Helicobacter pylori antibodies and their detection products and applications

Abstract

The present invention provides three pairs of Helicobacter pylori antibodies and their applications, and defines the amino acid sequences of the heavy chain complementary determining regions and light chain complementary determining regions of the antibodies. The Helicobacter pylori antibodies provided by the present invention have good specificity, high biological activity, strong stability, and high affinity with Helicobacter pylori, and can be used to prepare products for detecting Helicobacter pylori. The Helicobacter pylori detection product provided by the present invention has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection, economical and practical identification and typing, etc., and can perform rapid screening, rapid, accurate, and safe detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to Helicobacter pylori antibodies and applications thereof. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori Hp) is a spiral-shaped Campylobacter first isolated from a gastric mucosal biopsy specimen in 1982. It is also the only bacterium currently classified as a Class I carcinogen by the International Agency for Research on Cancer. If Hp infection is not intervened, it will lead to chronic inflammation of the stomach over time and eventually develop into gastric cancer. Hp infection is recognized as the main predisposing factor for gastric cancer. Hp is not only closely related to the occurrence and development of digestive system diseases, but can also cause diseases outside the digestive system. The infection rate of Hp in the natural population worldwide exceeds 50%. Helicobacter pylori is highly contagious. It can grow and reproduce in the human body and be excreted through saliva and feces. As the main source of infection of Helicobacter pylori, humans mainly transmit through: drinking contaminated water, close contact with Hp patients, eating unclean food from roadside stalls, reusing endoscopes, etc. In short, it is oral-to-mouth transmission, endoscopic transmission, and close contact transmission.

[0003] The pathogenic mechanism of Hp includes: Hp colonization, gastric mucosal damage caused by toxins, gastric mucosal damage mediated by the host's immune response, and abnormal gastric acid secretion caused by imbalance in gastrin and somatostatin regulation after Hp infection, etc., involving inflammation, immunity, acid secretion, oxidation and other aspects, and multiple Hp pathogenic factors such as virulence factors, cytokines, free radicals, and virulence genes are involved. There are many Hp virulence factors, mainly urease, flagellar motility, adhesin, superadhesion factor, lipopolysaccharide, and catalase of phospholipase A. At present, clinical isolates of Helicobacter pylori can be divided into two types according to whether they produce CagA protein. Type I bacteria contain CagA genes, can express CagA and VacA proteins, and produce vacuolar toxins, which are highly virulence strains; Type II bacteria do not contain CagA genes, do not express CagA and VacA proteins, do not produce vacuolar toxins, and are low-virulence strains; the rest are intermediate types, which have no CagA genes but can produce vacuolar toxins. After being infected, Helicobacter pylori type I can release strong toxicity and cause stomach diseases, such as duodenal ulcer, gastric cancer, etc. Helicobacter pylori type II does not produce cytotoxins. When it attacks, it usually manifests as indigestion. Although there are no obvious symptoms, you should cooperate with the doctor for treatment in time.

[0004] At present, the diagnostic methods of Hp are divided into two categories. One is invasive experiment: it uses gastric mucosal tissue obtained through endoscopy as the test material, and conducts microbiological culture, pathological histological detection, rapid urease test and genetic diagnosis. Although these methods are highly accurate, their scope of application is limited. They require medical workers to have a high level of professionalism and rich clinical experience and cannot be widely used. The other is non-invasive test: it does not require the acquisition of gastric mucosal tissue, and uses gastric juice, blood, saliva, feces and other specimens. The methods include fecal Hp antigen detection, serum Hp antibody detection, urea breath test and determination of Hp genes in feces and other specimens. Among them, urea breath test is the most commonly used method in clinical practice, but this method is easily affected by drugs and causes false negatives. Serum antibody detection Studies have shown that specific antibodies will only appear in the blood several weeks after the human body is infected with Hp, and the antibodies in the serum can be maintained for more than 6 months after Hp is eradicated. Therefore, positive serological antibodies cannot be used as the basis for current infection, and negative ones cannot be used as the basis for excluding infection. Because it may be in the infection, false negative results may occur when the early antibodies do not reach the detection threshold. Hp gene testing can be used to identify the type of Helicobacter pylori, but it is time-consuming and expensive, so it cannot be carried out in routine clinical practice. Hp-specific antigens in stool samples can be detected by immunochromatography and enzyme immunoassay. The advantages of this method are that samples are easy to collect and support self-testing chromatography methods, short test time, easy operation, low equipment dependence, and visualization of test results. Summary of the invention

[0005] The objects of the present invention are:

[0006] The first object of the present invention is to provide a method for preparing Helicobacter pylori antibodies and an immunogenic protein of Helicobacter pylori, which can prepare antibodies that specifically recognize Helicobacter pylori and achieve efficient antibody pairing.

[0007] A second object of the present invention is to provide an antibody against Helicobacter pylori, which can specifically recognize Helicobacter pylori.

[0008] The third object of the present invention is to provide the use of the above-mentioned Helicobacter pylori antibody in the preparation of Helicobacter pylori detection products.

[0009] A fourth object of the present invention is to provide a Helicobacter pylori detection product, preferably comprising a test paper and a test paper box.

[0010] In order to achieve the above object, the present invention provides a Helicobacter pylori antibody, including one of Helicobacter pylori antibody 1, Helicobacter pylori antibody 2 or Helicobacter pylori antibody 3.

[0011] Preferably, the Helicobacter pylori antibody 1 comprises antibody 1c and / or antibody 1n.

[0012] Any of the above is preferably that the antibody 1c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.10 to SEQ ID NO.12, and light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.13 to SEQ ID NO.15;

[0013] Preferably, any of the above items is that the antibody 1n comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NOs. 16 to 18, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NOs. 19 to 21.

