Single-chain antibody for detecting helicobacter pylori and application thereof

Abstract

The present application relates to the technical field of immune detection, in particular to a single-chain antibody for detecting Helicobacter pylori and application thereof. The present application constructs an avian scFv antibody library for Helicobacter pylori whole-bacterium lytic protein by using phage display technology, and screens a single-chain antibody for detecting Helicobacter pylori from the scFv antibody library, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with the amino acid sequences shown in SEQ ID NO: 1-3 respectively, and the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 with the amino acid sequences shown in SEQ ID NO: 4-6 respectively. The single-chain antibody has the characteristics of high detection accuracy and strong specificity, and has the potential for treating Helicobacter pylori. In addition, the present application can be expressed on a large scale and is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a single-chain antibody for detecting Helicobacter pylori and its application. Background Technology

[0002] Helicobacter pylori (H. pylori) is a Gram-negative microaerophilic bacterium that can survive in the gastric mucosa and cause various gastric diseases, including chronic gastritis, peptic ulcers, and even gastric cancer. Currently, methods for detecting H. pylori mainly include culture methods, urease tests, serological detection, and PCR technology. However, traditional detection methods suffer from insufficient sensitivity and specificity, as well as complex procedures. Therefore, developing new methods for detecting H. pylori with high specificity and sensitivity is of great significance.

[0003] Phage display technology refers to the use of genetic engineering techniques to insert the DNA sequence corresponding to a foreign polypeptide or protein into an appropriate position in the structural gene of the phage coat protein. The recombinant phage genome expresses the foreign polypeptide or protein as a fusion protein with the coat protein within the host bacteria, and displays the foreign polypeptide or protein onto the phage coat during phage self-assembly. Due to minimal steric hindrance, the foreign polypeptide or protein displayed on the phage coat maintains a relatively independent spatial structure and biological activity. In recent years, phage display technology has been widely used for the screening and optimization of monoclonal antibodies. Avian immune systems differ from mammalian immune systems, producing antibodies with higher affinity and broader antigen recognition capabilities. Therefore, screening for highly specific single-chain antibodies (scFv) against target antigens using phage display technology can be applied to numerous disciplines, including medicine, clinical diagnosis and treatment, and microbial detection. CN116693681A discloses a monoclonal antibody against Helicobacter pylori cytotoxin-associated protein A and its application. This monoclonal antibody can recognize and bind to Helicobacter pylori cytotoxin-associated protein A. CN117736320A discloses a method and application for detecting Helicobacter pylori based on HpaA and scFv antibodies. The single-chain antibody uses the HpaA protein of Helicobacter pylori as a binding target, which can effectively detect Helicobacter pylori with high specificity and high sensitivity.

[0004] Currently, the preparation of traditional monoclonal antibodies is time-consuming and costly. Compared with traditional monoclonal antibodies, recombinant antibodies have lower production costs and better uniformity. Given these limitations, it is necessary to further optimize phage display screening strategies to improve antibody specificity and affinity. Therefore, developing antibodies prepared using phage display technology and prokaryotic expression technology is both necessary and feasible. Summary of the Invention

[0005] To overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a single-chain antibody for detecting Helicobacter pylori. This antibody is obtained by screening an immune avian phage library and can specifically bind to Helicobacter pylori and its antigens, thereby detecting Helicobacter pylori with good specificity and accuracy.

[0006] Another object of the present invention is to provide the application of the above-described single-chain antibody for detecting Helicobacter pylori.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A single-chain antibody for detecting Helicobacter pylori comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO: 1-3, and the light chain variable region comprises amino acid sequences VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO: 4-6.

[0009] The amino acid sequence of the single-chain antibody used to detect Helicobacter pylori is shown in SEQ ID NO: 7.

[0010] The nucleotide sequence of the gene encoding a single-chain antibody for detecting Helicobacter pylori is shown in SEQ ID NO: 8.

[0011] A recombinant vector containing a gene encoding a single-chain antibody for detecting Helicobacter pylori is obtained by linking the nucleotide sequence of the gene encoding the single-chain antibody for detecting Helicobacter pylori to the vector.

[0012] The vector can be the phage vector pComb3xss or other vectors suitable for prokaryotic or eukaryotic expression.

[0013] A strain expressing a single-chain antibody for detecting Helicobacter pylori is obtained by transferring the aforementioned recombinant vector containing a gene encoding a single-chain antibody for detecting Helicobacter pylori into a host strain.

[0014] The host strain can be a prokaryotic expression strain such as Escherichia coli or other eukaryotic expression strains.

[0015] The Escherichia coli mentioned can be TOP10F'.

[0016] The method for preparing the single-chain antibody for detecting Helicobacter pylori includes the following steps:

[0017] The strain expressing the single-chain antibody for detecting Helicobacter pylori was induced to express the antibody, purified, and then used to obtain the single-chain antibody for detecting Helicobacter pylori.

[0018] The preferred conditions for inducing expression are: adding IPTG to the bacterial culture of the strain expressing a single-chain antibody for detecting Helicobacter pylori to induce expression.

[0019] The final concentration of IPTG is preferably 0.5 M.

[0020] The OD of the bacterial culture of the strain expressing single-chain antibodies for detecting Helicobacter pylori. 600 0.6-0.8.

[0021] The preferred conditions for inducing expression are 16℃ shaking culture for 12-18 h.

[0022] The purification is preferably performed using a Ni NTA Beads 6FF affinity chromatography column.

[0023] The preferred specific purification procedure is as follows:

[0024] The induced bacterial culture was lysed, centrifuged, and the supernatant was purified using a Ni NTA Beads 6FF affinity chromatography column to obtain a single-chain antibody for detecting Helicobacter pylori.

[0025] The use of the single-chain antibody or its conjugate for detecting Helicobacter pylori in the preparation of reagents or kits for detecting whole Helicobacter pylori or specific Helicobacter pylori antigens.

[0026] The use of the single-chain antibody or its conjugate for detecting Helicobacter pylori in the preparation of a medicament for treating Helicobacter pylori infection.

[0027] A reagent or kit for detecting Helicobacter pylori, comprising the single-chain antibody described above for detecting Helicobacter pylori.

