CN114113596A - Helicobacter pylori colloidal gold typing detection device and preparation method thereof - Google Patents
Helicobacter pylori colloidal gold typing detection device and preparation method thereof Download PDFInfo
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Abstract
本发明公开了一种幽门螺旋杆菌胶体金分型检测装置及其制备方法，检测装置包括底板，在所述底板的表面依次排列样品垫、胶体金结合垫、层析膜和吸收垫这四个部件，所述胶体金结合垫上包含胶体金标记抗人IgG的单抗；所述层析膜上设有包被有重组幽门螺杆菌细胞毒(CagA)抗原、重组幽门螺杆菌空泡毒(VacA)抗原和重组幽门螺杆菌尿素酶亚单位B蛋白(UreB)抗原的三条检测线和包被有羊抗鼠IgG多克隆抗体的质控线，且上述的三条检测线及质控线相互平行。该检测装置操作简单，适用范围广，检测成本低，适用于临床检测、现场调查、以及流行病学调查。
The invention discloses a colloidal gold typing detection device for Helicobacter pylori and a preparation method thereof. The detection device comprises a bottom plate, and four sample pads, colloidal gold binding pads, chromatographic membranes and absorption pads are sequentially arranged on the surface of the bottom plate. Parts, the colloidal gold binding pad contains colloidal gold-labeled anti-human IgG monoclonal antibody; the chromatographic membrane is coated with recombinant Helicobacter pylori cytotoxic (CagA) antigen, recombinant Helicobacter pylori vacuolar virus (VacA ) antigen and three detection lines of recombinant Helicobacter pylori urease subunit B protein (UreB) antigen and a quality control line coated with goat anti-mouse IgG polyclonal antibody, and the above three detection lines and quality control lines are parallel to each other. The detection device is simple to operate, has a wide range of applications, and has low detection cost, and is suitable for clinical detection, field investigation, and epidemiological investigation.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a helicobacter pylori colloidal gold typing detection device and a preparation method thereof.
Background
Helicobacter pylori (Hp) is a gram-negative, microaerophilic bacterium. The Hp infection is closely related to various diseases, including digestive system diseases such as chronic gastritis, peptic ulcer, gastric mucosa-related lymphoid tissue lymphoma, gastric cancer and the like. China is a high-incidence country of helicobacter pylori infection, the infection rate is more than 50%, but because the pathogenicity of helicobacter pylori is inconsistent (low toxicity type and high toxicity type), not all helicobacter pylori infections need to be treated. Therefore, the diagnosis of whether the helicobacter pylori is infected and which helicobacter pylori is infected have important guiding significance for clinical treatment.
The detection method of helicobacter pylori is classified into non-invasive and invasive tests depending on whether endoscopic assistance is required. Invasive tests include endoscopy, rapid urease tests, bacterial culture, histopathological examination. Invasive methods take biopsies mainly through an endoscope for further histological examination. However, these methods often cause invasive pain to patients, require certain conditions and techniques, are time-consuming and labor-consuming, and are not favorable for rapid clinical mass detection.
Non-invasive tests include urea breath tests, serological tests, fecal antigen testing. Non-invasive tests are effective in avoiding invasive pain, but these methods are either time consuming and costly, or fail to distinguish between active and past infections, or diagnostic accuracy is susceptible to sample storage, and none of the above methods are amenable to typing.
The existing mainstream technology for detecting helicobacter pylori can only judge whether helicobacter pylori is infected, and typing detection cannot be carried out, and the problem is not solved.
Disclosure of Invention
The invention provides a helicobacter pylori colloidal gold typing detection device and a preparation method thereof, which aim to overcome the defects of a helicobacter pylori detection technology in the prior art.
The technical scheme adopted by the invention is as follows: a colloidal gold typing detection device for helicobacter pylori comprises a bottom plate, wherein four components, namely a sample pad, a colloidal gold combination pad, a chromatographic membrane and an absorption pad, are sequentially arranged on the surface of the bottom plate, and the colloidal gold combination pad contains a monoclonal antibody of anti-human IgG marked by colloidal gold; the chromatographic membrane is provided with three detection lines coated with recombinant HP cytotoxic (CagA) antigen, recombinant HP vacuolating toxic (VacA) antigen and recombinant HP urease subunit B protein (UreB) antigen and a quality control line coated with goat anti-mouse IgG polyclonal antibody, and the three detection lines and the quality control line are parallel to each other.