[0014] Any of the above items is preferably that the Helicobacter pylori antibody 2 comprises antibody 2c and / or antibody 2n;

[0015] Any of the above is preferably that the antibody 2c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.22 to SEQ ID NO.24, and light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.25 to SEQ ID NO.27;

[0016] Preferably, any of the above items is that the antibody 2n comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.28 to SEQ ID NO.30, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.31 to SEQ ID NO.33.

[0017] Any of the above items is preferably that the Helicobacter pylori antibody three comprises antibody 3c and antibody 3n;

[0018] Any of the above is preferably that the antibody 3c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.34 to SEQ ID NO.36, and light chain complementary determining regions CDR1-VL, CDR2-VL, CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.37 to SEQ ID NO.39;

[0019] Preferably, any of the above items is that the antibody 3n comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.40 to SEQ ID NO.42, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.43 to SEQ ID NO.45.

[0020] Preferably, any of the above items is that the antibody 1c comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.46 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.47.

[0021] Preferably, any of the above items is that the antibody 1n comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.48 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.49.

[0022] Preferably, any of the above items is that the antibody 2c comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.50 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.51.

[0023] Preferably, any of the above items is that the antibody 2n comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.52 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.53.

[0024] Preferably, any of the above items is that the antibody 3c comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.54 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.55.

[0025] Preferably, any of the above items is that the antibody 3n comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO. 56 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO. 57.

[0026] Preferably, in any of the above items, the Helicobacter pylori antibody is of mouse origin.

[0027] Preferably, in any of the above items, the amino acid sequence of the light chain of antibody 1c is as shown in SEQ ID NO.58.

[0028] Preferably, in any of the above items, the amino acid sequence of the heavy chain of the antibody 1c is as shown in SEQ ID NO.59.

[0029] Preferably, in any of the above items, the amino acid sequence of the light chain of the antibody 1n is as shown in SEQ ID NO.60.

[0030] Preferably, in any of the above items, the amino acid sequence of the heavy chain of the antibody 1n is as shown in SEQ ID NO.61.

[0031] Preferably, in any of the above items, the amino acid sequence of the antibody 2c light chain is as shown in SEQ ID NO.62.

[0032] Preferably, in any of the above items, the amino acid sequence of the heavy chain of the antibody 2c is as shown in SEQ ID NO.63.

[0033] Preferably, in any of the above items, the amino acid sequence of the antibody 2n light chain is as shown in SEQ ID NO.64.

[0034] Preferably, in any of the above items, the amino acid sequence of the heavy chain of the antibody 2n is as shown in SEQ ID NO.65.

[0035] Preferably, in any of the above items, the amino acid sequence of the antibody 3c light chain is as shown in SEQ ID NO.66.

[0036] Preferably, in any of the above items, the amino acid sequence of the heavy chain of antibody 3c is as shown in SEQ ID NO.67.

[0037] Preferably, in any of the above items, the amino acid sequence of the antibody 3n light chain is as shown in SEQ ID NO.68.

[0038] Preferably, in any of the above items, the amino acid sequence of the heavy chain of the antibody 3n is as shown in SEQ ID NO.69.

[0039] Preferably, in any of the above items, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO.70.

[0040] Preferably, in any of the above items, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO.71.

[0041] The present invention also provides an immunogenic protein of Helicobacter pylori for preparing any of the above Helicobacter pylori antibodies, wherein the immunogenic protein of Helicobacter pylori comprises one of Helicobacter pylori immunogenic protein 1, Helicobacter pylori immunogenic protein 2 or Helicobacter pylori immunogenic protein 3.

[0042] Preferably, in any of the above items, the Helicobacter pylori immunogenic protein 1 comprises immunogenic protein 1C and / or immunogenic protein 1N.

[0043] Preferably, in any of the above items, the immunogenic protein 1C comprises the amino acid sequence shown in SEQ ID NO.4.

[0044] Preferably, in any of the above items, the antibody 1c is obtained by immunizing an animal with the immunogenic protein 1C.

[0045] Preferably, in any of the above items, the immunogenic protein 1N comprises the amino acid sequence shown in SEQ ID NO.5.

[0046] Preferably, in any of the above items, the antibody 1n is obtained by immunizing an animal with the immunogenic protein 1N.

[0047] Preferably, any of the above items is that the Helicobacter pylori immunogenic protein 2 comprises immunogenic protein 2C and / or immunogenic protein 2N.

[0048] Preferably, in any of the above items, the immunogenic protein 2C comprises the amino acid sequence shown in SEQ ID NO.6.

[0049] Preferably, any of the above items is that the antibody 2c is obtained by immunizing an animal with the immunogenic protein 2C.

[0050] Preferably, in any of the above items, the immunogenic protein 2N comprises the amino acid sequence shown in SEQ ID NO.7.

[0051] Preferably, in any of the above items, the antibody 2n is obtained by immunizing an animal with the immunogenic protein 2N.

[0052] Preferably, any of the above items is that the Helicobacter pylori immunogenic protein three comprises immunogenic protein 3C and / or immunogenic protein 3N.

[0053] Preferably, in any of the above items, the immunogenic protein 3C comprises the amino acid sequence shown in SEQ ID NO.8.

[0054] Preferably, any of the above items is that the antibody 3c is obtained by immunizing an animal with the immunogen protein 3C.

[0055] Preferably, in any of the above items, the immunogenic protein 3N comprises the amino acid sequence shown in SEQ ID NO.9.

[0056] Preferably, in any of the above items, the antibody 3n is obtained by immunizing an animal with the immunogenic protein 3N.

[0057] The present invention also provides a method for preparing the Helicobacter pylori antibody as described in any one of the above items, and the use of the Helicobacter pylori immunogenic protein as described in any one of the above items in preparing the Helicobacter pylori antibody.

