Reagent or kit for detecting monkeypox virus a29l protein by antibody sandwich method and application thereof

Abstract

The present application relates to the field of biotechnology, in particular to a reagent or kit for detecting monkeypox virus A29L protein by antibody sandwich method and application thereof. The present application provides a monkeypox virus antigen detection kit for monkeypox virus A29L protein and a preparation method thereof, providing a rapid, sensitive, simple, and economical detection method for screening monkeypox virus infection. Compared with traditional detection techniques, the present application detects the antigen of monkeypox virus instead of directly detecting the genetic material of the virus or culturing the virus. The antigen detection method is relatively simple, not only can obtain the detection result in a short time, but also can adapt to large-scale screening, especially in the outbreak of epidemic or high-risk population, which is helpful to realize early diagnosis and timely intervention.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to reagents or kits for detecting monkeypox virus A29L protein using an antibody sandwich method, and their applications. Background Technology

[0002] Traditional methods for detecting monkeypox virus, such as virus isolation and polymerase chain reaction (PCR) detection, generally require long experimental cycles and are complex to operate. Virus isolation methods typically require sophisticated laboratory facilities and long incubation times, and their sensitivity is low, especially for samples with low viral loads. While PCR detection has certain advantages in sensitivity, it requires specialized equipment, technical personnel, and long reaction times, and suffers from serious drawbacks such as low detection efficiency when screening high-load samples.

[0003] In the current field of monkeypox virus detection, various methods are used to detect monkeypox virus infection. Existing technologies mainly include virus isolation, polymerase chain reaction (PCR) detection, immunological methods (such as enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, etc.), and antigen detection methods. The following is a detailed introduction to these existing technologies:

[0004] 1. Virus isolation and culture

[0005] Virus isolation is the classic method for detecting monkeypox virus infection. This method involves isolating the monkeypox virus from patient samples and observing its growth and pathological changes in cell culture. However, virus isolation methods have significant limitations: 1) Time-consuming: The virus isolation process typically takes several days to several weeks to confirm infection; 2) Complex operation: It requires specialized laboratory facilities and technicians, and carries certain biosafety risks; 3) Limited sensitivity: If the viral load in the patient sample is low, the success rate of virus isolation may be reduced.

[0006] Therefore, although virus isolation methods can provide the most direct evidence of the virus, they are not suitable for rapid and efficient large-scale screening.

[0007] 2. Polymerase chain reaction (PCR)

[0008] PCR is a molecular biology technique widely used for detecting viral infections. The PCR method confirms viral infection by amplifying specific gene fragments of the virus (such as the A29 or B7R gene of monkeypox virus). This method has the following advantages and disadvantages: 1) High sensitivity: PCR can detect low amounts of viral nucleic acid with high sensitivity; 2) High specificity: Through primer design targeting specific genes of monkeypox virus, PCR has high specificity; 3) Operational complexity: Although PCR is sensitive and specific, it requires specialized equipment (such as a PCR instrument), skilled personnel, and relatively complex experimental procedures. Furthermore, the PCR process typically takes several hours to obtain results.

[0009] Although PCR is very effective in detecting monkeypox virus, its relatively long testing cycle and operational complexity make it unsuitable for large-scale, rapid screening, especially during outbreaks or when resources are limited.

[0010] 3. Enzyme-linked immunosorbent assay (ELISA)

[0011] Enzyme-linked immunosorbent assay (ELISA) is an immunological method commonly used for the detection of viral antibodies and antigens. In the diagnosis of monkeypox virus, ELISA can be used to detect antibody reactions in serum or directly detect viral antigens in samples. This method has the following characteristics: 1) High specificity and sensitivity: ELISA provides high specificity by utilizing the binding of antigens to specific antibodies; 2) Suitable for large-scale screening: Compared with PCR, ELISA is simpler to operate and does not require specialized molecular biology equipment, making it more suitable for large-scale screening; 3) Sensitivity issues: Although ELISA has high sensitivity, it can still be affected by factors such as sample quality and reagent quality. Furthermore, ELISA is mainly used to detect two types of indicators: antibodies (for detecting past infections) and antigens (for detecting current infections). Accuracy and sensitivity depend on the quality of the antigen or antibody.

[0012] Although ELISA is simpler to perform than PCR, it takes longer to get results and its sensitivity may be insufficient in some cases, especially when the viral load is low.