Preferably, the coating concentration of the recombinant HP cytotoxic (CagA) antigen, the recombinant HP vacuolating toxic (VacA) antigen and the recombinant HP urease subunit B protein (UreB) antigen coated on the mass detection line is 1-10 mg/mL; the optimal coating concentration of the goat anti-mouse IgG polyclonal antibody coated on the quality control line is 2-10 mg/mL.
Preferably, the chromatographic membrane is provided with three detection lines at one side closer to the sample adding end, and the quality control line at one side farther from the sample adding end.
Preferably, the distance between every two detection lines is 4-10 mm; the distance between the last detection line and the quality control line is 3-10 mm.
Preferably, the sample pad, the gold colloidal conjugate pad, the chromatographic carrier and the absorbent pad are arranged in four parts, and the edges of the four parts are overlapped.
The invention provides a preparation method of a helicobacter pylori colloidal gold typing detection device, which comprises the following steps:
A. preparing colloidal gold by a trisodium citrate reduction method;
B. coupling the mouse anti-human IgG monoclonal antibody and colloidal gold at the optimal pH value to prepare a colloidal gold-labeled mouse anti-human IgG monoclonal antibody solution, spraying the colloidal gold-labeled mouse anti-human IgG monoclonal antibody solution on a colloidal gold bonding pad, and drying for later use;
C. diluting the recombinant HP cytotoxic (CagA) antigen, the recombinant HP vacuolating toxin (VacA) antigen, the recombinant HP urease subunit B protein (UreB) antigen and the goat anti-mouse IgG polyclonal antibody, spraying the diluted recombinant HP cytotoxic (CagA) antigen, the recombinant HP vacuolating toxin subunit B protein (UreB) antigen and the recombinant HP anti-mouse IgG polyclonal antibody onto a chromatographic membrane to form a detection line and a quality control line respectively, and drying for later use;
D. and sequentially adhering the sample pad, the colloidal gold combined pad, the chromatographic membrane and the absorption pad on a bottom plate, cutting to obtain a detection reagent strip, and finally packaging the detection reagent strip into a plastic shell.
Preferably, the grain size of the colloidal gold in the step A of the preparation method of the helicobacter pylori colloidal gold typing detection device is 10-40 nm.
Preferably, the optimal pH value in the step B of the preparation method of the helicobacter pylori colloidal gold typing detection device is 8.0-9.5.
The Gold Immunochromatography (GICA) technique is a rapid diagnostic technique that applies gold colloid as a tracer to antigen-antibody reactions. The sample containing the substance to be detected is moved up along the membrane by the capillary action of the nitrocellulose membrane, and the receptor immobilized on the membrane binds thereto to cause a specific reaction. The antigen-antibody complex is accumulated continuously, and the colloidal gold labeled precipitate is developed, so that the detection purpose is achieved. The kit has the advantages of simplicity, rapidness, no need of special equipment, stable result and the like, is very suitable for field detection, and is widely applied to the fields of biology, medicines, foods and the like.
At present, the helicobacter pylori detection method based on the colloidal gold immunochromatography technology is mainly used for detecting IgG antibodies, can only judge whether helicobacter pylori is infected, and cannot carry out typing detection. The present invention designs and expresses different antigens of helicobacter pylori through gene engineering technology, fixes the antigens on a chromatographic membrane, and types the germs according to the difference of helicobacter pylori antibody types during detection so as to provide guidance for clinical treatment.
Compared with the existing method, the invention has the characteristics of simple and convenient operation, short detection time, high sensitivity, strong specificity, wide application range and the like. The product can quickly realize the diagnosis and typing detection of the helicobacter pylori, and is suitable for clinical detection, on-site investigation, epidemiological investigation and the like.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
In the figure: 1. a sample pad; 2. a colloidal gold bonding pad; 3. a chromatographic membrane; 4. an absorbent pad; 5. a base plate; t1, a first detection line; t2, a second detection line; t3, third detection line; C. and (4) quality control line.