[0028] A rapid method for detecting Helicobacter pylori includes the following steps:

[0029] (1) Coating: After inactivating the bacterial culture sample to be tested, place it in an ELISA plate for coating;

[0030] (2) Blocking: Discard the coating solution and wash with PBST; add skim milk powder to each well and block at 37℃ for 0.5-1h;

[0031] (3) Incubation: Discard the skim milk powder, wash with PBST; add the above single-chain antibody, and incubate at 37°C for 0.5-1 h;

[0032] (4) Washing: Discard the antibody solution and wash with PBST; add the diluted secondary antibody solution and incubate at 37°C for 0.5-1 h;

[0033] (5) Detection: Discard the diluted secondary antibody solution, wash with PBST; add TMB chromogenic solution, block at 37℃ for 15-30 min; then add H2SO4 solution to stop the color development, and read the OD value using a microplate reader. 450 Numerical values ​​are used to calculate the P / N value, and the P / N value is used to determine whether the bacterial culture sample is positive for Helicobacter pylori.

[0034] The concentration of the skim milk powder mentioned in step (3) is preferably 3% (W / V).

[0035] The secondary antibody mentioned in step (4) is preferably an HRP-labeled anti-HA tag antibody.

[0036] The preferred dilution ratio of the secondary antibody solution in step (4) is 1:10000.

[0037] The method for calculating the P / N value mentioned in step (5) is as follows:

[0038] P / N = (Experimental group OD) 450 - Blank group OD 450 ) / (negative group OD 450 - Blank group OD 450 ).

[0039] The method for judgment described in step (5) is preferred:

[0040] A P / N ratio ≥ 2.1 indicates a positive result for Helicobacter pylori, while a P / N ratio < 2.1 indicates a negative result for Helicobacter pylori.

[0041] The present invention has the following advantages and effects compared with the prior art:

[0042] (1) This invention utilizes phage display technology to construct an avian scFv antibody library containing fragmented whole-cell Helicobacter pylori protein for immunotherapy. This immunotherapy library can effectively screen for scFv antibodies against Helicobacter pylori. Figure 12 This provides a new and effective approach for further detection and treatment of Helicobacter pylori.

[0043] (2) The present invention provides a single-chain antibody for detecting Helicobacter pylori with high detection accuracy, strong specificity and simple acquisition method. The single-chain antibody can be used to construct a variety of detection platforms; and the antibody can be expressed on a large scale, is suitable for industrial production, and has the potential to treat Helicobacter pylori.

[0044] (3) This invention provides a method for rapid detection of Helicobacter pylori, which is simple to operate, highly accurate and specific. Attached Figure Description

[0045] Figure 1This is a 1% (w / w) agarose gel electrophoresis image of the light chain gene fragment (VL) and heavy chain gene fragment (VH) amplified by PCR in Example 1.

[0046] Figure 2 This is a 1% (w / w) agarose gel electrophoresis image of the overlapping VL and VH gene fragments in Example 1.

[0047] Figure 3 This is a diagram showing the results of comparing the antibody light chain and heavy chain CDR3 region sequences obtained by sequencing 15 randomly selected colonies on a plate after electroporation transformation in Example 1 with the antibody sequence fragments from avian antibody embryonic lineage.

[0048] Figure 4 This is an analysis chart of the phage-ELISA results after four rounds of affinity screening in Example 1, where R0 represents the result before screening, and R1-R4 correspond to the results of the four rounds of affinity screening, respectively.

[0049] Figure 5 This is an analysis diagram of the results of screening for positive monoclonal phages with specific single-chain antibodies in Example 1. Among them, 1-75 are positive monoclonal phages extracted from 75 monoclonal colonies randomly selected from the output plate after the third round of enrichment.

[0050] Figure 6 This is an ELISA result of the binding activity of monoclonal phage TL-14 to H. pylori in Example 1.

[0051] Figure 7 This is a diagram showing the amino acid sequence obtained from sequencing the prokaryotic clone of H. pylori using a single-chain antibody in Example 1.

[0052] Figure 8 This is the SDS-PAGE electrophoresis result of the detection of single-chain antibody against H. pylori in engineered E. coli in Example 1. In the figure, Mark: protein standard molecular weight; Before induction: bacterial culture before IPTG induction; After induction: bacterial culture after IPTG induction; Precipitation: precipitate obtained after bacterial lysis and centrifugation; Supernatant: supernatant obtained after bacterial lysis and centrifugation; Flow through: solution eluted from the supernatant after passing through the affinity chromatography column; Collection 1 and Collection 2: solutions for eluting the target protein, with elution buffer being 250 mM imidazole PBS solution (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

[0053] Figure 9 This is a graph showing the detection results of single-chain antibodies against different concentrations of Helicobacter pylori in Example 2.

[0054] Figure 10This is a graph showing the results of the single-chain antibody specificity analysis for detecting H. pylori in Example 2.

[0055] Figure 11 This is a diagram showing the molecular docking results.

[0056] Figure 12 This is a flowchart of the present invention. Detailed Implementation

[0057] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0058] The Helicobacter pylori standard strain SS1 used in this example was provided by Southern Medical University.

[0059] Example 1

[0060] 1. Immunizing hens with purified whole-cell Helicobacter pylori protein.

[0061] (1) The Helicobacter pylori SS1 strain frozen in an ultra-low temperature freezer (-80℃) was taken out and revived on Columbia solid medium plates containing 10% (v / v) sterile defibrinated sheep blood. It was then cultured at 37℃ in a microaerophilic environment (containing 85% N2, 10% CO2 and 5% O2) for 72 h. After the bacteria had grown to the top of the medium plate, they were scraped and resuspended in 10 mL of 0.85% (w / w) physiological saline using a disposable sterile spreader in a laminar flow hood. The resuspended bacteria were then inoculated onto 10 new Columbia solid medium plates containing 10% (v / v) sterile defibrinated sheep blood and cultured for another 72 h. The collected bacterial solution was then centrifuged in a high-speed centrifuge at 10000 rpm and 4℃ for 10 minutes. min; discard the supernatant, resuspend the bacterial pellet in 10 mL of 0.85% (w / w) physiological saline, centrifuge again and collect the bacterial pellet; repeat resuspension and centrifugation three times to remove residual culture medium; resuspend the collected bacterial pellet in 10 mL of PBS solution for sonication disruption, sonication power 300 W, sonication start 2 s, sonication interval 3 s, sonication time 5 min, sonication three times, the sonication process is kept in an ice water bath.