[0013] 4. Immunohistochemical staining

[0014] Immunohistochemical staining is used to detect monkeypox virus antigens in infected tissues, typically through tissue sections or cell smears. This method can confirm monkeypox virus infection at the histological level. The characteristics of this method are: 1) Localized observation: This method can provide local anatomical information of viral infection, helping to confirm the infected area; 2) Complex operation: Immunohistochemical staining requires multiple steps such as sample processing, staining, and microscopic observation, and requires operation by professional technicians; 3) Sensitivity issues: The sensitivity of immunohistochemistry is lower than that of PCR, and its detection effect is not good for early or low-viral-load infections.

[0015] 5. Antigen detection

[0016] Antigen testing is a rapid detection method that has emerged in recent years. It detects viruses by utilizing the binding of specific antibodies to monkeypox virus antigens. This method has the following characteristics: 1) Rapid: Antigen testing can usually produce results within 30 minutes to 1 hour, greatly improving the detection speed; 2) Convenient: The operation process is simple, suitable for rapid on-site testing, and can even be performed without a laboratory.

[0017] Lower sensitivity: Antigen detection is generally less sensitive than PCR, especially in samples with low viral load, which may lead to false negatives.

[0018] Several antigen detection kits are currently available on the market, primarily for detecting other viruses such as influenza and COVID-19. While there has been some research progress in antigen detection for monkeypox virus, its sensitivity and specificity still need improvement, especially when dealing with variant strains and different clinical samples. Summary of the Invention

[0019] In view of this, the purpose of this invention is to develop a highly sensitive and specific antigen detection kit for monkeypox virus infection. This invention provides a reagent or kit for detecting monkeypox virus A29L protein using an antibody sandwich method, and its application. By optimizing antigen binding efficiency and improving the specificity and sensitivity of the detection reagent, this invention enables rapid and accurate detection of monkeypox virus infection in various clinical and environmental samples.

[0020] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0021] The present invention provides a coated antibody comprising a heavy chain variable region and a light chain variable region; wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region have amino acid sequences as shown in SEQ ID NO.15, SEQ ID NO.16, and SEQ ID NO.17, respectively; and the LCDR1, LCDR2, and LCDR3 of the light chain variable region have amino acid sequences as shown in SEQ ID NO.12, WAS, and SEQ ID NO.18, respectively.

[0022] In some specific embodiments of the present invention, the coated antibody comprises: the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.41; and the light chain variable region having an amino acid sequence as shown in SEQ ID NO.43.

[0023] The present invention also provides a labeled antibody comprising a heavy chain variable region and a light chain variable region: the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region having amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and the LCDR1, LCDR2 and LCDR3 of the light chain variable region having amino acid sequences as shown in SEQ ID NO.4, LVS and SEQ ID NO.5 respectively.

[0024] In some specific embodiments of the present invention, the labeled antibody comprises: the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO.25; and the light chain variable region having an amino acid sequence as shown in SEQ ID NO.27.

[0025] Preferably, the labeled antibody is conjugated with a marker. More preferably, the marker comprises biotin.

[0026] The present invention also provides a paired monoclonal antibody for sandwich assay of monkeypox virus A29L protein, comprising the coating antibody and the labeling antibody.

[0027] The present invention also provides a nucleic acid molecule comprising a DNA fragment encoding any of the following: (I) the coating antibody; (II) the labeled antibody; or (III) the paired monoclonal antibody.

[0028] In some specific embodiments of the present invention, the heavy chain variable region of the coated antibody includes a nucleic acid molecule as shown in SEQ ID NO. 40; or the light chain variable region of the coated antibody includes a nucleic acid molecule as shown in SEQ ID NO. 42.

[0029] In some specific embodiments of the present invention, the heavy chain variable region of the labeled antibody includes a nucleic acid molecule as shown in SEQ ID NO. 24; or the light chain variable region of the labeled antibody includes a nucleic acid molecule as shown in SEQ ID NO. 26.

[0030] The present invention also provides an expression vector comprising the nucleic acid molecule.

[0031] The present invention also provides a host cell, including the expression vector.

[0032] The present invention also provides the use of any of the following in the preparation of a monkeypox virus A29L protein detection product: (I) the coating antibody; (II) the labeled antibody; or (III) the paired monoclonal antibody.

[0033] The present invention also provides a reagent or kit for detecting monkeypox virus A29L protein using an antibody sandwich method, comprising any one of the following: (I) the coating antibody; (II) the labeled antibody; or (III) the paired monoclonal antibody.

[0034] Preferably, the kit further includes any one or a combination of at least two of the following: standards, antibody diluent, labeled enzyme, stop solution, blocking solution, or washing solution.