Detailed Description
Referring to fig. 1, the helicobacter pylori colloidal gold typing detection device of the present invention comprises a base plate 5, wherein a sample pad 1, a colloidal gold binding pad 2, a chromatographic membrane 3 and an absorption pad 4 are sequentially adhered on the base plate 5; one end of the colloidal gold combining pad 2 is lapped with the sample sucking pad 1, the other end of the colloidal gold combining pad is lapped with one end of the chromatographic membrane 3, and the other end of the chromatographic membrane 3 is lapped with the sampling pad 4; a first detection line T1, a second detection line T2, a third detection line T3 and a quality control line C are arranged on the chromatographic membrane 3; the solid phase of the detection line T1 is provided with a purified HP urease subunit B protein (UreB) antigen, the solid phase of the detection line T2 is provided with a purified HP cytotoxic (CagA) antigen, and the solid phase of the detection line T3 is provided with a purified HP vacuolating toxic (VacA) antigen; the control line C is coated with goat anti-mouse IgG polyclonal antibody.
Example 1 core antigen expression of helicobacter pylori cytotoxic (CagA), vacuolating toxic (VacA) and urease subunit B protein (UreB)
The three gene sequences of helicobacter pylori cytotoxicity (CagA), vacuolating toxicity (VacA) and urease subunit B protein (UreB) are sequences of helicobacter pylori 43504 strains recorded by the applicant according to GenBank, epitope regions with strong antigenicity are preferably found to be used as target genes, the target genes are artificially synthesized by Jinwei Zhi company, and enzyme cutting site fragments are added at two ends of the genes according to requirements.
(1) The synthesized CagA and VacA gene sequences are subjected to BamHI and SacI double enzyme digestion; the synthesized core antigen gene sequence of UreB is subjected to EcoRI and XhoI double enzyme digestion. The double-digested DNA fragment was recovered and ligated with pET28a expression vector digested with the same enzyme to construct expression vectors pET28a-CagA, pET28a-VacA and pET28a-UreB, respectively.
(2) The ligation product was used to transform DH 5. alpha. competent cells, and the transformants were plated on ampicillin-resistant LB agar plates and cultured overnight at 37 ℃. Several monoclonal colonies were picked separately, and colony pcr was performed and sequenced. Culturing a large amount of positive clone strains, subpackaging, adding glycerol, and storing in an ultra-low temperature refrigerator at-80 ℃; extracting a large amount of positive cloning expression vectors, subpackaging and freezing at-20 ℃.
(3) The constructed expression plasmids pET28a-CagA, pET28a-VacA and pET28a-UreB are respectively transferred into a competent cell BL21(DE3) of escherichia coli for expression, the competent cell BL21 is coated on an LB plate containing ampicillin, the culture is carried out for 12-14 h at 37 ℃, a single colony is picked up and put into 4ml LB liquid culture medium containing ampicillin for culture overnight, and the bacteria are activated.
(4) The next day, the cells were inoculated into LB liquid medium containing ampicillin at a ratio of 1: 50, cultured until OD600 became about 0.5 to 0.6, IPTG was added to a final concentration of 0.1mM, and induced at 37 ℃ for 6 hours. Centrifuging at 12000rpm for 3min, and collecting bacteria. After the bacteria were washed with Phosphate Buffered Saline (PBS) at pH7.2, a lysate was added thereto, and after the cells were disrupted by sonication, the cells were centrifuged at 12000rpm for 15 minutes, and the supernatant was retained.
(5) Obtaining target protein by adopting a Ni column affinity chromatography method, analyzing the purity and the concentration of the protein by SDS-PAGE, and quantifying the protein by using a BCA kit (vazyme).
EXAMPLE 2 preparation of chromatographic membranes
Diluting the core antigen proteins of CagA, VacA and UreB to the coating concentration of 1-10 mg/mL by using a coating diluent; diluting the goat anti-mouse IgG polyclonal antibody to a coating concentration of 2-10 mg/mL by using a coating diluent; adjusting a machine, and respectively spraying the two coating liquids onto the chromatographic membrane 3 to form the detection lines T1, T2, T3 and a quality control line C; the coated chromatographic carrier 3 was dried at 37 ℃ for 3 hours and stored at room temperature. The coating diluent is 0.01M phosphate buffer solution with pH 7.2.
EXAMPLE 3 preparation of colloidal gold conjugate pad
(A) Preparation of colloidal gold
All available containers were soaked in a potassium dichromate tank for a while, 150mL of double distilled water was charged into a 250mL Erlenmeyer flask, heated to boiling with magnetic stirring for 1min, and discarded. Adding 99mL of double distilled water and 1mL of 1% tetrachloroauric acid solution, stirring uniformly, heating to boiling, rapidly adding 2mL of trisodium citrate solution while stirring, continuously stirring and heating for 15min after the solution color is changed into mauve and is not changed, cooling to room temperature, fixing the volume of the double distilled water to 100mL, and storing at 4 ℃.