[0062] (2) Centrifuge the bacterial solution after ultrasonic disruption in step (1) using a high-speed centrifuge. The centrifugation conditions are: 10,000 rpm, 4℃ for 30 min. Take the supernatant and desalt it through Sephadex-G25 cross-linked dextran gel (Shanghai McLean Biochemical Technology Co., Ltd.). Then concentrate it through a 15mL ultrafiltration tube with a 3KD cutoff (Millibo (Shanghai) Trading Co., Ltd.) to obtain the whole-cell disrupted and purified Helicobacter pylori protein.

[0063] (3) The concentration of the whole-cell broken and purified protein of Helicobacter pylori obtained in step (2) was determined by BCA. The protein was prepared into a solution with a concentration of 1 mg / mL using physiological saline. The solution was injected intramuscularly into four wing and abdominal sites of 20-week-old yellow-feathered laying hens, 250 μL at each site. After the first injection, 4 booster injections were given every 10 days with 0.5 mg / mL of immunizing reagent (whole-cell broken and purified protein of Helicobacter pylori). The injection method was the same as the first time. A total of 5 immunizations were performed.

[0064] 2. Total RNA extraction and cDNA synthesis from chicken spleen cells

[0065] Extract the spleen from the immunized hen in step 1, grind and separate the tissue cells using a cell sieve, add lymphocyte separation solution (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: 10771-100ML), and separate the erythrocytes and lymphocytes using density gradient centrifugation according to standard methods to obtain lymphocytes; extract total RNA from lymphocytes using Trizol reagent, and then reverse transcribe the RNA into cDNA using Thermo Scientific RT reverse transcription kit (Thermo Fisher Scientific, catalog number: K1622), and store at -80℃ for later use.

[0066] 3. Amplification of single-chain antibody (scFv) gene fragments

[0067] (1) The primers for amplifying the scFv antibody gene were synthesized by Shanghai Bioengineering Co., Ltd. The specific primer sequences are shown in Table 1.

[0068] Table 1 Amplification Primer Sequences

[0069]

[0070] Note: The underlined part marks the sfiI restriction site, and G-back is used for subsequent reverse sequencing.

[0071] (2) Using the cDNA obtained in step 2 as a template, the light chain variable region (VL) and heavy chain variable region (VH) of the scFv antibody were amplified respectively; VL-F and VL-R were the upstream and downstream primers for the light chain variable region, and VH-F and VH-R were the upstream and downstream primers for the heavy chain variable region. The reaction system and PCR conditions were set according to the instructions of PrimeSTAR HS (Premix) DNA polymerase (Baori Biotechnology (Beijing) Co., Ltd., catalog number: R040Q) to amplify the VL gene fragment and VH gene fragment.

[0072] (3) The amplification effect of the PCR products obtained in step (2) was analyzed by 1% (w / w) agarose gel electrophoresis, and the PCR products were excised and recovered to obtain the VL gene fragment and the VH gene fragment. Figure 1 ).

[0073] (4) Using the VL and VH gene fragments obtained in step (3) as templates, the VL and VH gene fragments were ligated using PrimeSTAR HS (Premix) DNA polymerase. The ligation primers were CSC-F and CSC-R in Table 1. The reaction setup and PCR conditions were set according to the instructions for use of PrimeSTAR HS (Premix) DNA polymerase.

[0074] (5) The amplification effect of the PCR product obtained in step (4) was analyzed by 1% (w / w) agarose gel electrophoresis, and the PCR product was excised and recovered to obtain the scFv antibody gene fragment containing the sfiI restriction site. Figure 2 The concentration and purity of the DNA products were determined using a NanoDrop micro spectrophotometer.

[0075] 4. Construction and purification of pComb3xss-scFV recombinant plasmid

[0076] (1) The pComb3xss plasmid (Wuhan Miaoling Biotechnology Co., Ltd.) and the scFv antibody gene fragment containing the sfiI restriction site obtained in step 3 were digested simultaneously using the NEB restriction endonuclease SfiI enzyme (New England Biotechnology (Beijing) Co., Ltd.). The digestion conditions were as per the instructions. After digestion, 22.5 μL of 0.5 MEDTA solution was added to every 200 μL of the system to inactivate the SfiI enzyme. The digested products were analyzed by 1% (w / w) agarose gel electrophoresis and recovered by gel excision. The concentration and purity of the recovered DNA products were determined by NanoDrop micro spectrophotometer.

[0077] (2) The pComb3xss plasmid and scFv gene fragment recovered from enzyme digestion in step (1) were ligated using NEB T4 DNA ligase (purchased from New England Biotechnology (Beijing) Co., Ltd.) to construct a recombinant plasmid. The PCR ligation conditions for T4 ligase were as specified in the instruction manual. After ligation, the PCR product was placed at 65℃ for 10 min to inactivate the T4 ligase.

[0078] (3) Subsequently, 3 M sodium acetate (pH 5.2) was added to the ligation product (recombinant plasmid) obtained in step (2). 20 μL of 3 M sodium acetate (pH 5.2) was added to every 200 μL of system, and the mixture was thoroughly mixed. Then, 660 μL of anhydrous ethanol was added, and the mixture was placed at -20°C overnight. The next day, the mixture was centrifuged at 4°C and 12,000 rpm for 30 min, and the supernatant was discarded. The product was resuspended in 75% (v / v) ethanol, centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was discarded. The centrifuge tube was inverted and placed in a fume hood to dry any residual ethanol. 30 μL of sterile water was added to reconstitute the product, and the mixture was placed at -20°C for later use. The purpose of the above steps is to remove ions from the recombinant plasmid, preparing it for the construction of an electroporation competent phage single-chain antibody gene library.

