A monoclonal antibody against Escherichia coli O55:B5 LPS and its application

Abstract

This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against *Escherichia coli* O55:B5 LPS and its applications. The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of CDR1 in the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3; the amino acid sequence of CDR1 in the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is LTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:5. Applying this monoclonal antibody to immunoassay techniques enables rapid, sensitive, and specific quantitative detection of *Escherichia coli* O55:B5 LPS or the entire bacterium in complex samples, demonstrating significant practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against Escherichia coli O55:B5 LPS and its application. Background Technology

[0002] Endotoxins are characteristic structural components of the outer membrane of Gram-negative bacterial cell walls, chemically composed of lipopolysaccharide (LPS). LPS accounts for 75% of the total surface area of ​​the outer membrane of Gram-negative bacteria, serving not only as a barrier maintaining bacterial structural integrity but also as a key molecular basis for their pathogenicity. When bacteria lyse or die, large amounts of LPS are released into the bloodstream or into the site of infection, potentially triggering severe pathological reactions such as fever, microcirculatory disturbances, disseminated intravascular coagulation (DIC), endotoxic shock, and even death.

[0003] Escherichia coli ( Escherichia coli LPS (Liver Blood Pulmonates) is one of the most common Gram-negative opportunistic pathogens in clinical practice. The O55:B5 serotype is a key representative strain of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC), and is closely associated with infantile diarrhea, hemorrhagic enteritis, and hemolytic uremic syndrome (HUS). LPS derived from O55:B5 is highly pyrogenic; even extremely low doses (nanogram levels) can trigger a pyrogenic reaction.

[0004] Existing technologies have reported monoclonal antibodies targeting the LPS core polysaccharide or lipid A. For example, a monoclonal antibody against Brucella LPS has been used in colloidal gold immunochromatographic assay (limit of detection 1000 CFU), and a humanized monoclonal antibody against Pseudomonas aeruginosa serotype IATSO11. However, due to the hydrophobicity and structural conservation of lipid A and the core polysaccharide, antibodies targeting these regions often exhibit low affinity or nonspecific binding. Although antibodies targeting specific serotype O-antigens have been reported, there are currently no monoclonal antibodies specifically targeting Escherichia coli O55:B5, a clinically common LPS serotype. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a monoclonal antibody against Escherichia coli O55:B5 LPS and its application.

[0006] The purpose of this invention is to obtain a monoclonal antibody capable of precisely binding to Escherichia coli O55:B5 LPS, thereby filling the gap in existing technologies regarding the lack of highly specific recognition tools for this particular serotype. To achieve the above objective, this invention employs the following technical solution: In a first aspect, the present invention provides a monoclonal antibody against Escherichia coli O55:B5 LPS, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region; The amino acid sequence of CDR1 in the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3. The amino acid sequence of CDR1 in the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is LTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:5.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:6; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:7.

[0008] A second aspect of the present invention provides a biological material comprising any one of the following (1) to (4): (1) Nucleic acid encoding the monoclonal antibody described in the first aspect; (2) An expression vector containing the nucleic acid described in (1); (3) A host containing the expression vector described in (2); (4) Hosts whose genomes contain the nucleic acids described in (1).

[0009] Furthermore, the present invention also provides an expression module comprising the nucleic acid, the expression module comprising a promoter, a terminator, and the nucleic acid described in the present invention.

[0010] Furthermore, the expression module also includes an expression module formed by combining one or more nucleic acids described in this invention in tandem, fusion expression or other feasible ways, and this invention does not limit this.

[0011] Furthermore, the present invention also provides a transcription unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, transmembrane signal peptides, and homologous recombination sites operatively linked to a nucleic acid sequence, such as promoter enhancers, ITR sequences, polyA, MIS signal peptides, etc.

[0012] Furthermore, the host cells described in this invention can be derived from plants, animals, microorganisms, or viruses, and this invention does not limit the source. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, thereby enabling the transformed host cells to replicate the protein-encoding vector or express the desired protein.