[0035] This invention provides the following beneficial effects:

[0036] Compared with the best existing technologies, the detection method involved in this invention has significant advantages in several aspects, including the following:

[0037] 1. Improve detection sensitivity and specificity

[0038] Existing technologies: While most existing monkeypox virus detection methods (such as virus isolation and culture, PCR, etc.) can provide relatively accurate results, their time, sensitivity, and specificity may be limited by factors such as operating conditions, sample quality, and viral load.

[0039] Advantages of this invention: By optimizing the antigen detection method and using monoclonal antibodies, this invention improves the sensitivity and specificity of detection, enabling accurate identification of monkeypox virus at low viral loads and reducing the occurrence of false positive or false negative results.

[0040] 2. Rapid detection and easy operation

[0041] Existing technology: Traditional PCR detection methods require a long time for sample preparation, amplification and analysis, usually several hours, and depend on laboratory equipment and professional operators, which limits their application at the grassroots level and in the field.

[0042] Advantages of this invention: This invention can quickly produce test results in a short time and is easy to operate. It not only shortens the test time, but can also be widely used in environments such as hospitals, clinics, and epidemic areas, and can respond quickly, especially during an outbreak.

[0043] 3. Low cost and high efficiency

[0044] Existing technologies: Current high-sensitivity virus detection technologies, such as PCR, typically require expensive equipment and consumables, and operators also require highly specialized training.

[0045] Advantages of this invention: This invention reduces reliance on high-cost equipment, while optimizing reagents and operating procedures, thus lowering the overall cost and making it suitable for large-scale deployment and addressing the economic burden of sudden epidemics.

[0046] 4. Greater adaptability and widespread applicability

[0047] Existing technologies: Current monkeypox virus detection technologies are often limited by equipment, environment, or technical level, resulting in low applicability in different regions and under different conditions;

[0048] Advantages of this invention: The detection technology of this invention not only improves the accuracy and speed of detection, but also has strong adaptability, low requirements for operating environment and equipment, and has broad market and application potential. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0050] Figure 1 This is a standard curve graph. Detailed Implementation

[0051] This invention discloses reagents or kits for detecting monkeypox virus A29L protein using an antibody sandwich method, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV) infection. It has become a new public health challenge worldwide. However, existing virus detection methods are relatively few and have problems such as insufficient sensitivity, complicated operation, and long detection cycle.

[0053] The main technical problem to be solved by this invention is to optimize existing detection methods and improve diagnostic accuracy, and to provide an efficient, convenient and accurate monkeypox virus antigen detection tool so as to quickly diagnose monkeypox virus infection in clinical, public health or animal surveillance, and at the same time provide assistance for disease control and prevention.

[0054] This invention develops a monkeypox virus antigen detection kit targeting the A29L protein of monkeypox virus and its preparation method, aiming to provide a rapid, sensitive, simple, and economical detection method for screening monkeypox virus infection. Compared to traditional detection techniques, the key innovation of this invention lies in its detection of the monkeypox virus antigen, rather than directly detecting the virus's genetic material or performing virus culture. The antigen detection method is relatively simple, not only obtaining results in a short time but also being suitable for large-scale screening, especially in outbreaks or high-risk populations, facilitating early diagnosis and timely intervention.

[0055] This invention optimizes antigen binding efficiency and enhances the specificity and sensitivity of the detection reagent, enabling rapid and accurate detection of monkeypox virus infection in various clinical and environmental samples. This innovative method effectively compensates for the shortcomings of existing detection techniques, providing a highly efficient and reliable tool for global public health. It helps health departments at all levels respond quickly to monkeypox outbreaks, implement precise prevention and control measures, reduce the risk of virus transmission, and ultimately protect public health. Through the development and widespread application of this novel antigen detection kit, the diagnostic efficiency and accuracy of monkeypox virus will be greatly improved in multiple fields such as disease prevention, epidemic monitoring, and animal quarantine, providing strong technical support for the global response to monkeypox virus transmission.

[0056] 1. Alternative: Traditional PCR detection method

[0057] Technical solution: Use polymerase chain reaction (PCR) technology for nucleic acid detection of monkeypox virus.

[0058] Advantages: High sensitivity: PCR is currently the recognized gold standard detection method with extremely high sensitivity, capable of detecting very low concentrations of the virus; Wide application: PCR equipment is commonly found in many laboratories, and the technology is mature and widely accepted.