[0058] The present invention also provides a marker protein of Helicobacter pylori, including one of marker protein 1 of Helicobacter pylori, marker protein 2 of Helicobacter pylori or marker protein 3 of Helicobacter pylori, and the Helicobacter pylori antibody recognizes the marker protein of Helicobacter pylori.

[0059] Preferably, any one of the above items is selected from bacterial urease proteins.

[0060] Preferably, any of the above items is that the marker protein of Helicobacter pylori comprises the amino acid sequence shown in SEQ ID NO.1.

[0061] Preferably, any of the above mentioned proteins is selected from the group consisting of bacterial cytotoxin associated gene A (CagA).

[0062] Preferably, any one of the above items is that the Helicobacter pylori marker protein 2 comprises the amino acid sequence shown as SEQ ID NO.2.

[0063] Any of the above is preferably a protein selected from bacterial vacuolating toxin-associated gene A (VacA).

[0064] Preferably, any of the above items is that the marker protein three of Helicobacter pylori comprises the amino acid sequence shown as SEQ ID NO.3.

[0065] The present invention also provides use of any of the above Helicobacter pylori antibodies in the preparation of a Helicobacter pylori detection product.

[0066] The present invention also provides a Helicobacter pylori detection test strip, which includes a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked in sequence on the base plate, wherein the marking pad contains the Helicobacter pylori antibody according to any one of claims 1 to 4, and the Helicobacter pylori antibody is marked with a marker.

[0067] Preferably, in any of the above items, the marker comprises at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

[0068] Preferably, any of the above items has a particle size of colloidal gold of 40 to 100 nm; more preferably, 40, 50, 60, 80, 100 nm and ranges therebetween.

[0069] Preferably, any of the above items has a particle size of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0070] Preferably, any of the above items has a particle size of 100-300 nm of the time-resolved fluorescent microspheres; more preferably, 100, 200, 300 nm and ranges therebetween.

[0071] Preferably, any of the above items has a particle size of the quantum dot microspheres of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0072] Preferably, in any of the above items, the mass ratio of the myoglobin antibody to colloidal gold is: (0.04-0.32):1; more preferably, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0073] Preferably, in any of the above items, the mass ratio of myoglobin antibody to colored microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0074] Preferably, in any of the above items, the mass ratio of myoglobin antibody to time-resolved fluorescent microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0075] Preferably, in any of the above items, the mass ratio of myoglobin antibody to quantum dot microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0076] Preferably, any one of the above items is that a detection line and a quality control line are provided on the detection pad.

[0077] Preferably, any of the above items is that the test lines are coated with 0.5-5 mg / mL of the Helicobacter pylori antibody, and more preferably, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg / mL and ranges therebetween.

[0078] Preferably, any of the above items is that the quality control line is coated with 0.5-5 mg / mL of goat anti-mouse IgG polyclonal antibody; more preferably, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg / mL and ranges therebetween.

[0079] Any of the above is preferably that the diluent for the Helicobacter pylori antibody and the goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose. Further preferably, the concentration of the PB buffer is 10, 20, 30, 40, 50 mM and the range therebetween; further preferably, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g and the range therebetween of trehalose.

[0080] Any of the above items is preferably that the detection pad is provided with a detection line and a quality control line; the detection line is coated with the Helicobacter pylori antibody; the quality control line is coated with sheep anti-chicken IgY; the marking pad contains another Helicobacter pylori antibody, the Helicobacter pylori antibody on the marking pad is marked with a marker, and the marker is marked with chicken IgY.

[0081] Preferably, any of the above items is that a detection line and a quality control line are provided on the detection pad; the detection lines are 3, which are respectively coated with at least one of the Helicobacter pylori antibodies 1, at least one of the Helicobacter pylori antibodies 2, and at least one of the Helicobacter pylori antibodies 3.

[0082] Preferably, any of the above items is that the Helicobacter pylori antibody on the labeling pad is different from the Helicobacter pylori antibody on the detection pad.

[0083] In a preferred embodiment of the present invention, there are three detection lines, namely, a Ure detection line, a Cag detection line, and a Vac detection line.

[0084] Preferably, if the Helicobacter pylori antibody on the Ure test line is antibody 1c, then the Helicobacter pylori antibody on the marking pad is antibody 1n; if the Helicobacter pylori antibody on the Ure test line is antibody 1n, then the Helicobacter pylori antibody on the marking pad is antibody 1c.

[0085] Preferably, if the Helicobacter pylori antibody on the Cag detection line is antibody 2c, then the Helicobacter pylori antibody on the marking pad is antibody 2n; if the Helicobacter pylori antibody on the Cag detection line is antibody 2n, then the Helicobacter pylori antibody on the marking pad is antibody 2c.

[0086] Preferably, if the Helicobacter pylori antibody on the Vac test line is antibody 3c, then the Helicobacter pylori antibody on the marking pad is antibody 3n; if the Helicobacter pylori antibody on the Vac test line is antibody 3n, then the Helicobacter pylori antibody on the marking pad is antibody 3c.

[0087] The present invention also provides a Helicobacter pylori detection kit, comprising any of the above-mentioned Helicobacter pylori detection test strips and a shell, wherein the Helicobacter pylori detection test strip is arranged inside the shell.

[0088] Preferably, in any of the above items, the housing comprises an upper cover and a lower cover which are detachably connected.

[0089] Preferably, any one of the above items is that the upper cover is provided with an observation window and a sample addition hole.