[0079] 5. Amplification of helper phages

[0080] (1) Take out Escherichia coli ER2738 strain (Lucigen, USA) from the -80℃ freezer, streak it onto LB agar plates containing 20 μg / mL tetracycline, and incubate overnight at 37℃. The next day, pick a single colony and inoculate it into 5 mL of LB liquid medium containing 20 μg / mL tetracycline. Incubate at 37℃ and 250 rpm for about 8 h. Take 200 μL of bacterial solution every 1 h to measure OD. 600 until OD 600 It reaches around 0.8.

[0081] (2) Dilute 1 μL of M13KO7 helper phage (New England Biotechnology (Beijing) Co., Ltd.) with sterile PBS solution at a concentration of 10 μL. 5 10 6 10 7 2 μL of the diluted M13KO7 helper phage was added to 98 μL of the Escherichia coli ER2738 bacterial culture obtained in step (1) and incubated at 37°C for 15 min to obtain the optimal dilution factor.

[0082] (3) After melting the top layer of LB agar, take 3 mL into a sterile centrifuge tube and place it in a 50℃ constant temperature water bath for preheating.

[0083] (4) Add the bacterial solution after infection in step (2) to the LB top agar preheated in step (3), mix thoroughly, quickly pour onto LB plates at 37°C, invert the plates, and incubate overnight at 37°C.

[0084] (5) Pick plaques from the plate and inoculate them into 5 mL of freshly cultured Escherichia coli ER2738 in step (1). Incubate at 37°C for 15 min, then shake at 250 rpm for 2 h. Transfer the bacterial culture to 95 mL of LB liquid medium and shake at 37°C and 250 rpm for 1 h. Add kanamycin sulfate to a final concentration of 70 μg / mL and continue shaking and culturing overnight.

[0085] 6. Recover bacteriophages

[0086] (1) Centrifuge the bacterial culture obtained in step 5 at 4°C and 10,000 rpm for 30 min, and collect the supernatant I;

[0087] (2) Add one-quarter of the supernatant volume of 5×PEG / NaCl solution (2.5 M) and mix well. Let stand on ice for 2 h to precipitate the phage. Then centrifuge at 4℃ and 10000 rpm for 30 min, discard the supernatant, and air dry the remaining solution to allow the PEG / NaCl solution in the tube to flow out completely.

[0088] (3) Add one-fifth volume of PBS to the supernatant I to resuspend the precipitate in step (2), add one-quarter volume of 5×PEG / NaCl solution to the supernatant I, and repeat step (2); after air drying the residual solution, add 1 mL of PBS to resuspend the precipitate, take 2 μL for titer determination, and store the rest at -80℃ for later use.

[0089] 7. Phage titer determination

[0090] The phages obtained in step 6 were serially diluted using sterile PBS solution. 2 μL of the diluted phages were added to 198 μL of freshly cultured *E. coli* ER2738, and incubated at 37°C for 15 min. 100 μL of the incubated phages was then spread onto LB agar plates containing 50 μg / mL of the appropriate antibiotic (kanamycin sulfate for determining helper phage titers; ampicillin for determining phage libraries), and incubated overnight at 37°C. The following day, the phage titer was calculated by counting the lysing spots formed on the LB agar plates, expressed as pfu / mL.

[0091] Phage titer = (dilution factor × 100 × number of counts) / phage stock solution volume (mL)

[0092] 8. Construction of phage single-chain antibody gene libraries (E. coli libraries and phage libraries containing target antibody fragments)

[0093] (1) Transfer the TG1 electroporation competent cells (New England Biotechnology (Beijing) Co., Ltd.) from the -80℃ freezer to an ice box to thaw, and pre-cool the electroporation cuvette (0.2 cm). Add 50 ng of the pComb3xss-scFV recombinant plasmid purified in step 4 (volume should not exceed 5 μL) to 50 μL of electroporation competent cells, and gently tap with your fingernail (avoid vigorous shaking to avoid generating air bubbles). Add the above mixture to the pre-cooled electroporation cuvette, insert the BioRad electroporator and electroporate, using the default recommended parameters (C=25 μF, PC=200 Ω, V=1.8 kV). After electroporation, immediately add 1 mL of 37℃ preheated SOC medium, rinse the electroporation cuvette, and aspirate the competent cells.

[0094] (2) Perform 10 electroporation transformation experiments consecutively according to step (1). Collect all competent cells into a 50 mL sterile tube and culture at 37℃ and 220 rpm for 1 h with shaking. Take 2 μL of bacterial culture for library capacity determination. The specific determination method is as follows: the bacterial culture is serially diluted 10-fold with SOC medium, and 100 μL of each gradient is spread on LB plates containing 50 μg / mL ampicillin and cultured overnight at 37℃. The next day, calculate the number of clones on the plate, randomly select 15 single clones for bacterial culture PCR, and send the successfully PCR-tested bacterial culture for sequencing to calculate the original library capacity.

[0095] The final results showed that the E. coli library containing the target antibody fragment had a library size of 2 × 10⁻⁶. 6 CFU / mL, 15 randomly selected clones all successfully amplified the target band by PCR, and the sequencing results are as follows. Figure 3 As shown, the left side represents the CDR3 region of the antibody light chain, and the right side represents the CDR3 region of the antibody heavy chain. The CDR3 regions of the antibody light chain and heavy chain are different, indicating that the sample insertion rate is 100%, reflecting that the constructed antibody library has good sequence diversity.

[0096] (3) The remaining bacterial culture was inoculated into 96 mL of LB liquid medium (containing 50 μg / mL ampicillin) and cultured at 37℃ with shaking at 220 rpm for 2 h; 1 mL of 1×10 12 The M13KO7 helper phage obtained in step 6 was incubated at 37°C for 30 min, followed by shaking culture at 250 rpm for 2 h. Kanamycin sulfate was added to the bacterial culture to a final concentration of 70 μg / mL, and the culture was continued overnight with shaking. The phage was recovered according to step 6, and the resulting solution is the phage display single-chain antibody library.

[0097] 9. Anti-H. pylori phage panning

[0098] (1) Coating: Following step 1, culture Helicobacter pylori standard strain SS1, add 2.5% (w / v) glutaraldehyde to the freshly cultured Helicobacter pylori standard strain SS1, centrifuge at 4000 g for 10 min, discard the supernatant, and resuspend the precipitate in carbonate buffer (CBS) to prepare OD. 600 Add 100 μL of bacterial solution to each well of the ELISA plate with a concentration of 0.2 μL, cover with sealing film, and incubate overnight at 4°C.

