CN111077310A - Enzyme linked immunosorbent assay kit for detecting fever with thrombocytopenia syndrome virus antigen - Google Patents
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Abstract
The invention discloses an enzyme linked immunosorbent assay kit for detecting a fever with thrombocytopenia syndrome virus antigen, which comprises: reagent A: ELISA coated plates coated with capture antibody; and (3) reagent B: PBS buffer solution; and (3) reagent C: antigen NP solution with concentration of 15.6 ng/mL; and (3) reagent D: AuNP-Ab with gold nano particle concentration of 100 mu g/mL2-HRP probe concentrate; and (3) reagent E: 0.01M PBS buffer pH7.4 containing 5% BSA as probe diluent; and (3) reagent F: skimmed milk powder; reagent G: 3,3',5,5' -tetramethylbenzidine; and (3) reagent H: 0.1M HCL. The invention has high sensitivity and strong specificity, and enhances the concentration of enzyme aggregation on each immune sandwich structure. The absorbance value under specific wavelength is measured, and the fever with thrombocytopenia syndrome virus antigen NP can be qualitatively and quantitatively detected.
Description
Technical Field
The invention relates to the technical field of disease detection, in particular to an enzyme linked immunosorbent assay kit for detecting a virus antigen of fever with thrombocytopenia syndrome.
Background
Severe febrile illnesses associated with gastrointestinal symptoms, thrombocytopenia and leukopenia were present in the eastern and middle parts of china between 2007 and 2010 with a high mortality rate. This disease is known as fever with thrombocytopenia syndrome (SFTS) and is caused by the newly discovered bunyavirus (SFTSV). Subsequently, SFTS was sequentially confirmed in korea, japan. SFTSV is reported to be a negative-strand segmented RNA virus consisting of three segments (L, M and S). The L, M and S segments encode RNA-dependent RNA polymerase, precursors of glycoproteins (Gn and Gc), Nucleocapsid Protein (NP) and nonstructural protein (NS), respectively. The Nucleocapsid Protein (NP) is closely associated with viral replication, is highly immunogenic and conserved, and the N protein is often selected as a target for antigen and antibody detection. Therefore, the detection of the surface antigen of the fever with thrombocytopenia syndrome is very significant.
Nanotechnology refers to the science and technology of studying the properties and interactions of substances, such as atoms and molecules, on the nanometer scale (between 1nm and l00 nm), and the multidisciplinary intersection of these properties. When the size of a substance is as small as 1-100 nm, the quantum effect, the substance locality and the huge surface and interface effect of the substance cause a plurality of properties of the substance to be changed qualitatively, and a plurality of singular phenomena different from a macroscopic object and a single isolated atom are presented. Gold nanoparticles have simple and efficient surface modifications that are compatible with biomolecules, and have high surface areas that allow them to carry many biomolecules (e.g., antibodies, enzymes, DNA, and other biomolecules) to produce significant signal enhancement.
Horse radish peroxidase is the labeling enzyme which is most widely applied in ELISA so far, mainly because the horse radish peroxidase is easy to extract and relatively low in price; on the other hand, the compound has stable property, and the activity is rarely lost after the compound is coupled with antigen or antibody. The substrate TMB (3,3',5,5' -tetramethyl benzidine) of horseradish peroxidase is a group with strong fat solubility, is easy to form polymer, generates coarse and deep blue precipitate at the active site of HRP, and then is added with sulfuric acid or phosphoric acid, has maximum absorbance at 450nm, and turns yellow. TMB is very sensitive and if too much protein or antibody is used, a significant background signal may be generated. Compared with other HRP substrates, TMB is oxidized more rapidly, and thus color development is faster. This makes TMB a good chromophore in combinatorial experiments. Meanwhile, the precipitation of reaction products enables active sites of HRP to be more exposed, and the enzymatic oxidation reaction is facilitated.
SFTS and other infectious diseases are difficult to diagnose clinically without microbial detection. Therefore, SFTS diagnostics require laboratory testing. Several methods of genome amplification for SFTS diagnosis have been reported, however, the genome amplification technology is limited by the requirement of expensive equipment and technical expertise thereof. As is well known, ELISA has good specificity, low cost and simple reading method, and is becoming the gold standard for laboratory and clinical analysis. The minimum detection limit of ELISA was 0.1ngmL-1To 1mg mL-1. However, the concentration of relevant biomarkers in various diseases at early stages is sometimes below the detection limit of ELISA, and therefore, development of an ultra-sensitive detection method for different types of biomarkers is urgently required, and increasing the amount of horseradish peroxidase (HRP) immobilized on signal particles has also been proven to be an effective means for improving ELISA signals.