[0090] Compared with the prior art, the present invention has the following beneficial effects:

[0091] The present invention provides an antibody preparation method in which one of the immunogenic proteins (antibody 1c, antibody 1n) contains an epitope that is highly conservative and not prone to missed detection, and is a dominant epitope that is easy to produce high-affinity antibodies; the other two immunogenic proteins (antibody 2c, antibody 2n and antibody 3c, antibody 3n) are highly immunogenic and are epitopes that determine strain typing and are easy to produce specific antibodies. The antibody combination provided by the present invention improves the detection breadth and sensitivity of the Helicobacter pylori detection product.

[0092] The Helicobacter pylori antibody provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with Helicobacter pylori, and can be used to prepare products for detecting Helicobacter pylori.

[0093] The Helicobacter pylori detection kit provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection and economical and practical, and is of great significance for the typing diagnosis of HP infection, prediction of disease progression, judgment of prognosis and screening of drug efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 It is the upper cover of the Helicobacter pylori detection kit in the preferred embodiment 2 of the present invention.

[0095] Figure 2 It is the lower cover of the Helicobacter pylori detection kit in the preferred embodiment 2 of the present invention.

[0096] Figure 3 This is a structural diagram of the Helicobacter pylori detection test strip in preferred embodiment 2 of the present invention.

[0097] Figure 4 This is the electrophoresis diagram of the anti-Helicobacter pylori monoclonal antibody in the preferred embodiment 1 of the present invention.

[0098] Figure 5 This is a display diagram of the detection results in preferred embodiment 3 of the present invention.

[0099] Icons: 1-Observation window; 2-Sample loading hole; 3-Test card strip area; 4-Bottom plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample pad; 9-Quality control line; 10-Test line one; 11-Test line two; 12-Test line three. DETAILED DESCRIPTION

[0100] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0101] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy chain or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0102] "Framework" or "FR" regions mean the regions of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0103] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0104] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0105] The immunogenic protein of Helicobacter pylori, characterized in that it is selected from at least one of marker protein 1, marker protein 2 or marker protein 3, and comprises immunogenic protein C and immunogenic protein N;

[0106] The immunogen protein 1C, immunogen protein 2C, and immunogen protein 3C include amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8; the immunogen protein 1N, immunogen protein 2N, and immunogen protein 3N include amino acid sequences as shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9.

[0107] In the first aspect, the present invention provides marker protein 1, marker protein 2, and marker protein 3 of Helicobacter pylori, and the amino acid sequences of the marker proteins are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; in addition, the present invention provides 6 immunogenic proteins of Helicobacter pylori, including immunogenic protein 1C, immunogenic protein 2C, immunogenic protein 3C and immunogenic protein 1N, immunogenic protein 2N, and immunogenic protein 3N, and the amino acid sequences of the immunogenic protein 1C, immunogenic protein 2C, and immunogenic protein 3C are as shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8, and the amino acid sequences of the immunogenic protein 1N, immunogenic protein 2N, and immunogenic protein 3N are as shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ IDNO.9.

[0108] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.9 are shown in Table 1.

[0109] Table 1

[0110]

[0111]

[0112] In a second aspect, the present invention provides three pairs of Helicobacter pylori antibodies, namely antibody 1c and antibody 1n, antibody 2c and antibody 2n, antibody 3c and antibody 3n:

[0113] The antibody 1c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NO.10 to SEQ ID NO.12, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NO.13 to SEQ ID NO.15;

[0114] The antibody 1n includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NO.16 to SEQ ID NO.18, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NO.19 to SEQ ID NO.21.

[0115] The antibody 2c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NO.22 to SEQ ID NO.24, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NO.25 to SEQ ID NO.27;

[0116] The antibody 2n includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NO.28 to SEQ ID NO.30, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NO.31 to SEQ ID NO.33.

[0117] The antibody 3c comprises heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are sequentially shown in SEQ ID NO.34 to SEQ ID NO.36, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are sequentially shown in SEQ ID NO.37 to SEQ ID NO.39;

[0118] The antibody 3n includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH, whose amino acid sequences are shown in SEQ ID NO.40 to SEQ ID NO.42, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL, whose amino acid sequences are shown in SEQ ID NO.43 to SEQ ID NO.45.

[0119] The amino acid sequences shown in SEQ ID NO.10 to SEQ ID NO.45 are shown in Table 2.

[0120] Table 2

[0121]

[0122]

[0123] The Helicobacter pylori antibody provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with Helicobacter pylori, and can be used to prepare products for detecting Helicobacter pylori.

[0124] In some preferred embodiments of the present invention, the antibody 1c comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.46 and SEQ ID NO.47, respectively;

[0125] The antibody 1n comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.48 and SEQ ID NO.49, respectively;

[0126] The antibody 2c comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.50 and SEQ ID NO.51, respectively;

[0127] The antibody 2n comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.52 and SEQ ID NO.53, respectively;

[0128] The antibody 3c comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.54 and SEQ ID NO.55, respectively;

[0129] The antibody 3n comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.56 and SEQ ID NO.57, respectively;

[0130] Preferably, the Helicobacter pylori antibody is of murine origin.

[0131] The amino acid sequences shown in SEQ ID NO.46 to SEQ ID NO.57 are shown in Table 3.

[0132] Table 3

[0133]

[0134] In some preferred embodiments of the present invention, the amino acid sequences of the antibody 1c and antibody 1n, the antibody 2c and antibody 2n, the antibody 3c and antibody 3n, and the antibody light chain constant region and heavy chain constant region are as shown in Table 4.

[0135] Table 4

[0136]

[0137]

[0138]

[0139]

[0140] In a third aspect, the present invention provides the use of the Helicobacter pylori antibody in the preparation of a Helicobacter pylori detection product.