[0013] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0014] A third aspect of the invention provides a labeled antibody, comprising a marker and the monoclonal antibody described in the first aspect.

[0015] Furthermore, the markers include chemical markers and biological markers; the chemical markers include isotopes and / or chemical drugs; the biological markers include biotin, avidin, or enzymes.

[0016] Furthermore, the enzyme includes horseradish peroxidase or alkaline phosphatase.

[0017] A fourth aspect of the invention provides a conjugate comprising a conjugation medium and the monoclonal antibody described in the first aspect.

[0018] Furthermore, the coupling medium includes a solid medium or a semi-solid medium.

[0019] Furthermore, the coupling medium is selected from colloidal gold, polystyrene sheets, or beads.

[0020] A fifth aspect of the present invention provides the use of the monoclonal antibody described in the first aspect, the biological material described in the second aspect, the labeled antibody described in the third aspect, or the conjugate described in the fourth aspect in the preparation of a product for detecting Escherichia coli O55:B5 LPS.

[0021] A sixth aspect of the present invention provides a product for detecting Escherichia coli O55:B5 LPS, comprising the monoclonal antibody described in the first aspect, the biological material described in the second aspect, the labeled antibody described in the third aspect, or the conjugate described in the fourth aspect.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention provides a monoclonal antibody targeting LPS derived from *E. coli* O55:B5. By immunizing mice with LPS and performing fusion screening, a monoclonal antibody capable of recognizing native LPS conformation with high affinity was successfully obtained, effectively recognizing LPS specifically derived from *E. coli* O55:B5. This monoclonal antibody exhibits high affinity and specificity, effectively neutralizing LPS-induced inflammatory responses. Applying this monoclonal antibody to immunoassay techniques enables rapid, sensitive, and specific quantitative detection of *E. coli* O55:B5 LPS or the entire bacterium in complex samples (such as food, body fluids, and cell culture supernatants). It can be used on various detection platforms such as ELISA, immunochromatography, and immunofluorescence, providing a new intervention method for Gram-negative bacterial infections. Attached Figure Description

[0023] Figure 1 The results are for specific detection of LPS monoclonal antibody derived from Escherichia coli O55:B5.

[0024] Figure 2 The standard curves for LPS monoclonal antibodies derived from Escherichia coli O55:B5 are shown, where A represents the binding results of the LPS monoclonal antibody derived from Escherichia coli O55:B5 with the antigen, and B represents the standard curve. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0028] Example 1: Preparation of Monoclonal Antibodies 1. Mouse immunization Female Balb / C mice aged 6-8 weeks were immunized via multiple dorsal injections. For the initial immunization, 2 mg / mL LPS was emulsified with an equal volume of Freund's adjuvant to a final concentration of 1 mg / mL, with an immunization volume of 100 μL per mouse. Subsequent immunizations used Freund's incomplete adjuvant for fusion, with an immunization volume of 50 μL per mouse. Immunizations were administered 21-30 days apart, and serum samples were collected from the tail tip 7-10 days post-immunization for serum analysis. Serum titers were monitored using ELISA to assess immunization efficacy at different stages. Selected mice to be fused received a pulse immunization at half the dose of the booster immunization. The immunogen was diluted with physiological saline to 200 μL and gently injected into the left abdominal cavity of the mice using a 1 mL syringe.

