterium tuberculosis detection method, the scheme of the invention has the advantages of obviously improving the aspects of simplicity, rapidity, sensitivity and/or specificity and the like.
Detailed Description
The technical solution of the present invention is described in detail below. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
EXAMPLE 1 pretreatment of samples
And (3) taking 1ml of positive sample, subpackaging the positive sample into 10 centrifuge tubes, wherein each centrifuge tube is 100ul, and adding a proper amount of the sample treatment solution into each centrifuge tube to ensure that the pH value is 1-2. The resulting mixture was milky white due to denaturation of the protein. The denatured protein was centrifuged at 13,000 rpm in a high-speed centrifuge for 5 minutes, 75. mu.l of the supernatant was discarded, and the pH was adjusted to 6.0 to 8.0 with 2M potassium carbonate solution. The sample was cooled to 4 ℃ for 30 minutes to increase the precipitation rate, and the solution was returned to room temperature (25 ℃) for further use before analysis.
EXAMPLE 2 preparation of lipoarabinomannans
Lipoarabinomannans are commercially available (e.g., from Hangzhou Wakura Kingbo Biotech, Inc.) or are self-prepared (see: CN 2010105436856).
Example 3 preparation of coated reaction plate
Preparing a coating solution: adding 0.5mL of sodium carboxymethylcellulose mother liquor into a 200mL reagent bottle, adding 10mL of Tris-HCL mother liquor, adding 10mL of trehalose mother liquor, and supplementing pure water to 100 g.
Taking 15mL of a centrifuge tube, diluting the lipoarabinomannan antigen and the polypeptide (the polypeptide shown in SEQ ID No1, or the combination of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No 2 or the polypeptide shown in SEQ ID No 3 according to the molar ratio of 2-5: 1) by using the coating diluent according to the proper concentration, preparing coating mixed liquor with the proper concentration, taking a luminescent plate for enzyme-linked immunosorbent assay, taking 125 mu l of the luminescent plate per hole, and standing overnight at 2-8 ℃; washing with washing solution for 2 times; sealing the enzyme label plate with 5% BSA blocking solution, wherein each hole is 220 μ l, and the temperature is kept between 2 ℃ and 8 ℃ overnight; and (5) sucking and drying the sealing liquid in the reaction hole, and performing vacuum drying for 3 hours for later use.
Example 4 detection
Diluting the treated sample with a sample diluent (containing a blocking agent) to prepare a detection solution, respectively adding 100 mu l of the detection solution, a positive control substance and a negative control substance into sample adding holes of a detection reaction plate, arranging blank holes, adding 100 mu l of the sample diluent, waiting for 30 minutes, washing for 5 times with a washing solution, adding 150 mu l of an enzyme-labeled antibody, reacting for 30 minutes, washing for 5 times, adding 50 mu l of luminescent substrate, measuring with an enzyme-linked immunosorbent assay, reading, and recording the detection result. Wherein,
cutoff value calculation: COV-negative control mean + 0.12.
The OD value of the specimen is more than or equal to COV and is positive, and the OD value of the specimen is less than COV and is negative.
The negative control OD was calculated to be less than 0.06 and greater than 0.06 as the actual OD.
Samples from the same subjects were also subjected to a control test using conventional dot-blot filtration, and the results are detailed in Table 1.
TABLE 1
In the table, n.a. indicates that the item of data was not available.
The results show that the scheme of the embodiment has higher positive detection rate for tuberculosis patients with positive sputum detection compared with the common dot-blot filtration method. And meanwhile, the serum of healthy adults is detected, so that the false positive result is lower. Meanwhile, the detection of serum samples of hepatitis C positive patients, hepatitis B positive patients and syphilis positive patients shows that the scheme of the embodiment has higher specificity compared with the common dot infiltration method.
Therefore, the scheme provided by the embodiment has higher sensitivity and higher specificity for detecting the mycobacterium tuberculosis.
Sequence listing
<110> Shanghai Rongsheng biopharmaceutical Co., Ltd
<120> lipoarabinomannan detection sensitizer, composition and kit thereof
<141> 2019-08-06
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<213> Artificial Sequence
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His Gln Cys Ala Arg Phe Gln Met Trp His Trp Arg Leu Ile Arg Cys
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<213> Artificial Sequence
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His Gln Cys Ala Arg Phe Gln Met Arg His Trp Arg Leu Ile Arg Cys
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Claims (10)
1. A mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer is characterized by comprising a polypeptide shown in SEQ ID No 1.
2. The mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer according to claim 1, further comprising a polypeptide shown in SEQ ID No 2 or a polypeptide shown in SEQ ID No 3, and forming a composition with the polypeptide shown in SEQ ID No1, wherein the molar ratio of the polypeptide shown in SEQ ID No1 to the polypeptide shown in SEQ ID No 2 or the polypeptide shown in SEQ ID No 3 is 2-5: 1.
3. A mycobacterium tuberculosis lipoarabinomannan antibody detection composition is characterized in that a polypeptide and a lipoarabinomannan antigen are coated together for improving the sensitivity and specificity of lipoarabinomannan antibody detection, the polypeptide is selected from a polypeptide shown in SEQ ID No1, or a mixture of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No 2 according to the molar ratio of 2-5: 1, and the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No 3 according to the molar ratio of 2-5: 1.
4. The Mycobacterium tuberculosis lipid arabinomannan antibody assay composition of claim 3, wherein the amount of the Mycobacterium tuberculosis lipid arabinomannan antigen coated on the assay reaction plate is from 0.5 μ g/well to 2 μ g/well.
5. The Mycobacterium tuberculosis lipid arabinomannan antibody assay composition of claim 3, wherein the amount of the Mycobacterium tuberculosis lipid arabinomannan antigen coated on the assay plate is 1 μ g/well.
6. The Mycobacterium tuberculosis lipoarabinomannan antibody assay composition of claim 3, wherein the polypeptide is coated at a concentration of 0.5 μ g/well to 1 μ g/well on the assay plate.
7. A kit for detecting a Mycobacterium tuberculosis lipoarabinomannan antibody, which comprises a sample treatment solution, a Mycobacterium tuberculosis lipoarabinomannan antigen, a Mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer according to any one of claims 1-2 and a sample diluent, wherein the Mycobacterium tuberculosis lipoarabinomannan antigen and the Mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer according to any one of claims 1-2 are coated on a detection plate.
8. The kit for detecting the antibody against the Mycobacterium tuberculosis lipoarabinomannan of claim 7, wherein the concentration of the blocking agent added to the sample diluent is 10 μ g/ml to 100 μ g/ml.
9. The kit for detecting the antibody against the mycobacterium tuberculosis lipoarabinomannan according to claim 7, wherein the sample treatment solution is an acidic solution selected from one or more of perchloric acid, hydrochloric acid, dilute nitric acid, dilute sulfuric acid and trifluoroacetic acid, and is used for treating the sample to be detected and controlling the final pH value of the sample to be detected to be 6.0-8.0.
10. The kit for detecting the antibody against the mycobacterium tuberculosis lipoarabinomannan of claim 7, wherein the kit further comprises a coated plate, an enzyme conjugate working solution, a substrate developing solution A, a substrate developing solution B, a washing solution, a negative and positive control and a stop solution.
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