(B) Preparation of colloidal gold-labeled monoclonal antibody
Adjusting the pH value of the colloidal gold to 8.0-9.5 by 0.1M potassium carbonate, adding an anti-human IgG monoclonal antibody into the colloidal gold according to the ratio of 5-10 ug antibody/ml, magnetically stirring for 40min, adding BSA while stirring to the final concentration of 0.4%, stirring for 10min at room temperature, continuously adding 10% PEG2000 solution to the final concentration of 0.2%, stirring for 10min at room temperature, centrifuging the mixture of the gold-labeled antibodies at 10000rpm and 4 ℃ for 30min, discarding the supernatant, resuspending the gold-labeled antibody precipitate with a resuspension solution, sterilizing the gold-labeled antibody precipitate with a final volume of 1/10 of the original volume by a microfiltration membrane of 0.45 mu M, and storing at 4 ℃.
Wherein the heavy suspension is Tris-HCl solution containing 1% BSA and 4% sucrose.
(C) Preparation of colloidal gold bonding pad
Soaking the colloidal gold bonding pad in a sealing solution for 1h, and drying at 37 ℃. The prepared gold-labeled antibody was then spread evenly on a gold-labeled pad at 20 cm per ml of solution, freeze-dried, packaged, and stored at 4 ℃.
Wherein the blocking solution is 2% Bovine Serum Albumin (BSA), 1% Tween-20, 2.5% sucrose, 0.05% NaN3, 0.01M pH7.2PBS solution.
EXAMPLE 4 preparation of sample pad
And soaking the sample pad in the sample pad treatment solution for 30min, and drying at 37 ℃ for later use. The sample pad treatment solution included 0.1M Tris-HCl, 0.5% BSA, 3% skim milk, 1% Tween-20.
EXAMPLE 5 Assembly
And (3) sequentially adhering the pretreated sample pad, the colloidal gold bonding pad, the chromatographic membrane and the absorption pad on a bottom plate, cutting to obtain a detection reagent strip, and finally packaging the detection reagent strip into a plastic shell.
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and variations of the present invention are possible to those skilled in the art. Any modification, equivalent replacement, improvement and the like of the present invention shall be included in the protection scope of the present invention.
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Cited By (5)
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| CN115290889A (en) * | 2022-07-25 | 2022-11-04 | 上海昌润生物科技有限公司 | Helicobacter pylori HP typing fluorescence immunochromatographic assay reagent and preparation and detection methods thereof |
| CN115960222A (en) * | 2022-10-21 | 2023-04-14 | 青海大学 | Preparation and application of a nanobody for Helicobacter pylori typing detection |
| CN116047077A (en) * | 2022-12-31 | 2023-05-02 | 武汉睿奇生物工程有限公司 | Reagent strip for rapidly detecting HP antibody typing in urine and kit comprising reagent strip |
| CN118652346A (en) * | 2024-06-28 | 2024-09-17 | 杭州华葵金配生物科技有限公司 | Antibodies targeting Helicobacter pylori type B urease and their applications |
| CN118652346B (en) * | 2024-06-28 | 2025-04-15 | 杭州华葵金配生物科技有限公司 | Antibodies targeting Helicobacter pylori type B urease and their applications |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115290889A (en) * | 2022-07-25 | 2022-11-04 | 上海昌润生物科技有限公司 | Helicobacter pylori HP typing fluorescence immunochromatographic assay reagent and preparation and detection methods thereof |
| CN115960222A (en) * | 2022-10-21 | 2023-04-14 | 青海大学 | Preparation and application of a nanobody for Helicobacter pylori typing detection |
| CN115960222B (en) * | 2022-10-21 | 2024-05-24 | 青海大学 | Preparation and application of nanoantibody for Helicobacter pylori typing detection |
| CN116047077A (en) * | 2022-12-31 | 2023-05-02 | 武汉睿奇生物工程有限公司 | Reagent strip for rapidly detecting HP antibody typing in urine and kit comprising reagent strip |
| CN118652346A (en) * | 2024-06-28 | 2024-09-17 | 杭州华葵金配生物科技有限公司 | Antibodies targeting Helicobacter pylori type B urease and their applications |
| CN118652346B (en) * | 2024-06-28 | 2025-04-15 | 杭州华葵金配生物科技有限公司 | Antibodies targeting Helicobacter pylori type B urease and their applications |
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