[0141] In a fourth aspect, the present invention provides a Helicobacter pylori detection test strip, the detection test strip comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked in sequence on the base plate;

[0142] The marking pad contains the Helicobacter pylori antibody, and the Helicobacter pylori antibody is marked with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0143] The Helicobacter pylori detection test strip provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, ability to identify and type, suitability for on-site rapid detection, and economical and practical, and is of great significance for the early diagnosis of HP infection, prediction of disease progression, judgment of prognosis, and screening and evaluation of the efficacy of anti-HP drugs.

[0144] In some preferred embodiments, the marker includes but is not limited to at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres, or quantum dot microspheres;

[0145] Preferably, the particle size of the colloidal gold may be, for example, but not limited to, 40 nm, 60 nm, 80 nm or 100 nm;

[0146] Preferably, the particle size of the colored microspheres, time-resolved fluorescent microspheres and quantum dot microspheres may be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm;

[0147] Preferably, the mass ratio of Helicobacter pylori antibody coupled to colloidal gold is: (0.04-0.32):1; the mass ratio of Helicobacter pylori antibody to colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres is: (0.1-0.4):1.

[0148] By adjusting the particle size and dosage of the marker, the sensitivity of the test strip can be made higher.

[0149] In some preferred embodiments, a detection line (T line) and a quality control line (C line) are provided on the detection pad;

[0150] The test line is coated with 0.5-5 mg / mL of the Helicobacter pylori antibody;

[0151] The quality control line was coated with 0.5~5 mg / mL of goat anti-mouse IgG polyclonal antibody;

[0152] Preferably, the diluent for the Helicobacter pylori antibody and goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose.

[0153] In some preferred embodiments, the Helicobacter pylori antibody on the labeling pad is different from the Helicobacter pylori antibody on the detection pad.

[0154] For example, the protein on the labeling pad is HP and the antibody is antibody 1c, wherein antibody 1c is Helicobacter pylori monoclonal antibody 1, and the protein on the detection pad is HP antibody 1n, wherein antibody 1n is Helicobacter pylori monoclonal antibody 2.

[0155] In a preferred embodiment of the present invention, there are three detection lines, namely, a Ure detection line, a Cag detection line, and a Vac detection line.

[0156] Preferably, if the Helicobacter pylori antibody on the Ure test line is antibody 1c, then the Helicobacter pylori antibody on the marking pad is antibody 1n; if the Helicobacter pylori antibody on the Ure test line is antibody 1n, then the Helicobacter pylori antibody on the marking pad is antibody 1c.

[0157] Preferably, if the Helicobacter pylori antibody on the Cag detection line is antibody 2c, then the Helicobacter pylori antibody on the marking pad is antibody 2n; if the Helicobacter pylori antibody on the Cag detection line is antibody 2n, then the Helicobacter pylori antibody on the marking pad is antibody 2c.

[0158] Preferably, if the Helicobacter pylori antibody on the Vac test line is antibody 3c, then the Helicobacter pylori antibody on the marking pad is antibody 3n; if the Helicobacter pylori antibody on the Vac test line is antibody 3n, then the Helicobacter pylori antibody on the marking pad is antibody 3c.

[0159] The test strip provided by the present invention is based on the principle of antigen-antibody reaction, and after labeling Helicobacter pylori monoclonal antibodies with colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and quantum dots, the marker is solidified on a glass cellulose membrane. Another monoclonal antibody of Helicobacter pylori is coated on a detection pad (such as an NC membrane), and based on the principle of antigen-antibody reaction, it can be detected by the naked eye or a matching instrument within the detection time. If Helicobacter pylori exists in the sample, a double antibody sandwich structure is formed, forming a strip visible to the naked eye or a light intensity signal in the instrument. If Helicobacter pylori is not in the sample, no strip appears on the NC membrane or there is no light intensity signal in the instrument. Negative or positive judgment is made based on the presence or absence of a signal, or the viral load is predicted based on the intensity of the light intensity signal.

[0160] By using the test strip provided by the present invention for testing, the test results can be obtained within 10-30 minutes of the entire process, which is fast and efficient, and helps medical personnel to obtain the test results in a timely manner, make comprehensive judgments and take timely measures based on the results, avoid panic, and reduce the spread of the epidemic.

[0161] In a fifth aspect, the present invention provides a Helicobacter pylori detection kit, comprising the Helicobacter pylori detection test strip and a shell, wherein the Helicobacter pylori detection test strip is arranged inside the shell.

[0162] The Helicobacter pylori detection kit provided by the present invention contains a Helicobacter pylori detection test strip, and thus has all the beneficial effects of the Helicobacter pylori detection test strip.

[0163] In some preferred embodiments, the housing includes an upper cover and a lower cover that are detachably connected;

[0164] The upper cover is provided with an observation window and a sample adding hole.

[0165] The shapes of the observation window and the sample addition hole are not specifically limited in the present invention. The observation window can be, for example, square and located above the test line and the quality control line of the test strip for observing the test results. The sample addition hole can be, for example, a circular hole with a diameter of 0.5-1 cm and located above the sample pad.

[0166] Test kit sample loading:

[0167] It should be noted that, unless otherwise specified in the following examples, the diluent for the Helicobacter pylori antibody and goat anti-mouse IgG polyclonal antibody is a 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose.

[0168] Example 1

[0169] Helicobacter pylori antibodies include: Helicobacter pylori monoclonal antibody 1, Helicobacter pylori monoclonal antibody 2, Helicobacter pylori monoclonal antibody 3, Helicobacter pylori monoclonal antibody 4, Helicobacter pylori monoclonal antibody 5, and Helicobacter pylori monoclonal antibody 6. The antibodies are antibody 1c and antibody 1n, antibody 2c and antibody 2n, antibody 3c and antibody 3n described in the present invention. The antibody variable regions and light chain and heavy chain sequences are shown in Tables 3 and 4.