At present, an enzyme linked immunosorbent assay kit for detecting the antigen of the fever with thrombocytopenia syndrome virus based on a horseradish peroxidase and antibody double-labeled gold probe detects the surface antigen of the fever with thrombocytopenia syndrome virus, and the detection is not reported.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an enzyme linked immunosorbent assay kit for detecting a virus antigen of fever with thrombocytopenia syndrome.
An enzyme linked immunosorbent assay kit for detecting a fever with thrombocytopenia syndrome virus antigen comprises:
reagent A: ELISA coated plates coated with capture antibody;
and (3) reagent B: 30 × 0.3M pH7.4 PBS buffer;
and (3) reagent C: antigen NP solution with concentration of 15.6 ng/mL;
and (3) reagent D: AuNP-Ab with gold nano particle concentration of 100 mu g/mL2-HRP probe concentrate;
and (3) reagent E: 0.01M PBS buffer pH7.4 containing 5% BSA as probe diluent;
and (3) reagent F: skimmed milk powder;
reagent G: 3,3',5,5' -tetramethylbenzidine;
and (3) reagent H: 0.1M HCL.
The capture antibody coated ELISA coated plate is prepared by the following method: diluting rabbit polyclonal antibody of NP antigen of SFTSV to 40 mu g/mL by using 0.05M sodium carbonate buffer solution with pH being 9.6, adding a 96-well enzyme label plate, keeping stand at 4 ℃ for 16h, and washing by using a washing buffer solution; adding 5% of skimmed milk by mass, sealing at 37 deg.C for 2 hr, washing with washing buffer, drying on clean absorbent paper, placing into a sealed bag, vacuumizing, and standing at 4 deg.C; SFTSV is an abbreviation for fever with thrombocytopenia syndrome virus, and NP is an abbreviation for nucleoprotein.
An antigen NP solution at a concentration of 15.6ng/mL was prepared as follows: cloning nucleotide sequence of NP of fever-associated-thrombocytopenia syndrome virus GENBANK MF574213.1 into pET30a expression vector, transferring into BL21(DE3) competent cell, selecting correct clone, culturing, adding IPTG, culturing, purifying, taking out 1. mu.L, diluting to 15.6ng/mL with PBS (pH 7.4), adding BSA to make mass content be 1%, obtaining antigen NP solution with concentration of 15.6 ng/mL.
AuNP-Ab with gold nano particle concentration of 100 mu g/mL2The HRP probe concentrate was prepared by the following method: regulating the pH of a newly prepared gold nanoparticle aqueous solution with the concentration of 50 mu g/mL to 7.0-8.0 by using a 0.2M potassium carbonate aqueous solution, adding an antibody labeled by horseradish peroxidase with the final concentration of 15 mu g/mL, stirring at 4 ℃ for 12h after mixing, adding PEG20000 with the final mass concentration of 0.2%, stirring at room temperature for 30min, and centrifuging at 10000rpm for 40min to obtain AuNP-Ab supernatant2Washing with double distilled water (pH7.0-8.0) for 2 times by using an HRP probe, and redissolving in a storage buffer solution to prepare the AuNP-Ab with the gold nanoparticle concentration of 100 mu g/mL2HRP probe concentrate was stored at 4 ℃ until use.
The invention has the advantages that:
1. the horseradish peroxidase is an enzyme which is easy to extract, low in price and stable in property. The substrate TMB (3,3',5,5' -tetramethyl benzidine) is very sensitive, and compared with other HRP substrates, the oxidation speed of the TMB is higher, so that the color development is faster.
2. The traditional detection technology of enzyme-linked immunosorbent assay (ELISA) with the advantages of high sensitivity, strong specificity and the like is adopted, and the detection efficiency is favorably improved.
3. Horse radish peroxidase HRP labeled fever with thrombocytopenia syndrome virus antibody Ab by electrostatic adsorption2Preparation of enzyme-linked detection probe AuNP-Ab connected to gold nano-particle2HRP, which enhances the concentration of enzyme aggregation on each immunological sandwich. By obtaining the absorbance value of the reaction product of the sandwich structure and the substrate TMB under a specific wavelength (450nm), the fever with thrombocytopenia syndrome virus antigen NP can be qualitatively and quantitatively detected.