[0059] Disadvantages: Long time: PCR testing usually takes several hours, including sample extraction, amplification and testing, which is not suitable for rapid screening; High equipment requirements: PCR requires complex equipment and a professional laboratory environment, which is not suitable for field or grassroots applications; High cost: PCR equipment, reagents and consumables are relatively expensive, and the cost is higher compared to some emerging testing technologies.

[0060] 2. Alternative solution: Detection based on microfluidic chips

[0061] Technical solution: Utilizing microfluidic chip technology, sample processing and virus detection are performed on the chip, combined with biosensor technology to achieve monkeypox virus detection.

[0062] Advantages: High efficiency: Microfluidic chips can complete sample processing and analysis in a small volume, with high efficiency and fast response; Integration: Multiple steps such as sample processing, virus detection and signal analysis can be integrated on the same chip, reducing manual operation and sample contamination; Portability: This technology can be used in portable devices, suitable for on-site testing.

[0063] Disadvantages: Equipment complexity: Although the chip itself is small, its manufacturing process may be relatively complex and requires relatively sophisticated equipment to operate and analyze; High cost: The initial equipment cost is high, which may limit its widespread application in some low-resource areas.

[0064] The amino acid sequence information of the CDR region of the antibody described in this invention is shown in Table 1:

[0065] Table 1. Amino acid sequence of the CDR region of the antibody

[0066]

[0067] MPXV Antibody #5-VH

[0068] DNA sequence (SEQ ID NO.24):

[0069] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGTCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGAGCATTAATCCTACCTATGGTGATACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0070] amino acid sequence:

[0071] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.25):

[0072] QVQLQQPGAELVKPGSSVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGS INPTYGDT NFTEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYC TRWDY WGQGTTLTVSS

[0073] MPXV Antibody #5-VL

[0074] DNA sequence (SEQ ID NO.26):

[0075] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGTTGGCAAGGTACACATTTTCCCCAAACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0076] amino acid sequence:

[0077] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.27):

[0078] DVVMTQTPLTLSVTIGQPASISCKSSQ SLLESDGKTY LNWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQT FGGGTKLEIK

[0079] MPXV Antibody #25-VH

[0080] DNA sequence (SEQ ID NO.28):

[0081] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGGGGATTAATCCTACCAGTGGTGCTACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGTTCAGCCTACCTGCAACTCAGCAGCCTGGCATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0082] amino acid sequence:

[0083] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.29):

[0084] QVQLQQPGAELVKPGASVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGG INPTSGAT NFTEKFKTKATLTVDKSSSSAYLQLSSLASEDSAVYYC TRWDY WGQGTTLTVSS

[0085] MPXV Antibody #25-VL

[0086] DNA sequence (SEQ ID NO.30):

[0087] GGTGTTGTGATGACCCAGACTCCACTCACTTTGTCGATTACCCTTGGACAACCAGCCTCCATTTCTTGCAAGTCAGGTCAGAGCCTCTTAGATAGTAATGGAAAGGCATACTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0088] amino acid sequence:

[0089] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.31):

[0090] GVVMTQTPLTLSITLGQPASISCKSG QSLLDSNGKAY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGTHFPHT FGGGTKLEIK

[0091] MPXV Antibody #28-VH

[0092] DNA sequence (SEQ ID NO.32):

[0093] CAGGTTATTCTGAAAGAGTCTGGCCCTGGAATATTGCAGCCCTCTCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTTAATACTTATAATACAGCTGTGAACTGGATTCGTCAGCCTTCTGGAAAGGGTCTGGAGTGGTTGGCACTAATCGGGTCAGATGATAATGAACTCTATAACCCATTTCTGAAAAGTCGAATCACAATCTCCAAGGATGCCTCCAACAGCCAGGTATTCCTCAAGATCACTAGTGTGGACACTCAAGATTCTGCCACATACTACTGTGTTAACGGGGGATTACGACGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0094] amino acid sequence:

[0095] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.33):

[0096] QVILKESGPGILQPSQTLSLTCSFS GFSLNTYNTA VNWIRQPSGKGLEWLAL IGSDDNE LYNPFLKSRITISKDASNSQVFLKITSVDTQDSATYYC VNGGLRRAWFAY WGQGTLVTVSA

[0097] MPXV Antibody #28-VL

[0098] DNA sequence (SEQ ID NO.34):

[0099] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATTAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTATAGTCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTTACAGGGAGTGGATATGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0100] amino acid sequence:

[0101] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.35):