[0029] 2. Cell fusion and screening On the day of fusion, mice were euthanized by cervical dislocation and immediately immersed in 75% alcohol for 5-10 minutes. They were then transferred to a clean bench, and the spleen was removed by cutting open the right abdomen with scissors. The spleen was gently ground on a 200-mesh stainless steel sieve and continuously rinsed with RPMI 1640 culture medium. The spleen cell suspension was collected in a 50 mL centrifuge tube, centrifuged at 1200 r / min for 8 minutes, the supernatant was discarded, and the cells were gently dispersed by tapping the bottom of the centrifuge tube. The spleen cells were resuspended in RPMI 1640 culture medium and transferred to a new 50 mL centrifuge tube. This centrifugation was repeated three times. Then, 15 mL of RPMI 1640 culture medium was added and mixed thoroughly. Cell fusion was performed using the traditional polyethylene glycol (PEG) fusion method. Myeloma cells and spleen cells were mixed evenly at a 1:5 ratio, centrifuged at 1200 r / min for 8 minutes, the supernatant was completely discarded, and the cells were gently dispersed by tapping the bottom of the centrifuge tube. Cell fusion was then prepared. Within the first minute, add 1 mL of PEG (37°C) dropwise to the mixed cells, gently shaking the centrifuge tube during the addition. Within the second minute, hold the centrifuge tube and let it stand. Within the third and fourth minutes, slowly add 2 mL of RPMI 1640 culture medium at a rate of 1 mL / min to the centrifuge tube. Within the fifth and sixth minutes, slowly add 4 mL of RPMI 1640 culture medium at a rate of 2 mL / min to the centrifuge tube. Within the seventh minute, add RPMI 1640 culture medium at a rate of 6 mL / min. Finally, add RPMI 1640 culture medium to a final volume of 20 mL, gently shaking the centrifuge tube throughout the process. Place the centrifuge tube in an incubator and let it stand for 5 minutes, then centrifuge at 800 r / min and 37°C for 8 minutes. Discard the supernatant and gently tap the bottom of the centrifuge tube with your thumb to disperse the cells. Resuspend the fused hybridoma cells in HAT selective medium and mix well. Then, add the 0.5 × 10⁻⁶ cells to the centrifuge tube. 5 The concentration was set at 200 μL per well, and the cells were cultured in a CO2 cell incubator.

[0030] On day 7 of fusion, the cell supernatant was positively detected using ELISA. 50 μL of cell culture medium was added to an LPS-coated and blocked ELISA plate, and subsequent ELISA procedures were performed until OD was measured. 450Limiting dilution was used for cell subcloning. Wells with high positivity and good cell condition were selected for subcloning, prioritizing single-cluster cells while maintaining high positivity and good inhibition. Cell condition and quantity were monitored periodically based on the dilution ratio. When the cell count reached a certain level, the supernatant was evaluated promptly. After 3-4 subcloning cycles, the best-performing cell clusters were selected for expansion culture. A portion was cryopreserved for later use, while the remainder was used to prepare large quantities of monoclonal antibodies.

[0031] The process of using LPS coating to perform ELISA screening on the culture supernatant is as follows: a) Dilute LPS with coating buffer to a final concentration of 1 μg / mL, with a volume of 100 μL per well. Incubate overnight at 4°C. After incubation, wash three times with PBST (containing 0.05% Tween-20) buffer.

[0032] b) Block with 5% BSA, add 200 μL to each well, incubate at 37°C for 2 hours, then wash three times with PBST buffer, pat dry and set aside.

[0033] c) Add primary antibody (cell culture supernatant), 50 μL per well, incubate at 37°C for half an hour, and wash four times with PBST buffer and pat dry.

[0034] d) Dilute the HRP-labeled goat anti-mouse antibody 5K times and add it to the above ELISA plate, 100 μL per well. Incubate at 37°C for half an hour. After incubation, wash four times with PBST buffer and pat dry.

[0035] e) Add the finished color developer and place at 37℃ for 5-10 minutes to develop the color.

[0036] f) Add the stop solution to terminate the reaction.