[0099] (2) Blocking: The next day, discard the coating solution, wash 3 times with PBST, pat dry on absorbent paper, add 350 μL of 3% (W / V) skim milk powder to each well, and block at 37℃ for 1 h.

[0100] (3) Incubation of the library: Discard the skim milk powder, wash 3 times with PBST, pat dry on absorbent paper, add 100 μL of the phage display single-chain antibody library solution constructed in step 8 to each well, and record the phage library titer as R1 Input. Cover with sealing film and incubate at 37℃ for 1.5 h.

[0101] (4) Washing: Carefully aspirate the phage solution, add 200 μL of 0.05% (V / V) Tween-20 PBS solution to each well, and vigorously pipette to wash. Repeat this step 10 times, and pat dry on absorbent paper.

[0102] (5) Elution: Add 100 μL of glycine-HCl solution (0.1 M, pH 2.2) for acid elution, let stand for 2 min, and then add 50 μL of Tris-HCl solution (1 M, pH 8.8) for neutralization.

[0103] (6) Collect the liquid in the well, take 2 μL for phage titer determination, and record it as R1 Output. Transfer the remaining liquid to 2 mL of freshly cultured Escherichia coli ER2738 bacterial culture, incubate at 37℃ for 30 min, add 6 mL of preheated LB liquid medium and ampicillin with a final concentration of 50 μg / mL, and continue to shake culture for 2 h.

[0104] (7) Add 1 mL of 1×10 12 Add pfu / mL M13KO7 helper phage, supplement with LB liquid medium to 100mL, and add ampicillin to a concentration of 50 μg / mL. Continue to culture in a shaker at 37°C for 2 h. Add kanamycin sulfate to a final concentration of 70 μg / mL, and culture in a shaker overnight. Then, recover the phage according to step 6.

[0105] (8) Following the above method, perform the next round of affinity panning. A total of 4 rounds of affinity panning are performed. In each round, the concentration of the coated bacterial solution gradually decreases, while the concentration of the Tween-20 PBS solution used for washing and the number of washes gradually increase. The panning conditions are shown in Table 2.

[0106] Table 2 Selection criteria for anti-H. pylori phage

[0107]

[0108] 10. Evaluation of the selection results

[0109] After four rounds of screening, polyclonal phage-ELISA was used to preliminarily assess the enrichment of Input-positive phages obtained in each round. The steps are briefly described below:

[0110] (1) Coating: Following step 1, culture Helicobacter pylori standard strain SS1, add 2.5% (w / v) glutaraldehyde to the freshly cultured Helicobacter pylori standard strain SS1, centrifuge at 4000 g for 10 min, discard the supernatant, and resuspend the precipitate in carbonate buffer (CBS) to prepare OD. 600 Dilute the bacterial suspension with a concentration of 0.2 μL to 10 times, add 100 μL of the diluted bacterial suspension to each well of the ELISA plate, cover with sealing film, and incubate overnight at 4°C.

[0111] (2) Blocking: The next day, discard the coating solution, wash 3 times with PBST, pat dry on absorbent paper, add 350 μL of 3% (W / V) skim milk powder to each well, and block at 37℃ for 1 h.

[0112] (3) Incubation: Discard the skim milk powder, wash 3 times with PBST, pat dry on absorbent paper, and add 100 μL of the Input positive phage solution obtained in each round of step 9 (1×10⁻⁶) to each well. 11 (pfu / mL), cover with sealing film, and incubate at 37℃ for 1.5h.

[0113] (4) Washing: Carefully aspirate the phage solution, wash 3 times with PBST, pat dry on absorbent paper, add 100 μL of HRP-labeled anti-M13 phage secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 11973-MM05) diluted in sterile PBS (1:10000) to each well, and block at 37℃ for 1 h.

[0114] (5) Detection: Discard the diluent, wash 3 times with PBST, and pat dry on absorbent paper; add 100 μL of standard TMB colorimetric solution (freshly prepared) to each well, and incubate at 37℃ for 15 min; add 50 μL of H2SO4 (2M) solution to each well to stop the colorimetric reaction, and read the OD value using a microplate reader. 450 Numerical value.

[0115] During the screening process, the concentration of the coating antigen used in each round decreased sequentially, while the concentration of detergent and the number of washes increased, making the screening conditions progressively more stringent. This helped to enrich positive phages with high binding affinity. After each round of increasingly stringent screening conditions, phages with weak binding affinity were eliminated, leaving only polyclonal phages that bound strongly to the Helicobacter pylori standard strain SS1, such as... Figure 4 As shown, the third round of Input-positive phage OD 450 The highest value indicates that the positive phages were effectively enriched after the third round of screening.

[0116] 11. Screening for monoclonal phages that are positive for specific single-chain antibodies

[0117] (1) Seventy-five single colonies were randomly selected from the R3 Output titer plate after the third round of enrichment and inoculated into 2 mL of LB liquid medium containing 50 μg / mL ampicillin. After incubation at 37℃ and 250 rpm for about 6 h, 200 μL of bacterial solution was taken every 1 h to measure OD. 600 until OD 600 It reaches around 0.8.

[0118] (2) Add 20 μL of M13KO7 helper phage (1×10⁻⁶) 12 (pfu / mL), incubate at 37℃ for 15 min, then shake at 250 rpm for 2 h. Add kanamycin sulfate to the bacterial culture to a final concentration of 70 μg / mL, and continue shaking and incubating overnight.

[0119] (3) The next day, the bacterial solution was centrifuged at 10,000 g for 25 min at 4℃, the supernatant was collected and filtered through a 0.22 μm filter membrane for later use.

[0120] (4) Evaluate the effectiveness of the positive monoclonal phages obtained in step (3) according to the method in step 10. Determination of positive clones: (Experimental group OD) 450 - Blank group OD 450 ) / (negative group OD 450 - Blank group OD 450 ≥2.1, meaning P / N is greater than or equal to 2.1. The larger the value, the better the effect.