[0170] A 12% SDS-PAGE gel was prepared according to the conventional method, and 5 μg of the above antibodies were loaded and electrophoresed using a protein molecular weight standard as a reference. The results showed that the six anti-Helicobacter pylori monoclonal antibodies all showed two characteristic bands of about 55Kd and 25Kd, which were the light chain and heavy chain of IgG ( Figure 4 ). After scanning and analysis, the antibody content of the bands was above 90%.

[0171] Figure 4 Among them, 1 is anti-Helicobacter pylori monoclonal antibody one; 2 is anti-Helicobacter pylori monoclonal antibody two; 3 is anti-Helicobacter pylori monoclonal antibody three; 4 is anti-Helicobacter pylori monoclonal antibody four; 5 is anti-Helicobacter pylori monoclonal antibody five; 6 is anti-Helicobacter pylori monoclonal antibody six.

[0172] The antibodies used in the following examples are the same as those in Example 1.

[0173] Example 2

[0174] A Helicobacter pylori detection kit, such as Figure 1~Figure 3 As shown, it includes a Helicobacter pylori detection test strip and a shell, and the Helicobacter pylori detection test strip is arranged inside the shell. The Helicobacter pylori detection test strip includes a bottom plate 4 and a sample pad 8, a marking pad 7, a detection pad 5 and a sample suction pad 6 which are stacked on the bottom plate in sequence; the detection pad is provided with a detection line 10, a detection line 2 11, a detection line 3 12 and a quality control line 9; the shell includes a detachably connected upper cover and a lower cover; the upper cover is provided with an observation window 1 and a sample addition hole 2, and the lower cover is provided with a detection card strip area 3.

[0175] Example 3

[0176] Example 3 provides a Helicobacter pylori antigen detection kit prepared by using colloidal gold labeled antibodies.

[0177] 1 Main Materials

[0178] 1.1 Antibodies: Mouse monoclonal antibodies are all Helicobacter pylori antibodies described in the present invention, and are labeled and coated respectively; Sheep anti-mouse IgG antibody: a product of Shenzhen Feipeng Biological Co., Ltd., used for coating the nitrocellulose membrane quality control line.

[0179] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius.

[0180] 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs; commonly used reagents are all analytical grade reagents.

[0181] 1.4 Acquisition of recombinant antigens: Based on the data of Helicobacter pylori published by the NCBI database, protein recombinant antigens were commercially synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. In the present invention, the amino acid sequences of the synthetic protein recombinant antigens are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0182] 2 Methods

[0183] 2.1 Preparation of colloidal gold labeling pad:

[0184] The steps for preparing the colloidal gold labeling pad are as follows:

[0185] (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and adjust the pH to 8.5 with 0.2 M K2CO3;

[0186] (2) 25 μg of monoclonal antibody 1 (antibody 1c), monoclonal antibody 3 (antibody 2c), and monoclonal antibody 5 (antibody 3c) were added respectively. The mass ratio of antibody 1c, antibody 2c, and antibody 3c to colloidal gold particles was 0.1:1, respectively. The rotary shaker was adjusted to a certain speed. After rotating and labeling at room temperature for 1.5 h, 20 μL of blocking solution was added.

[0187] (3) Centrifuge at 12,000 rpm for 15 min and discard the supernatant;

[0188] (4) Add 100 μL of colloidal gold solution;

[0189] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C for 2 h for later use;

[0190] 2.2 NC membrane coating:

[0191] Use 0.02M PB containing 0.5% trehalose to dilute monoclonal antibody 2 (antibody 1n), monoclonal antibody 4 (antibody 2n), monoclonal antibody 6 (antibody 3n) to 1.5 mg / mL, and goat anti-mouse IgG antibody to 2 mg / mL; then use a film sprayer to draw the detection line T and the quality control line C on the nitrocellulose membrane respectively. After coating, dry the NC membrane in an oven at 37°C for 24 h for use.

[0192] 2.3 Assembly of the kit:

[0193] Place the coated nitrocellulose membrane in the middle of the plastic support plate in a drying room and stick it. Overlap the marker colloidal gold pad (1 / 3 of the colloidal gold pad) on one side of the T line of the nitrocellulose membrane and stick it. Overlap the sample pad (1 / 5 of the colloidal gold pad) on the other side of the colloidal gold pad. Overlap the sample pad (1 / 10 of the sample pad) on one side of the C line of the nitrocellulose membrane. Use a cutting machine to cut the pasted plastic plate into test strips of a certain width and then load them into the test card to form a Helicobacter pylori antigen detection kit.

[0194] 2.4 Detection:

[0195] Step 1, take out the test kit and the sample to be tested and equilibrate to room temperature;

[0196] Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table;

[0197] Step 3: Figure 1 Add 2 drops of sample (about 80-100 μL) to the sample well;

[0198] Step 4: Start the timer and read the result after 10 minutes. Note that if the sample does not undergo lateral chromatography or diafiltration within 1 minute after adding the sample, it may be because the sample is too viscous and needs to be pretreated with saline.

[0199] 3 Results

[0200] Under the action of lateral flow chromatography, when there is type I Helicobacter pylori in the sample, the test line will show color, and the quality control line will also show color ( Figure 5 a in Figure 5 b in the figure); when there is type II Helicobacter pylori in the sample, the test line will show color, and the quality control line will also show color ( Figure 5 c in the figure); when there is intermediate Helicobacter pylori in the sample, the test line will show color, and the quality control line will also show color ( Figure 5 d in ); when there is no Helicobacter pylori in the sample, the test line does not show color, but the quality control line shows color ( Figure 5 e in the figure); after loading, if the quality control line does not show color, the result is considered invalid regardless of whether the test line shows color or not ( Figure 5 f in Figure 5 In the g, Figure 5 h in Figure 5 i in Figure 5 (j in )

[0201] 4 Clinical Results

[0202] The colloidal gold-labeled antibody obtained in Example 3 was used to prepare a Helicobacter pylori antigen detection kit, and the patients infected with Helicobacter pylori were tested and all the results were positive, indicating that the Helicobacter pylori antibody provided by the present invention can be used in the preparation of Helicobacter pylori detection products, and the Helicobacter pylori detection marker provided by the present invention can indicate Helicobacter pylori positivity.