[0037] 3. Ascites preparation and purification Seven to ten days before hybridoma cell injection, each mouse was injected with 500 μL of sterile paraffin oil to reduce its immune response. Cells were collected using RPMI 1640 medium and centrifuged at 1200 r / min for 8 min. The supernatant was discarded, and the pellet was resuspended in culture medium. This process was repeated three times to remove FBS from the culture medium, resulting in a 500 μL cell suspension. The mouse abdomen was gently wiped with a cotton ball soaked in 75% alcohol, and the cell suspension was injected into the left lower abdomen. After 7 to 10 days, once the mouse abdomen was swollen, ascites was aspirated using a 5 mL syringe, with the abdomen gently massaged to facilitate drainage. The ascites was centrifuged at 8000 r / min for 10 min to remove blood, fat, and other tissues. The yellow ascites was then collected in centrifuge tubes and stored at -20°C.

[0038] Antibody purification was performed using the octanoic acid-saturated ammonium sulfate method, and the specific steps are as follows: (1) Take 2 mL of mouse ascites and centrifuge at 9000 r / min for 50 min at 4°C. Take the supernatant and add 2 times the volume of sodium acetate solution (0.01 M, pH=4) to a glass bottle. Adjust the pH to 4.5 with 1 M HCl. After stirring, add a certain volume of octanoic acid and continue stirring. Stop stirring after 30 min and let stand for 2 h. (2) Centrifuge the above solution at 9000 r / min for 50 min at 4°C, take the supernatant, add 200 μL of 0.01 M PBS, adjust the pH to 7.4 with 1 M NaOH, and then slowly add (NH4)2SO4 to a final concentration of 0.277 g / mL at 4°C (stirring while adding, and add within 30 min), and let stand overnight at 4°C to allow for complete precipitation; (3) Centrifuge the above solution at 8000 r / min for 15 min at 4℃, discard the supernatant, reconstitute with 2 mL of 0.01 M PBS, and dialyze the reconstituted solution at 4℃ for 72 h. After dialysis, aliquot and store at -20℃ for later use.

[0039] Example 2 Antibody Affinity Assay 1. Coating: Coat the ELISA plate with 100 μL of antigen at a concentration of 1 μg / mL and incubate overnight at 4°C. After the incubation, wash the plate three times with PBST buffer and pat dry.

[0040] 2. Blocking: Add blocking solution (5% BSA) to block non-specific binding sites, incubate at 37°C for 2 hours, and then wash three times with PBST buffer and pat dry.

[0041] 3. Pre-conjugation (key step): The antigen was serially diluted with PBS (50, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0001 μg / mL), and each diluted antigen was mixed with 10 μg / mL of antibody. The mixture was incubated overnight at 4°C to allow the reaction to reach equilibrium.

[0042] 4. Competitive Binding: Add the mixture from step 3 (containing the antigen-antibody complex and free antibody) to the antigen-coated plate and incubate at 37°C for half an hour. At this time, the antigen on the plate will capture the remaining free antibody in the solution.

[0043] 5. Detection: After washing, add enzyme-labeled secondary antibody and corresponding substrate for color development, and measure the absorbance value.

[0044] 6. Calculation: Substitute different absorbance values ​​into the following formula to obtain the affinity constant K.

[0045] A0 / (A0-A)=1+(K / a0), where A0 represents the OD when there are no competitors (i.e., the free antigen concentration is 0). 450 A represents the OD when a specific concentration of free antigen is present. 450 , where a0 represents the total concentration of free antigen added in the experiment.

[0046] After calculation, K = 0.043 nM.

[0047] The selected antibodies were then sequenced to obtain their complementarity-determining region (CDR) sequences, as shown in Table 1. Table 1 Antibody Sequences

[0048] Example 3 Specificity Detection Lipid A, Escherichia coli ( E. coli O55: LPS and Escherichia coli derived from B5 E. coli LPS from O111:B4, Salmonella enteritidis serotype Salmonella typhimurium ( Salmonella enterica serotype typhimurium LPS and Pseudomonas aeruginosa from LPS ( ) Pseudomonas aeruginosa LPS and Klebsiella pneumoniae (from) Klebsiella pneumoniae LPS and Escherichia coli from ) E. coli LPS from J5 was diluted and analyzed, and absorbance values ​​were measured. Three replicates were performed for each concentration. The specific steps are as follows: a) Coating: LPS from different sources was diluted (0.1, 1, 10, 100 μg / mL) and coated onto an ELISA plate. The plate was incubated overnight at 4°C.