[0102] DIVMTQSHKFMSTLVGDRVSITCKAS QDVGTA IVWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGYGTDFTLTISNVQSEDLADYFC QQYSSYPLT FGSGTKLEIK

[0103] MPXV Antibody #87-VH

[0104] DNA sequence (SEQ ID NO.36):

[0105] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGTCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTATATGTACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGGAGCATTAATCCTACCTATGGTGATACTAACTTCACTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGATGGGATTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0106] amino acid sequence:

[0107] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.37):

[0108] QVQLQQPGAELVKPGSSVKLSCKAS GYTFTSYY MYWVKQRPGQGLEWIGS INPTYGDT NFTEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYC TRWDY WGQGTTLTVSS

[0109] MPXV Antibody #87-VL

[0110] DNA sequence (SEQ ID NO.38):

[0111] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGAAAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGTTGGCAAGGTACACATTTTCCCCAAACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0112] amino acid sequence:

[0113] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.39):

[0114] DVVMTQTPLTLSVTIGQPASISCKSS QSLLESDGKTY LNWLLQRPGQSPKRLIY LVS KVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPQT FGGGTKLEIK

[0115] MPXV Antibody #94-VH

[0116] DNA sequence (SEQ ID NO.40):

[0117] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGAGCTATGGTTACGACGGGGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0118] amino acid sequence:

[0119] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.41):

[0120] EVQLVESGGGLVKPGGSLKLSCAAS GFTFSDYY MYWVRQTPEKRLEWVAT ISDGGSYT YYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYC ARELWLRRGWFAY WGQGTLVTVSA

[0121] MPXV Antibody #94-VL

[0122] DNA sequence (SEQ ID NO.42):

[0123] GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATTTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAGCAGCTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0124] amino acid sequence:

[0125] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.43):

[0126] DIVMTQSHKFMSTSVGDRVSITCKAS QDVGTA VAWYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGFGTDFTLTISNVQSEDLADYFCQ QYSSYLT FGAGTKLELK

[0127] MPXV Antibody #111-VH

[0128] DNA sequence (SEQ ID NO.44):

[0129] CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAACAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACTTATACTGGTAATACAAACTACAACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGGGGAATCTACAATGACTACGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0130] amino acid sequence:

[0131] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.45):

[0132] QVQLQQSGPELVRPGVSVKISCKGS GYTFTDYA MHWVKQSHAKSLEWIGV ISTYTGNT NYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYC ARGESTMTTTY WGQGTLVTVSA

[0133] MPXV Antibody #111-VL

[0134] DNA sequence (SEQ ID NO.46):

[0135] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGGGAATATTCACAATTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGAACACAATATTCTCTCAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAATTATTACTGTCAACATTTTTGGAGAACTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0136] amino acid sequence:

[0137] - Variable region sequence (CDRs are the underlined parts, SEQ ID NO.47):

[0138] DIQMTQSPASLSASVGETVTITCRAS GNIHNY LAWYQQKQGKSPQLLVY NAK TLADGVPSRFSGSGSGTQYSLKINSLQPEDFGNYYC QHFWRTPPT FGGGTKLEIK

[0139] Unless otherwise specified, the reagents or kits for detecting monkeypox virus A29L protein using the antibody sandwich method provided by this invention, as well as the raw materials and reagents used in their application, are all commercially available.

[0140] The present invention will be further illustrated below with reference to the embodiments:

[0141] Example 1: Determination of antibody titer

[0142] 1. Main reagents

[0143] All antibodies used in this invention are independently developed, and the main antibodies include: ①MPXVab#5 (ab5), ②MPXVab#25 (ab25), ③MPXVab#28 (ab28), ④MPXVab#87 (ab87), ⑤MPXVab#94 (ab94), and ⑥MPXVab#111 (ab111).

[0144] The A29L antigen protein sequence is as follows (SEQ ID NO.48):

[0145] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKL VCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKHNDEVLFRLENHAETLRAAMISLAKKIDVQTGRRPYEEFGGSHHHHHH

[0146] (1) Coating: A29L antigen, concentration 2μg / mL, 100μL / well, 37℃, 2h.

[0147] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0148] (3) Washing: PBST, 300 μL / well, twice.

[0149] (4) Add primary antibody: Dilute Ab-5, Ab-25, Ab-28, Ab-87, Ab-94, and Ab-111 (ELISA universal antibody diluent, purchased from Wuhan BOSTER Biotechnology Co., Ltd., catalog number: AR1106-2) with primary antibody diluent to 1 μg / mL to 0.015 μg / mL, 100 μL / well, 37℃, 1h.