[0121] according to Figure 5 It can be seen that the number of monoclonal phages is the largest, meaning that phage 14 is the most effective, and it is named TL-14.

[0122] 12. Identification of the binding activity of specific single-chain antibody-positive monoclonal phages to H. pylori

[0123] The most effective monoclonal phage, TL-14, was selected from the positive monoclonal phage efficacy evaluation. 200 μL of TL-14 was added to 2 mL of freshly cultured ER2738 *E. coli*. The culture was incubated at 37°C for 30 min, followed by the addition of 6 mL of preheated LB broth and ampicillin at a final concentration of 50 μg / mL. The culture was then continued with shaking for 2 h. Finally, 1 mL of 1×10⁻⁶ thiophanate-methyl was added. 12 M13KO7 helper phage (pfu / mL) was added to LB broth to a final volume of 100 mL, and ampicillin was added to a final concentration of 50 μg / mL. The mixture was then incubated at 37°C with shaking for 2 h. Kanamycin sulfate was then added to a final concentration of 70 μg / mL, and the mixture was incubated overnight with shaking. Phage recovery was performed according to step 6. 2 μL of the phage was used for titer determination, and the remainder was stored at -80°C. The binding activity of TL-14 with H. pylori was assessed according to step 10, using the following method:

[0124] ① Coating: Following step 1, culture Helicobacter pylori standard strain SS1. Add 2.5% (w / v) glutaraldehyde to the freshly cultured Helicobacter pylori standard strain SS1, centrifuge at 4000g for 10 min, discard the supernatant, and resuspend the precipitate in carbonate buffer (CBS) to prepare OD. 600 Dilute the bacterial suspension at 0.2 μL to 10-fold and add 100 μL of the diluted bacterial suspension to each well in each column of the ELISA plate. Make 3 replicates, cover with sealing film, and incubate overnight at 4°C.

[0125] ② Blocking: The next day, discard the coating solution, wash 3 times with PBST, pat dry on absorbent paper, add 350μL of 3% (W / V) skim milk powder to each well, and block at 37℃ for 1 hour.

[0126] ③Incubation: Discard the skim milk powder, wash 3 times with PBST, and pat dry on absorbent paper; dilute the positive monoclonal phage TL-14 to 1×10⁻⁶. 11 Add 100 μL of pfu / mL solution to each well of the microplate, using PBS as a blank control, and incubate at 37°C for 1 h.

[0127] ④ Washing: Carefully aspirate the phage solution, wash 3 times with PBST, and pat dry on absorbent paper. Add 100 μL of HRP-labeled anti-M13 phage secondary antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 11973-MM05) diluted in sterile PBS (1:10000) to each well and incubate at 37℃ for 1 h.

[0128] ⑤ Detection: Discard the diluent, wash 3 times with PBST, and pat dry on absorbent paper; add 100 μL of standard TMB chromogenic solution (freshly prepared) to each well, and incubate at 37℃ for 15 min; add 50 μL of H2SO4 (2M) solution to each well to stop the color development, and read the OD value using a microplate reader. 450 Numerical value.

[0129] Plot a standard curve with Helicobacter pylori concentration (log) on ​​the x-axis and absorbance on the y-axis. (See reference) Figure 6 .from Figure 6 As can be seen from the ELISA detection standard curve, the anti-Helicobacter pylori monoclonal phage provided in this application can effectively bind to Helicobacter pylori, and within a certain concentration range, the absorbance shows a good linear relationship with the bacterial concentration, thereby specifically achieving the detection of Helicobacter pylori.

[0130] 13. Prokaryotic expression of anti-H. pylori single-chain antibody

[0131] (1) Select the best-performing monoclonal phage TL-14 from the positive monoclonal phage effect evaluation, add 20 μL to 2 mL of freshly cultured Escherichia coli ER2738 bacterial solution, incubate at 37℃ for 15 min, then shake at 250 rpm for 2 h, take 1 mL of bacterial solution for sequencing, centrifuge the remaining bacterial solution at 4℃ and 4000g for 5 min, and extract the monoclonal phage particles according to the instructions of Omega EZNA® Plasmid DNA Mini Kit (purchased from Shanghai Sobo Biotechnology Co., Ltd.).

[0132] (2) TOP10F' heat-shock competent cells (Shanghai Angyu Biotechnology Co., Ltd.) were transferred from a -80℃ freezer to an ice box to thaw for 10 min. 2 μL of monoclonal phage particles were added, gently tapped to mix, and then quickly inserted into the ice box for incubation for 30 min. TOP10F' heat-shock competent cells were placed in a 42℃ water bath for 60 s heat shock, then quickly inserted into the ice box for incubation for 3 min. Subsequently, 700 μL of LB liquid medium (37℃) was added, and the cells were cultured in a constant temperature shaking incubator at 37℃ and 220 rpm for 1 h. 100 μL of bacterial suspension was spread on an LB solid plate (containing 50 μg / mL ampicillin) and cultured overnight. Monoclonal bacteria were picked up with a sterile toothpick and placed in 5 mL of LB liquid medium (containing 50 μg / mL ampicillin) and cultured overnight at 37℃ and 220 rpm. A single-clone bacterial culture was inoculated into 500 mL LB medium at an inoculum rate of 1% (v / v) for expansion culture. The culture was then incubated at 37°C with shaking at 220 rpm until OD reached [the desired growth rate]. 600 It is approximately 0.8.

[0133] (3) Add 500 μL of IPTG solution (0.5 M) to the bacterial culture in step (2) and culture overnight at 16°C and 220 rpm in a constant temperature shaking incubator; collect the bacterial culture and centrifuge at 4°C and 4000 g for 10 min; weigh the new centrifuge tube, discard the supernatant after centrifugation, resuspend the bacterial culture in PBS, collect it in the weighed new centrifuge tube, centrifuge at 4°C and 4000 g for 10 min; discard the PBS, weigh the centrifuge tube, and subtract the weight of the centrifuge tube before and after resuspension to obtain the weight of the wet bacterial cells.

[0134] (4) Bacterial lysis was performed according to the instructions of ACE lysis solution (Nanjing Aisiyi Biotechnology Co., Ltd., catalog number: BR0005-02) based on the weight of wet bacterial cells. The lysis solution was centrifuged at 8500 g for 15 min at 4℃. The supernatant was collected, and the lysed bacterial precipitate was retained for subsequent SDS-PAGE electrophoresis analysis.