[0203] Example 4

[0204] Example 4 provides a Helicobacter pylori antigen detection kit prepared by using time-resolved fluorescent microspheres labeled with antibodies.

[0205] The three pairs of Helicobacter pylori monoclonal antibodies provided by the present invention are used to prepare a Helicobacter pylori antigen detection kit. The kit comprises a detection card and a test strip. The detection card is divided into an upper cover and a lower bottom. The test strip is embedded with monoclonal antibody 1 (antibody 1c), monoclonal antibody 3 (antibody 2c), and monoclonal antibody 5 (antibody 3c) labeled with time-resolved fluorescent microspheres on a fluorescent pad. The detection line is coated with monoclonal antibody 2 (antibody 1n), monoclonal antibody 4 (antibody 2n), and monoclonal antibody 6 (antibody 3n). The Helicobacter pylori antigen in the sample is quantitatively detected by a double antibody sandwich method.

[0206] 1 Kit preparation operation process:

[0207] The monoclonal antibody 1 (antibody 1c), monoclonal antibody 3 (antibody 2c), and monoclonal antibody 5 (antibody 3c) prepared by the method of the present invention are labeled on the surface of time-resolved fluorescent microspheres. Specific examples are as follows:

[0208] Time-resolved fluorescent microsphere antibody labeling: 1 mL of 1% carboxyl time-resolved fluorescent microspheres, add 9 mL of MES buffer, then add 25 μL of EDC solution (10 mg / mL) and 25 μL of NHS solution (10 mg / mL), shake at room temperature for 30 min, and collect the precipitate by centrifugation. Add HEPES reconstitution solution, ultrasonically disperse evenly, add 1 mL of 1 mg / mL monoclonal antibody 1, shake at room temperature for 120 min, and collect the precipitate by centrifugation. Then add 1 mL of blocking solution, shake at room temperature for 120 min. Collect the microsphere precipitate by centrifugation and reconstitute with reconstitution solution.

[0209] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with the microsphere complex solution, and the fluorescent pads were sprayed with a gold spray film instrument at a spraying rate of 3 μL / cm and a spraying interval of 6 mm. After spraying, they were placed in a 37°C low humidity (<30%) oven for 2 h.

[0210] NC membrane coated CT line: Detection line 1 used monoclonal antibody 4 (antibody 2n) at a concentration of 1.5 mg / mL, detection line 2 used monoclonal antibody 6 (antibody 3n) at a concentration of 1.5 mg / mL, detection line 3 used monoclonal antibody 2 (antibody 1n) at a concentration of 1.5 mg / mL, and line C used goat anti-mouse IgG antibody at a concentration of 1 mg / mL. 1 μL / cm was used for drawing the lines, and then the samples were placed in a 37°C low humidity (<30%) oven for 24 hours.

[0211] Sample pad treatment: The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20 mM Tris buffer, and each 100 mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0212] Test strip assembly: NC film, sample suction pad, fluorescent pad, and sample pad are sequentially pasted on the PVC board. The sample suction pad and fluorescent pad are pressed 1~2mm against the NC film, and the sample pad is pressed 1~2mm against the fluorescent pad. After assembly, the test strip is cut into a width of 4±0.4mm and loaded into a card shell. The card shell and desiccant are placed in an aluminum foil bag and sealed. After labeling and boxing, the finished test card is obtained.

[0213] Test sample preparation: The commercially synthesized protein recombinant antigen produced by Sangon Biotech (Shanghai) Co., Ltd. was tested after gradient dilution. The gradient dilution results are shown in Table 5.

[0214] Table 5:

[0215]

[0216] 2 Kit testing process

[0217] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample adding end of the test card, and set up a PBS control group at the same time.

[0218] The standard curve of the test strip was introduced. After 10 minutes, the fluorescent immunoassay analyzer was used to scan the detection area to obtain the fluorescent signal. After the test, the concentration value corresponding to the Helicobacter pylori antigen was displayed. The test results are shown in Tables 6 and 7 below.

[0219] Table 6 Test results of dilution samples

[0220]

[0221] Table 7 Recombinant antigen test results

[0222]

[0223] Example 5

[0224] Example 5 provides a Helicobacter pylori antigen detection kit prepared by using colored microspheres labeled with antibodies.

[0225] The three pairs of Helicobacter pylori monoclonal antibodies independently developed by our company are used to prepare Helicobacter pylori antigen detection test strips. The test strips have monoclonal antibodies 1, 3, and 5 labeled with colored microspheres embedded on the marking pad, and the detection line is coated with monoclonal antibodies 2, 4, and 6 to qualitatively detect Helicobacter pylori antigens in the sample using the double antibody sandwich method.

[0226] 1. Preparation of test strips:

[0227] 1.1 Colored microspheres labeled with antibodies

[0228] (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 mol / L K2CO3;

[0229] (2) Monoclonal antibody 1 labeled microspheres: Take 1 mL of the above pH adjusted solution, add 30 μg each of monoclonal antibody 1 (antibody 1c), monoclonal antibody 3 (antibody 2c), and monoclonal antibody 5 (antibody 3c), and the mass ratio of antibody 1c, antibody 2c, and antibody 3c to colored microspheres is 0.3:1, respectively. After reacting at room temperature for 1 hour, centrifuge and discard the supernatant;

[0230] Chicken IgY antibody-labeled microspheres: add 5 μg of chicken IgY to 1 mL of the pH-adjusted solution, with the mass ratio of antibody to colored microspheres being 0.05:1. After reacting at room temperature for 1 hour, centrifuge and discard the supernatant.