[0150] (5) Washing: PBST, 300 μL / well, 3 times.

[0151] (6) Add secondary antibody: Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG Fc (purchased from Proteintech, catalog number: SA00001-1), 1:10000 (diluted using universal ELISA antibody diluent, purchased from Wuhan BOSTER Biotechnology Co., Ltd., catalog number: AR1106-2), 100 μL / well, 37℃, 30 min.

[0152] (7) Washing: PBST, 300 μL / well, 3 times.

[0153] (8) Add TMB substrate: 100 μL / well, 37 °C, 10 min.

[0154] (9) Termination: 2 mol HCl, 50 μL / well.

[0155] (10) Microplate reader reading: OD450-OD620.

[0156] Results Interpretation: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance was eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm. Dilution ratios were calculated from 1μg / mL to 0.015μg / mL. Wells with OD values ​​more than twice that of the blank control were considered positive. The dilution factor of the lightest-colored positive well represents the titer of the target antibody in that serum sample.

[0157] Numerically, the lightest-colored well (0.015 μg / mL) had an OD value more than twice that of the blank control Blank, so this well can be judged as positive and used as the target antibody titer.

[0158] Table 2. Antibody titer ratio

[0159]

[0160] Example 2 Biotin binding efficiency

[0161] (1) Coating: A29L antigen, concentration 2μg / mL, 100μL / well, 37℃, 2h.

[0162] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0163] (3) Washing: PBST, 300 μL / well, twice.

[0164] (4) Add primary antibody: Biotin-conjugated antibodies (Bio-5 (Biotin-conjugated ab5), Bio-25 (Biotin-conjugated ab25), Bio-28 (Biotin-conjugated ab28), Bio-87 (Biotin-conjugated ab87), Bio-94 (Biotin-conjugated ab94), or Bio-111 (Biotin-conjugated ab111)) were prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120). The dilution buffer (ELISA universal antibody dilution buffer, purchased from Wuhan BOSTER Biotechnology Co., Ltd., catalog number: AR1106-2) was 1 μg / mL to 0.015 μg / mL, 100 μL / well, 37℃, 1h.

[0165] (5) Washing: PBST, 300 μL / well, 3 times.

[0166] (6) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0167] (7) Add TMB substrate: 100 μL / well, 37℃, 10 min.

[0168] (8) Washing: PBST, 300 μL / well, 3 times.

[0169] (9) Termination: 2 mol HCl, 50 μL / well.

[0170] (10) Microplate reader reading: OD450-OD620.

[0171] Interpretation of Results:

[0172] Read the OD value; 450nm is the specific absorbance, and 620nm is the non-specific absorbance. Non-specific absorbance can be removed by subtracting the absorbance at 620nm from the absorbance at 450nm. Convert the absorbance to the corresponding dilution ratio according to concentrations from 1μg / mL to 0.015μg / mL. The following values ​​are obtained: wells with an OD value higher than twice that of the blank control are considered positive; the dilution factor of the lightest-colored positive well is the target antibody titer in that serum sample.

[0173] The biotin-streptavidin system is an amplified labeling technique, and its sensitivity is improved after the addition of biotin for optimization.

[0174] Table 3 Biotin binding efficiency

[0175]

[0176] Negative control experiment:

[0177] (1) Coating: Non-specific antigen (non-specific antigen A is human adenovirus, B is EV71, C is SFTSV, D is human rotavirus, E is influenza virus, F is human herpes zoster virus, G is hepatitis B virus, antigens are stored in the laboratory.), concentration 2 μg / mL, 100 μL / well, 37℃, 2h.

[0178] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0179] (3) Washing: PBST, 300 μL / well, twice.

[0180] (4) Add primary antibody: Biotin-conjugated antibodies (Bio-5, Bio-25, Bio-28, Bio-87, Bio-94 or Bio-111) were prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120). The dilution was 1 μg / mL to 0.015 μg / mL, 100 μL / well, 37℃, 1h.

[0181] (5) Washing: PBST, 300 μL / well, 3 times.

[0182] (6) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0183] (7) Add TMB substrate: 100 μL / well, 37℃, 10 min.

[0184] (8) Washing: PBST, 300 μL / well, 3 times.

[0185] (9) Termination: 2 mol HCl, 50 μL / well.

[0186] (10) Microplate reader reading: OD450-OD620.