Drawings
FIG. 1 AuNP-Ab prepared according to the invention2Schematic representation of HRP probe.
FIG. 2 is an ELISA standard curve for detecting the antigen of the fever with thrombocytopenia syndrome virus prepared by the reagent of the present invention.
FIG. 3 is an ELISA specificity curve of the reagent prepared by the invention for detecting the surface antigen of fever with thrombocytopenia syndrome virus.
Detailed Description
In view of the importance of serological detection in virus detection, the unique signal amplification characteristics of nanotechnology and the advantages of enzyme-linked immunoassay. Two technologies of enzyme-linked immunosorbent assay and nanotechnology are combined, and gold nanoparticles are coupled with horseradish peroxidase-labeled antibody molecules to prepare a detection probe. The probe, the virus antigen and a fever with thrombocytopenia syndrome virus antibody Ab1 embedded in an enzyme-linked immunosorbent assay (ELISA) kit form a sandwich-like structure through the action of the antibody antigens, and finally, the virus antigen is quantitatively detected through the absorbance of a reaction product of enzyme and a substrate TMB (3,3',5,5' -tetramethylbenzidine) at a specific wavelength (450nm), so that a new method for serological detection is established, and the detection sensitivity is greatly improved.
3,3',5,5' -Tetramethylbenzidine (Beijing Solebao biotech Co., Ltd., Cat. PR1200)
The present invention will be further illustrated by the following specific examples.
Example 1
An ELISA coated plate coated with capture antibody was prepared by the following method: diluting rabbit polyclonal antibody (Kyoto Biotechnology Co., Ltd.) against NP antigen of SFTSV to 40. mu.g/mL with 0.05M sodium carbonate buffer solution with pH of 9.6, adding 96-well enzyme-labeled plate, standing at 4 deg.C for 16h, and washing with washing buffer solution; adding 5% of skimmed milk by mass, sealing at 37 deg.C for 2 hr, washing with washing buffer, drying on clean absorbent paper, placing into a sealed bag, vacuumizing, and standing at 4 deg.C; SFTSV is an abbreviation for fever with thrombocytopenia syndrome virus, NP is an abbreviation for nucleoprotein.
The washing buffer was 0.01M PBS buffer, pH7.4.
Example 2
An antigen NP solution at a concentration of 15.6ng/mL was prepared as follows:
artificially synthesizing a nucleotide sequence of NP of fever-associated-thrombocytopenia syndrome virus (GENBANK: MF 574213.1), cloning the nucleotide sequence to a pET30a expression vector (commercial product), transferring to BL21(DE3) competent cells (commercial product), uniformly coating the cells on a solid LB culture medium containing 50 mu g/mL kanamycin resistance, after overnight culture at 37 ℃, selecting correct clones, inoculating the correct clones to a 2 XYT culture medium containing 50 mu g/mL kanamycin resistance, culturing at 37 ℃ until the OD value reaches 0.6, and adding IPTG to the final concentration of 1mM, 200rpm and overnight culture at 30 ℃; centrifuging at 4 ℃, 8000rpm for 20min, discarding the culture supernatant, resuspending the bacterial solution with PBS, centrifuging, and discarding the supernatant; resuspending the bacterial solution with PBS, carrying out ice-bath ultrasonic crushing with the power of 800W, carrying out ultrasonic treatment for 5s, stopping the ultrasonic treatment for 5s, and repeating the steps for 200 times; centrifuging at 4 deg.C and 10000rpm for 3h, and collecting supernatant; purifying protein by using His Trap FF nickel ion purification column and matching with AKTA instrument; eluting the target protein with elution buffer solution, concentrating by using an ultrafiltration concentration tube, simultaneously changing the elution buffer solution into 1 XPBS, identifying NP by SDS-PAGE, subpackaging and freezing at-80 ℃; mu.L of the thus-obtained sample was diluted with PBS to 15.6ng/mL, and BSA was added thereto so that the mass content became 1%, whereby an antigen NP solution having a concentration of 15.6ng/mL, that is, reagent C, was obtained.