[0049] b) Blocking: Block with 5% BSA at 37°C for 2 hours.

[0050] c) Add primary antibody: Dilute the antibody to 5 μg / mL and add 100 μL to each well of enzyme-labeled enzyme with different concentrations of coating material. Incubate at 37°C for 30 minutes.

[0051] d) Add secondary antibody: Dilute HRP-labeled goat anti-mouse secondary antibody 5K times, add 100 μL to each well, and incubate for 30 minutes.

[0052] e) Color development: Add the finished color developer and incubate at 37°C for 5-10 minutes.

[0053] f) Termination: Add 50 μL of 2M H2SO4 to each well and read the OD value.

[0054] The results are as follows Figure 1As shown, only LPS from E. coli O55:B5 showed color development, while LPS from other sources did not, indicating that the antibody has good specificity.

[0055] Example 4: Confirmation of Antigen Detection Range Standard curve of LPS monoclonal antibody derived from Escherichia coli O55:B5.

[0056] a) Coating: The antigen was serially diluted with PBS (50, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0001 μg / mL), and 100 μL was added to each well. The mixture was incubated overnight at 4°C.

[0057] b) Blocking: Block with 5% BSA at 37°C for 2 hours.

[0058] c) Add primary antibody: Dilute the antibody to 5 μg / mL, add 100 μL to each well, and incubate at 37°C for 30 minutes.

[0059] d) Add secondary antibody: Dilute the HRP-labeled goat anti-mouse secondary antibody 5000 times, 100 μL per well.

[0060] g) Color development: Add the finished color developer and incubate at 37°C for 5-10 minutes.

[0061] h) Termination: Add 50 μL of 2M H2SO4 to each well and read the OD value.

[0062] The results are as follows Figure 2 As shown in A and B, the antigen detection range is 0.01-10 μg / mL, R 2 =0.995, EC50=0.355μg / mL.

[0063] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against Escherichia coli O55:B5 LPS, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of CDR1 in the heavy chain variable region is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of CDR1 in the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of CDR2 is LTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

5.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:7; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

8.

3. A biomaterial, characterized in that, Includes any one of the following (1) to (4): (1) The nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 2; (2) An expression vector containing the nucleic acid described in (1); (3) A host containing the expression vector described in (2); (4) Hosts whose genomes contain the nucleic acids described in (1).

4. A labeled antibody, characterized in that, Includes markers and monoclonal antibodies as described in any one of claims 1 to 2.

5. The labeled antibody as described in claim 4, characterized in that, The markers include chemical markers and biological markers; the chemical markers include isotopes and / or chemical drugs; the biological markers include biotin, avidin, or enzymes.

6. The labeled antibody as described in claim 5, characterized in that, The enzymes include horseradish peroxidase or alkaline phosphatase.

7. A coupling, characterized in that, Includes the coupling medium and the monoclonal antibody as described in any one of claims 1 to 2.

8. The coupling as described in claim 7, characterized in that, The coupling medium includes a solid medium or a semi-solid medium.

9. The use of the monoclonal antibody according to any one of claims 1 to 2, the biological material according to claim 3, the labeled antibody according to any one of claims 4 to 6, or the conjugate according to any one of claims 7 to 8 in the preparation of a product for detecting Escherichia coli O55:B5LPS.

10. A product for detecting Escherichia coli O55:B5 LPS, characterized in that, The product includes the monoclonal antibody as described in any one of claims 1 to 2, the biomaterial as described in claim 3, the labeled antibody as described in any one of claims 4 to 6, or the conjugate as described in any one of claims 7 to 8.

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