[0231] (3) Add 1 mL of 20% BSA to block for 2 h, then centrifuge and discard the supernatant;

[0232] (4) After re-dissolving the above microspheres with 100 μL of pH 8.0 reconstitution solution, the two microspheres were mixed at a ratio of 5:1, and the mixture was diluted with the reconstitution solution at a ratio of 15%;

[0233] 1.2 Preparation of labeling pad

[0234] The dilution of the labeled colored microsphere antibody complex was sprayed on the marking pad using a gold spray film instrument at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, it was placed in a 37°C low humidity (<30%) drying room for 2 hours.

[0235] 1.3 Sample pad preparation

[0236] The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0237] 1.4 C / T line coating

[0238] Test line 1, test line 2, and test line 3 use monoclonal antibody 4 (antibody 2n), monoclonal antibody 6 (antibody 3n), and monoclonal antibody 2 (antibody 1n) at a concentration of 1.5 mg / mL (line C uses goat anti-chicken IgY antibody at a concentration of 2 mg / mL, and 1 μL / cm is used for line drawing. After the test, it is placed in a 37℃ low humidity (<30%) oven for drying for 24 hours.

[0239] 1.5 Test strip assembly

[0240] On the PVC board, the sample suction pad, NC film, and marking pad are sequentially pasted, the sample pad, the sample suction pad and the marking pad are pressed 1~2mm by the NC film, and the sample pad is pressed 1~2 mm by the marking pad. After assembly, the test strip is cut into a width of 4±0.4mm and loaded into the cartridge. The cartridge and the desiccant are placed in an aluminum foil bag. The finished test kit is obtained by labeling and boxing.

[0241] 1.6 Test sample preparation: The commercially synthesized protein recombinant antigen produced by Shanghai Bioengineering Co., Ltd. was gradient diluted for testing. The gradient dilution results are shown in Table 8.

[0242] Table 8:

[0243]

[0244] 2. Detection

[0245] Place the test card on a clean, flat surface, draw 80-100 μL of the prepared recombinant antigen sample and drop it into the sample end of the test card, and set up a PBS control group. Observe the test card window after 10 minutes. There are test lines 1, 2, and 3 in the window, which are positive for type I Helicobacter pylori, and only one C line is negative. The test results are as follows: The results in Table 9 show that the antigen compliance rate of the three pairs of monoclonal antibody assembled test strips is 100%;

[0246] Table 9:

[0247]

[0248] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Helicobacter pylori antibody, including one of Helicobacter pylori antibody 1, Helicobacter pylori antibody 2 or Helicobacter pylori antibody 3, characterized in that: The Helicobacter pylori antibody 1, Helicobacter pylori antibody 2 or Helicobacter pylori antibody 3 is an antibody pair combination; wherein The Helicobacter pylori antibody 1 is composed of antibody 1c and antibody 1n; The amino acid sequence of the light chain of the antibody 1c is shown in SEQ ID NO.58; the amino acid sequence of the heavy chain of the antibody 1c is shown in SEQ ID NO.59; The amino acid sequence of the light chain of the antibody 1n is shown in SEQ ID NO.60; the amino acid sequence of the heavy chain of the antibody 1n is shown in SEQ ID NO.61; The Helicobacter pylori antibody 2 is composed of antibody 2c and antibody 2n; The light chain amino acid sequence of the antibody 2c is shown in SEQ ID NO.62; The amino acid sequence of the heavy chain of antibody 2c is shown in SEQ ID NO.63; The amino acid sequence of the light chain of the antibody 2n is shown in SEQ ID NO.64; the amino acid sequence of the heavy chain of the antibody 2n is shown in SEQ ID NO.65; The Helicobacter pylori antibody 3 is composed of antibody 3c and antibody 3n; The amino acid sequence of the light chain of the antibody 3c is shown in SEQ ID NO.66; the amino acid sequence of the heavy chain of the antibody 3c is shown in SEQ ID NO.67; The amino acid sequence of the light chain of the antibody 3n is shown in SEQ ID NO.68; the amino acid sequence of the heavy chain of the antibody 3n is shown in SEQ ID NO.

69.

2. The Helicobacter pylori antibody according to claim 1, characterized in that The antibody 1c comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.46 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.47; The antibody 1n comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.48 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO.

49.

3. The Helicobacter pylori antibody according to claim 1, characterized in that The antibody 2c comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.50 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.51; The antibody 2n comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.52 and a light chain variable region as shown in SEQ ID NO.

53.

4. The Helicobacter pylori antibody according to claim 1, characterized in that The antibody 3c comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.54 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.55; The antibody 3n includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO. 56 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

57.

5. Use of the Helicobacter pylori antibody according to any one of claims 1 to 4 in the preparation of a Helicobacter pylori detection product.

6. A Helicobacter pylori detection test strip, comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate in sequence, characterized in that: The marking pad contains the Helicobacter pylori antibody according to any one of claims 1 to 4, and the Helicobacter pylori antibody is marked with a marker; a detection line and a quality control line are arranged on the detection pad; the detection lines are 3, which are respectively coated with at least one of the Helicobacter pylori antibodies one, at least one of the Helicobacter pylori antibodies two, and at least one of the Helicobacter pylori antibodies three; the Helicobacter pylori antibody on the marking pad is different from the Helicobacter pylori antibody on the detection pad.

7. A Helicobacter pylori detection kit, characterized in that: It comprises the Helicobacter pylori detection test strip as claimed in claim 6 and a shell, wherein the Helicobacter pylori detection test strip is arranged inside the shell.

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