Example 3
AuNP-Ab with gold nano particle concentration of 100 mu g/mL2The HRP probe concentrate was prepared by the following method:
regulating pH of newly prepared Gold nanoparticle aqueous solution (Gold nanoparticles, AuNPs) of 50 μ g/mL with 0.2M potassium carbonate aqueous solution to 7.0 (or any value between 7.0-8.0), adding horseradish peroxidase-labeled antibody of 15 μ g/mL, mixing, stirring at 4 deg.C for 12h, adding PEG20000 of 0.2% of final mass concentration, stirring at room temperature for 30min, centrifuging at 10000rpm for 40min, discarding supernatant to obtain AuNP-Ab2HRP probe, washed 2 times with double distilled water (pH7.0 (can be any value between 7.0-8.0)), and redissolved in storage buffer to prepare AuNP-Ab with gold nanoparticle concentration of 100 μ g/mL2HRP probe concentrate, stored at 4 ℃ for future use;
the horseradish peroxidase-labeled antibody was a monoclonal antibody (1H10) against NP antigen of SFTSV (Kyoto Biotechnology Co., Ltd.)
The storage buffer was 0.01M water pH7.4, BSA 0.5 mass%, sucrose 2.0 mass%, glycerol 50 mass%.
Example 4
An enzyme linked immunosorbent assay kit for detecting a fever with thrombocytopenia syndrome virus antigen comprises:
reagent A: ELISA coated plates coated with capture antibody; example 1 preparation
And (3) reagent B: 30 × concentrated 0.3M pH7.4 PBS buffer;
and (3) reagent C: antigen NP solution with concentration of 15.6 ng/mL; example 2 preparation
And (3) reagent D: 20 Xgold nanoparticle concentration of 100. mu.g/mL AuNP-Ab2-HRP probe concentrate; example 3 preparation
And (3) reagent E: 0.01M PBS buffer pH7.4 containing 5% BSA as probe diluent;
and (3) reagent F: skimmed milk powder;
reagent G: 3,3',5,5' -tetramethylbenzidine (Beijing Solebao Biotechnology Co., Ltd., Cat. PR 1200);
and (3) reagent H: 0.1M HCL.
Example 5
The usage of the enzyme linked immunosorbent assay kit for detecting the virus antigen of the fever with thrombocytopenia syndrome:
temperature conditions of 37 ℃ and shaking conditions of 30rpm were required throughout the process. The reagent B was diluted 30 times with double distilled water to obtain 1 Xreagent B, and the reagent E was diluted 20 Xreagent D to obtain 1 Xreagent D. Adding 200. mu.L of 5% reagent F (solvent is 1 Xreagent B) to reagent A, blocking for 1h, and washing 6 times with 1 Xreagent B; diluting reagent C with 5% reagent F (solvent is 1 × reagent B) by gradient to obtain antigen dilution solution with concentration of 3.9, 1.0, 0.25, 0.0625, 0.0156, 0.004, 0.001, 0.00025 ng/mL;
simultaneously selecting purified proteins of BSA and Hemagglutinin (HA) and a purified capsid protein (VP1) of human enterovirus D68 type as unrelated proteins to carry out specificity experiments, and diluting the three unrelated proteins by using a reagent F (a solvent is 1 multiplied by a reagent B) with the mass concentration of 5% to ensure that the final concentrations are all 200 ng/mL;
sequentially adding 100 mu L of antigen diluent with different concentrations and three kinds of unrelated protein diluent with 100 mu L of each concentration into the treated reagent A, shaking and incubating for 1h, and washing for 6 times by using 1 multiplied by the reagent B; adding 100 μ L of 1 × reagent D, washing 6 times with 1 × reagent B, adding 100 μ L of reagent G, reacting at room temperature for 15 min, terminating the reaction with 100 μ L of reagent H, reading absorbance of 96-well ELISA plate with microplate reader, establishing standard curve, and qualitatively and quantitatively detecting NP, as shown in FIG. 2. The specificity of the kit is shown in FIG. 3. The four parameter equation for the standard curve is: y ═ (0.07204-5.02809E6)/(1+ (x/7080030)0.99963)+5028090. For the detection of real samples, different from the step of adding the antigen, the virus culture supernatant is cracked by 1% Triton X-100 for 40min, diluted by 640 times by using a reagent F (the solvent is 1 multiplied by the reagent B) with the mass concentration of 5%, then added into a reagent A (100 mu L), the rest steps are the same as the steps, the OD value obtained after color development is 0.54775, the OD value is substituted into a standard curve to obtain the antigen content of 0.70379ng/mL, and the product is multiplied by the dilution times to obtain the antigen content of a sample of 450.4256 ng/mL.