[0187] Results Interpretation: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance was eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm. Dilution ratios were calculated from 1μg / mL to 0.015μg / mL. Wells with OD values ​​more than twice that of the blank control were considered positive. The dilution factor of the lightest-colored positive well represents the titer of the target antibody in that serum sample.

[0188] In ELISA experiments, a negative control is a sample that does not contain the target protein. The main purpose of a negative control is to verify whether false positives have occurred and to estimate the level of nonspecific binding. By setting up a negative control, the background signal when the specific target is not added can be understood, thereby ensuring the reliability of the experimental results.

[0189] Table 4 Negative Control Experiment

[0190]

[0191] Example 3 Antibody Pairing

[0192] (1) Coating: Primary antibody (Ab-5, Ab-25, Ab-28, Ab-87, Ab-94, Ab-111), 2 μg / mL, 100 μL / well, 37℃, 2h.

[0193] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0194] (3) Washing: PBST, 300 μL / well, twice.

[0195] (4) Add antigen: A29L, 1μg / mL, 100μL / well, 1h.

[0196] (5) Washing: PBST, 300 μL / well, 3 times.

[0197] (6) Add secondary antibody: Biotin-conjugated antibodies (Bio-5, Bio-25, Bio-28, Bio-87, Bio-94 or Bio-111) were prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120), 0.5 μg / mL, 100 μL / well, 37℃, 1h.

[0198] (7) Washing: PBST, 300 μL / well, 5 times.

[0199] (8) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0200] (9) Add TMB substrate: 100 μL / well, 37 °C, 10 min.

[0201] (10) Washing: PBST, 300 μL / well, 3 times.

[0202] (11) Termination: 2 mol HCl, 50 μL / well.

[0203] (12) Microplate reader reading: OD450-OD620.

[0204] Results Interpretation: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance can be eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm. Antibody pairing was performed using a checkerboard method, a process of selecting the optimal antibody pair. Three pairs of antibodies were found to be ideal, so further pairing was performed.

[0205] Table 5 Antibody pairing results

[0206]

[0207] Negative control experiment:

[0208] (1) Coating: Primary antibody (Ab-5, Ab-25, Ab-28, Ab-87, Ab-94, Ab-111), 2 μg / mL, 100 μL / well, 37℃, 2h.

[0209] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0210] (3) Washing: PBST, 300 μL / well, twice.

[0211] (4) Add antigen: non-specific antigen human herpes zoster virus (the non-specific antigen is stored in the laboratory), concentration 1 μg / mL, 100 μL / well, 1h.

[0212] (5) Washing: PBST, 300 μL / well, 3 times.

[0213] (6) Add secondary antibody: Biotin-conjugated antibodies (Bio-5, Bio-25, Bio-28, Bio-87, Bio-94 or Bio-111) were prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120), 0.5 μg / mL, 100 μL / well, 37℃, 1h.

[0214] (7) Washing: PBST, 300 μL / well, 5 times.

[0215] (8) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0216] (9) Add TMB substrate: 100 μL / well, 37 °C, 10 min.

[0217] (10) Washing: PBST, 300 μL / well, 3 times.

[0218] (11) Termination: 2 mol HCl, 50 μL / well.

[0219] (12) Microplate reader reading: OD450-OD620.

[0220] Interpretation of results: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance can be eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm.

[0221] Table 6 Results of Antibody Paired Negative Controls

[0222]

[0223] Select the best-performing antibody pairs for a second pairing experiment:

[0224] (1) Coating: Primary antibody (ab5, ab28, ab94), 2 μg / mL, 100 μL / well, 37℃, 2h.

[0225] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0226] (3) Washing: PBST, 300 μL / well, twice.

[0227] (4) Add antigen: A29L 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, 1.5625 ng / mL, 100 μL / well, 1h.

[0228] (5) Washing: PBST, 300 μL / well, 3 times.

[0229] (6) Add secondary antibody: Biotin-conjugated antibodies (Bio-5, Bio-94, Bio-5) were prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120), 0.2 μg / mL, 100 μL / well, 37℃, 1h.

[0230] (7) Washing: PBST, 300 μL / well, 5 times.

[0231] (8) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0232] (9) Add TMB substrate: 100 μL / well, 37 °C, 10 min.

[0233] (10) Washing: PBST, 300 μL / well, 3 times.

[0234] (11) Termination: 2 mol HCl, 50 μL / well.

[0235] (12) Microplate reader reading: OD450-OD620.

[0236] Results Interpretation: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance can be eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm. In the second pairing, all three antibody pairs were replicated and averaged. It was found that AB94 as the primary antibody and Bio-5 as the secondary antibody yielded the best pairing results.