[0135] (5) The supernatant was collected and purified by using a Ni NTA Beads 6FF affinity chromatography column (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) to obtain scFv protein. The specific purification steps are described in the instruction manual.

[0136] Sequencing revealed that the nucleotide sequence of the gene encoding the scFv antibody in the monoclonal phage particle is shown in SEQ ID NO: 8, the amino acid sequence of the scFv antibody is shown in SEQ ID NO: 7, and the various frame regions are shown in Table 3 and... Figure 7 As shown.

[0137] See protein purification Figure 8 As shown in the figure, a distinct expression band of the target single-chain antibody appears at approximately 34 kDa. Because the expression vector contains a histidine tag, it can bind to and elute with imidazole via Ni-NTA affinity chromatography, thus purifying the target antibody protein. The purified single-chain antibody has a molecular weight of approximately 34 kDa and high purity.

[0138] The nucleotide sequence of the gene encoding the scFv antibody (SEQ ID NO: 8) and the nucleotide sequences of the His peptide tag and HA peptide tag are shown below:

[0139] GCCCTGACTCAGCCGTCCTCGGTGTCAGCGAACCTGGGAGGAACCGTCAAGATCACCTGCTCCGGGAGTAGCATCTACTATGACTGGCACCAGCAGAAGTCACCTGGCAGTGCCCCTGTCACTCTGATCTATAACAACAACAAGAGACCCTCAGACATCCCTTCACGATTCTCCGGTGCCCTATCCGGCTCCAC ACACACATTAACCATCACTGGGGTCCGAGCCGAGGACGAGGCTGTCTATTTCTGTGGGACCTTCGACAATTACGGTGATATTTTTGGGGCCGGGACAACCCTGACCGTCCTAGGTCAGTCCTCTAGATCTTCCGCCGTGACGTTGGACGAGTCCGGGGGCGGCCTCCAGACGCCCGGAGGAGGGCTCAGCCTCG TCTGCAAGGCCTCCGGGTTCACCTTCAGCAGTTACAGCATGGGTTGGGTGCGACAGGCGCCCGGCAAGGGGCTGGAGTGGGTCGCTGGTATCAGCGACACTGGTAGATATACAGGCTACGGGTCGGCGGTGAAGGGCCGTGCCACCATCTCGAGGGACAACGGGCAGAGCACAGTGAGGCTGCAGCTGAACAAC CTCCGGGCTGAGGACACCGCCACCTACTACTGCGCCAAAGAGTCTGGTAGTCCTTACTATTGGGATGCTGGTTCTATCGACGCATGGGGCCACGGGACCGAAGTCATCGTCTCCTCCACTAGTggccaggccggccagcaccatcaccatcaccatggcgcatacccgtacgacgttccggactacgcttcttag

[0140] Note: The lowercase portion at the 3' end of the sequence is the purified and identified His peptide tag + HA peptide tag, and the uppercase portion is the complete nucleotide sequence of the gene encoding the scFv antibody.

[0141] The amino acid sequence of the scFv antibody (SEQ ID NO: 7) and the amino acid sequences of the His peptide tag and HA peptide tag are shown below:

[0142] ALTQPSSVSANLGGTVKITCSGSSIYYDWHQQKSPGSAPVTLIYNNNKRPSDIPSRFSGALSGSTHTLTITGVRAEDEAVYFCGTFDNYGDIFGAGTTLTVLGQSSRSSAVTLDESG GGLQTPGGGLSLVCKASGFTFSSYSMGWVRQAPGKGLEWVAGISDTGRYTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTATYYCAKESGSPYYWDAGSIDAWGHGTEVIVSSTS GQAGQHHHHHHGAYPYDVPDYAS

[0143] Note: The underlined parts represent His peptide tags + HA peptide tags.

[0144] Table 3. Frame regions and their sequences

[0145]

[0146] Example 2

[0147] 1. Establishment of an enzyme-linked immunosorbent assay (ELISA) based on H. pylori-specific single-chain antibody

[0148] (1) Coating: Following step 1, culture Helicobacter pylori standard strain SS1, add 2.5% (w / v) glutaraldehyde to the freshly cultured Helicobacter pylori standard strain SS1, centrifuge at 4000 g for 10 min, discard the supernatant, and resuspend the precipitate in CBS to prepare OD. 600 Dilute the bacterial suspension at 0.2 μL to 10-fold and add 100 μL of the diluted bacterial suspension to each well in each column of the ELISA plate. Make 3 replicates, cover with sealing film, and incubate overnight at 4°C.

[0149] (2) Blocking: The next day, discard the coating solution, wash 3 times with PBST, pat dry on absorbent paper, add 350 μL of 3% (W / V) skim milk powder to each well, and block at 37℃ for 1 hour.

[0150] (3) Incubation: Discard the skim milk powder, wash 3 times with PBST, and pat dry on absorbent paper; dilute the single-chain antibody purified in Example 1 to 100 μg / mL, add 100 μL / well to the microplate, use PBS solution as blank control, and incubate at 37℃ for 1 h.

[0151] (4) Washing: Carefully aspirate the antibody solution, wash 3 times with PBST, and pat dry on absorbent paper. Add 100 μL of HRP-labeled anti-HA tag antibody (Abogen (Shanghai) Trading Co., Ltd.) diluted in sterile PBS (1:10000) to each well and incubate at 37°C for 1 h.

[0152] (5) Detection: Discard the diluent, wash 3 times with PBST, and pat dry on absorbent paper; add 100 μL of standard TMB colorimetric solution (freshly prepared) to each well, and incubate at 37℃ for 15 min; add 50 μL of H2SO4 (2M) solution to each well to stop the colorimetric reaction, and read the OD value using a microplate reader. 450 Numerical value. P / N = (Experimental group OD) 450 - Blank group OD 450 ) / (negative group OD 450 - Blank group OD 450 A standard curve was plotted with Helicobacter pylori concentration (log) on ​​the x-axis, absorbance on the left y-axis, and P / N on the right y-axis.