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting a fever with thrombocytopenia syndrome virus antigen is characterized by comprising:
reagent A: ELISA coated plates coated with capture antibody;
and (3) reagent B: 30 × 0.3M pH7.4 PBS buffer;
and (3) reagent C: antigen NP solution with concentration of 15.6 ng/mL;
and (3) reagent D: AuNP-Ab with gold nano particle concentration of 100 mu g/mL2-HRP probe concentrate;
and (3) reagent E: 0.01M PBS buffer pH7.4 containing 5% BSA as probe diluent;
and (3) reagent F: skimmed milk powder;
reagent G: 3,3',5,5' -tetramethylbenzidine;
and (3) reagent H: 0.1M HCL.
2. The kit according to claim 1, wherein the capture antibody-coated ELISA plate is prepared by the following method: diluting rabbit polyclonal antibody of NP antigen of SFTSV to 40 mu g/mL by using 0.05M sodium carbonate buffer solution with pH being 9.6, adding a 96-well enzyme label plate, keeping stand at 4 ℃ for 16h, and washing by using a washing buffer solution; adding 5% of skimmed milk by mass, sealing at 37 deg.C for 2 hr, washing with washing buffer, drying on clean absorbent paper, placing into a sealed bag, vacuumizing, and standing at 4 deg.C; SFTSV is an abbreviation for fever with thrombocytopenia syndrome virus, and NP is an abbreviation for nucleoprotein.
3. The kit of claim 1, wherein the antigen NP solution at a concentration of 15.6ng/mL is prepared by the following method: cloning nucleotide sequence of NP of fever-associated-thrombocytopenia syndrome virus GENBANK MF574213.1 into pET30a expression vector, transferring into BL21(DE3) competent cell, selecting correct clone, culturing, adding IPTG, culturing, purifying, taking out 1. mu.L, diluting to 15.6ng/mL with PBS (pH 7.4), adding BSA to make mass content be 1%, obtaining antigen NP solution with concentration of 15.6 ng/mL.
4. The kit of claim 1, wherein the gold nanoparticles are AuNP-Ab at a concentration of 100 μ g/mL2The HRP probe concentrate used was as followsThe method comprises the following steps: regulating the pH of a newly prepared gold nanoparticle aqueous solution with the concentration of 50 mu g/mL to 7.0-8.0 by using a 0.2M potassium carbonate aqueous solution, adding an antibody labeled by horseradish peroxidase with the final concentration of 15 mu g/mL, stirring at 4 ℃ for 12h after mixing, adding PEG20000 with the final mass concentration of 0.2%, stirring at room temperature for 30min, and centrifuging at 10000rpm for 40min to obtain AuNP-Ab supernatant2Washing with double distilled water (pH7.0-8.0) for 2 times by using an HRP probe, and redissolving in a storage buffer solution to prepare the AuNP-Ab with the gold nanoparticle concentration of 100 mu g/mL2HRP probe concentrate was stored at 4 ℃ until use.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733287A (en) * | 2020-06-22 | 2020-10-02 | 天津大学 | Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome |
| CN113249334A (en) * | 2021-03-16 | 2021-08-13 | 贵州省人民医院 | Hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-associated thrombocytopenia syndrome virus |
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| CN103399148A (en) * | 2013-08-06 | 2013-11-20 | 西南大学 | Human respiratory syncytial virus detection kit based on nanogold signal amplification |
| CN105510580A (en) * | 2015-12-01 | 2016-04-20 | 浙江普康生物技术股份有限公司 | Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus) |
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| CN103399148A (en) * | 2013-08-06 | 2013-11-20 | 西南大学 | Human respiratory syncytial virus detection kit based on nanogold signal amplification |
| CN105510580A (en) * | 2015-12-01 | 2016-04-20 | 浙江普康生物技术股份有限公司 | Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733287A (en) * | 2020-06-22 | 2020-10-02 | 天津大学 | Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome |
| CN111733287B (en) * | 2020-06-22 | 2022-10-11 | 天津大学 | Kit for detecting pathogenic nucleic acid of fever with thrombocytopenia syndrome |
| CN113249334A (en) * | 2021-03-16 | 2021-08-13 | 贵州省人民医院 | Hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-associated thrombocytopenia syndrome virus |
| CN113249334B (en) * | 2021-03-16 | 2023-12-08 | 贵州省人民医院 | Hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody |
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