[0237] Table 7 Results of the second antibody pairing

[0238]

[0239] The best pair was selected for a third pairing experiment:

[0240] (1) Coating: Primary antibody (ab94), 2 μg / mL, 100 μL / well, 37℃, 2h.

[0241] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37℃, 1.5h.

[0242] (3) Washing: PBST, 300 μL / well, twice.

[0243] (4) Add antigen: A29L 2000pg / mL, 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 100μL / well, 1h.

[0244] (5) Washing: PBST, 300 μL / well, 3 times.

[0245] (6) Add secondary antibody: Biotin-conjugated antibody (Bio-5) was prepared using the LinKine™ Biotin Conjugation Kit (purchased from Abbkine Scientific Co., Ltd., catalog number: KTL0120), 0.1 μg / mL, 100 μL / well, 37℃, 1h.

[0246] (7) Washing: PBST, 300 μL / well, 5 times.

[0247] (8) Add the labeling enzyme: Streptavidin-HRP, 100 μL / well, 37℃, 30 min.

[0248] (9) Add TMB substrate: 100 μL / well, 37 °C, 10 min.

[0249] (10) Washing: PBST, 300 μL / well, 3 times.

[0250] (11) Termination: 2 mol HCl, 50 μL / well.

[0251] (12) Microplate reader reading: OD450-OD620.

[0252] Interpretation of results: OD values ​​were read; 450nm represents specific absorption, and 620nm represents non-specific absorption. Non-specific absorbance can be eliminated by subtracting the absorbance at 620nm from the absorbance at 450nm.

[0253] Table 8 Results of the third antibody pairing

[0254]

[0255] A standard curve was plotted using a four-parameter fitting method with the fused tagged protein (fused GST tag) A29L as a standard (see [link]). Figure 1 ) Figure 1 Based on the data in the table, a standard curve was plotted using four-parameter fitting. R0 2 =0.9998 indicates that the standard curve is meaningful. The Ab94 / Bio-5 double antibody sandwich method was used to detect monkeypox virus, showing a concentration-dependent relationship within the A29L range of 0–2000 pg / mL, and can be used for the quantitative detection of A29L.

[0256] Table 9 Quantitative Detection of A29L

[0257]

[0258] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A coating antibody that binds to the A29L protein of monkeypox virus, characterized in that, The coating antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17 in turn; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO. 12, WAS and SEQ ID NO. 18 in turn.

2. The coating antibody of claim 1, wherein comprising: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 41; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

43.

3. A labeled antibody that binds to the monkeypox virus A29L protein, characterized in that, The labeling antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in turn; the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown in SEQ ID NO. 4, LVS and SEQ ID NO. 5 in turn.

4. The labeled antibody of claim 3, wherein the antibody is a monoclonal antibody. comprising: the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 25; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

27.

5. Pair of mAbs for the detection of monkeypox virus A29L protein by sandwich method, characterized in that, The coating antibody for binding to the A29L protein of monkeypox virus and the labeling antibody for binding to the A29L protein of monkeypox virus.

6. A nucleic acid molecule, characterized in that, comprising a DNA fragment encoding any one of: (I) the coating antibody for binding to the A29L protein of monkeypox virus according to claim 1 or 2; (II) the labeling antibody for binding to the A29L protein of monkeypox virus according to claim 3 or 4; or (III) the paired monoclonal antibody for detecting the A29L protein of monkeypox virus by sandwich method according to claim 5.

7. An expression vector, characterized in that, comprising the nucleic acid molecule according to claim 6.

8. A host cell characterized in that, comprising the expression vector according to claim 7.

9. Use of any one of: (I) the coating antibody for binding to the A29L protein of monkeypox virus according to claim 1 or 2; (II) the labeling antibody for binding to the A29L protein of monkeypox virus according to claim 3 or 4; or (III) the paired monoclonal antibody for detecting the A29L protein of monkeypox virus by sandwich method according to claim 5 in the preparation of a product for detecting the A29L protein of monkeypox virus.

10. A reagent or kit for detecting a monkeypox virus A29L protein by an antibody sandwich method, characterized by, comprising any one of: (I) the coating antibody for binding to the A29L protein of monkeypox virus according to claim 1 or 2; (II) the labeling antibody for binding to the A29L protein of monkeypox virus according to claim 3 or 4; or (III) the paired monoclonal antibody for detecting the A29L protein of monkeypox virus by sandwich method according to claim 5.

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