[0153] See results Figure 9 The ELISA standard curve shows that the anti-Helicobacter pylori single-chain antibody provided in this application can effectively bind to Helicobacter pylori. According to the reference P / N ≥ 2.1, a positive result is indicated. The minimum detection limit for the anti-Helicobacter pylori single-chain antibody is approximately 1 × 10⁻⁶. 4 cfu / mL.

[0154] 2. Specificity analysis based on H. pylori-specific single-chain antibody

[0155] (1) Coating: Escherichia coli ATCC 27853, Escherichia coli O157:H7, Staphylococcus aureus Mu50, Salmonella ATCC 14028, Bacillus cereus CICC 10352, and Acinetobacter baumannii ATCC 19606 preserved in our laboratory were heat-inactivated, centrifuged at 4000 g for 10 min, the supernatant was discarded, and the precipitate was resuspended in CBS to prepare OD. 600 Dilute the bacterial suspension (0.2 g / L) 10-fold and add 100 μL to each well to coat the microplate. Perform three replicates for each bacterial strain, and include a blank control. Cover with sealing film and incubate overnight at 4°C.

[0156] (2) Blocking: The next day, discard the coating solution, wash 3 times with PBST, pat dry on absorbent paper, add 350 μL of 3% (W / V) skim milk powder to each well, and block at 37℃ for 1 h.

[0157] (3) Incubation: Discard the skim milk powder, wash 3 times with PBST, and pat dry on absorbent paper; dilute the single-chain antibody purified in Example 1 to 100 μg / mL, add 100 μL / well to the microplate, use PBS solution as blank control, and incubate at 37℃ for 1 h.

[0158] (4) Washing: Carefully aspirate the antibody solution, wash 3 times with PBST, and pat dry on absorbent paper. Add 100 μL of HRP-labeled anti-HA tag antibody (Abogen (Shanghai) Trading Co., Ltd.) diluted in sterile PBS (1:10000) to each well and incubate at 37°C for 1 h.

[0159] (5) Detection: Discard the diluent, wash 3 times with PBST, and pat dry on absorbent paper; add 100 μL of standard TMB colorimetric solution (freshly prepared) to each well, and incubate at 37℃ for 15 min; add 50 μL of H2SO4 (2M) solution to each well to stop the colorimetric reaction, and read the OD value using a microplate reader. 450 Numerical value. Negative: OD450 of experimental group / OD450 of blank group ≥ 2.1.

[0160] according to Figure 10 The results showed that the OD450 values ​​of *Escherichia coli* ATCC 27853, *Escherichia coli* O157:H7, *Staphylococcus aureus* Mu50, *Salmonella* ATCC 14028, *Bacillus cereus* CICC 10352, and *Acinetobacter baumannii* ATCC 19606 bound to the *H. pylori*-specific single-chain antibody were all less than 0.36, indicating a negative result. This suggests that none of the above bacteria showed cross-linking with the *H. pylori*-specific single-chain antibody, demonstrating the specificity of the established method.

[0161] 3. Molecular docking

[0162] Based on the amino acid sequence of the scFV antibody (SEQ ID NO: 7), its protein structure was predicted using alphafold according to conventional methods, and its molecular docking with the HpaA protein was analyzed to assess the interaction between the two.

[0163] This invention performs molecular docking between the PDB file of the scFV antibody and the PDB file of the HpaA protein, which has the highest prediction accuracy using alphafold. The results show that the scFV antibody pocket binding site is located at the top, while the predicted scFV antibody binding site for the HpaA protein is located at the bottom right. Figure 11 This indicates that the target of the scFV antibody is not the HpaA protein.

[0164] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A single-chain antibody for detecting Helicobacter pylori, characterized in that... It includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains amino acid sequences VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO: 1-3, respectively. The light chain variable region contains amino acid sequences VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO: 4-6, respectively.

2. The single-chain antibody for detecting Helicobacter pylori according to claim 1, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

7.

3. The gene encoding the single-chain antibody for detecting Helicobacter pylori as described in claim 1 or 2, characterized in that... Its nucleotide sequence is shown in SEQ ID NO:

8.

4. A recombinant vector containing a gene encoding a single-chain antibody for detecting Helicobacter pylori, characterized in that: It is obtained by ligating the nucleotide sequence of the gene described in claim 3 to a vector.

5. A bacterial strain expressing a single-chain antibody for detecting Helicobacter pylori, characterized in that: It is obtained by transferring the recombinant vector described in claim 4 into the host strain.

6. The method for preparing a single-chain antibody for detecting Helicobacter pylori according to claim 1 or 2, characterized in that... It includes the following steps: The strain expressing the single-chain antibody for detecting Helicobacter pylori as described in claim 5 is induced to express the antibody, purified, and then used to obtain the single-chain antibody for detecting Helicobacter pylori.

7. The use of the single-chain antibody or its conjugate for detecting Helicobacter pylori as described in claim 1 or 2 in the preparation of reagents or kits for detecting whole Helicobacter pylori.

8. A reagent or kit for detecting Helicobacter pylori, characterized in that... It contains the single-chain antibody for detecting Helicobacter pylori as described in claim 1 or 2.

9. A method for detecting Helicobacter pylori for non-therapeutic and non-diagnostic purposes, characterized in that... It includes the following steps: (1) Coating: After inactivating the bacterial culture sample to be tested, place it in an ELISA plate for coating; (2) Blocking: Discard the coating solution and wash with PBST; add skim milk powder to each well and block at 37℃ for 0.5-1h; (3) Incubation: Discard the skim milk powder, wash with PBST; add the single-chain antibody as described in claim 1 or 2, and incubate at 37°C for 0.5-1 h; (4) Washing: Discard the antibody solution and wash with PBST; add the diluted secondary antibody solution and incubate at 37°C for 0.5-1 h; (5) Detection: Discard the diluted secondary antibody solution, wash with PBST; add TMB chromogenic solution, block at 37℃ for 15-30 min; then add H2SO4 solution to stop the color development, and read the OD value using an ELISA reader. 450 Numerical values ​​are used to calculate the P / N value, and the P / N value is used to determine whether the bacterial culture sample to be tested is positive for Helicobacter pylori.

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