CN120058919B

CN120058919B - Monoclonal antibody against Rift Valley fever virus Gn protein and its application in virus detection

Info

Publication number: CN120058919B
Application number: CN202510556661.0A
Authority: CN
Inventors: 刘景亮; 李建民; 郝勐; 胡雅婷; 郭萌萌; 陈一卉; 袁虹宇; 付广成
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center; Academy of Military Medical Sciences AMMS of PLA
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2025-04-29
Filing date: 2025-04-29
Publication date: 2025-07-18
Anticipated expiration: 2045-04-29
Also published as: CN120058919A

Abstract

The present invention discloses a monoclonal antibody against the Gn protein of the Rift Valley fever virus and its application in virus detection. The double antibody sandwich ELISA test is constructed using monoclonal antibodies A1 and C1, which can specifically detect the Gn antigen of the Rift Valley fever virus, and has no cross reaction with other Bunyavirus viruses such as the Gn antigen of the fever with thrombocytopenia syndrome virus. The monoclonal antibody sequence is clear, the monoclonal antibody used can be prepared by large-scale culture, has high sensitivity, and has a detection limit of 781 pg/mL.

Description

Monoclonal antibody of anti-split valley fever virus Gn protein and application thereof in virus detection

Technical Field

The invention belongs to the technical field of biology, and particularly relates to monoclonal antibodies of anti-split valley fever virus Gn protein and application thereof in virus detection.

Background

The heat of the valley is a mosquito-borne infectious disease of both animals and humans. Clinically, fever, headache and arthralgia are caused by the heat of the valley, and severe cases can involve multiple viscera, so that the death rate is high. There is no vaccine or specific therapeutic agent currently available to the market.

The causative agent of Setaria is Setaria virus (RIFT VALLEY FEVER virus; RVFV), the RVFV genome being a single negative strand RNA virus, consisting of L, M, S fragments, wherein the L fragment encodes an RNA polymerase. The M fragment encodes envelope glycoproteins Gn and Gc, gn being primarily responsible for viral binding to host cells, while Gc plays a key role in membrane fusion. After entering the organism, the virus is firstly replicated in the invaded local tissue, is transferred to the local lymph node through the lymphatic system for further replication, then enters the blood circulation to form viremia, generally lasts for 4-7 days, and can generate infection poisoning symptoms such as fever and the like and cause multi-organ focal infection.

Currently, laboratory diagnostic methods for viral infection mainly include virus isolation, nucleic acid detection, antigen detection, serological detection, and the like. Where virus isolation is the gold standard for diagnosis of viral infection, this method is demanding for laboratory conditions and requires a long incubation time. Although the detection method of viral nucleic acid is sensitive and quick compared with the traditional virus isolation culture method, the detection method has higher requirements on equipment and personnel. Serum IgM detection begins 5-6 days after infection with virus, igG occurs 14 days after infection with virus, and rapid detection cannot be achieved. The establishment of a simple and easy diagnostic reagent capable of specifically detecting early stage of the infection of the valley fever virus is imperative, and has significance for the rapid detection of suspected infected people.

The Gn protein of the rift valley fever virus is a relatively conserved envelope glycoprotein, is mainly highly expressed in infected cells, has strong antigenicity, exists in early peripheral blood of virus infected patients or infected animals, and can be used for early diagnosis of rift valley fever virus infection by detecting Gn antigen in serum of acute phase patients.

The Gn protein of the rift valley virus is encoded by the M gene of the rift valley virus and mediates the receptor recognition process of the virus. The Gn protein is a type I transmembrane protein, which is anchored to the membrane surface of the viral particle by a C-terminal helical structure. The extracellular region of the Gn protein can be divided into the head region (154-469 aa) and the stem region (470-582 aa). Research reports that the neutralizing epitope of Gn proteins is mainly concentrated in the head region, which consists of three regions, domain I, domain II and domain III, respectively. Currently, known split valley fever virus neutralizing antibodies have main binding regions of domain I and domain III.

Methods for detecting viruses by antigen capture ELISA methods can be divided into two types, one type is to use polyclonal antibodies as capture antibodies or detection antibodies, and the methods have great differences in antisera of different batches, so that the methods are difficult to repeat and realize laboratory standardization. Another class of detection of viruses using monoclonal antibodies, there is currently no commercial kit for detection of rift valley fever virus using monoclonal antibodies against Gn.

The invention application with publication number of CN114409770A discloses a rift valley fever virus humanized monoclonal antibody and application thereof, wherein 8 humanized monoclonal antibodies are disclosed, which can effectively treat mice infected by RVFV and prevent the RVFV from infecting the mice. However, the monoclonal antibodies are useful in therapy and are not disclosed as being useful in ELISA assays.

Disclosure of Invention

The invention provides a method for detecting a rift valley fever virus Gn antigen by establishing a double-antibody sandwich ELISA method based on 2 high-binding activity monoclonal antibodies A1 and C1. In the kit, the monoclonal antibodies A1 and C1 are immunoglobulins of the subclass human IgG1, and both monoclonal antibodies can specifically bind to the Gn antigen of the rift valley fever virus. The monoclonal antibody adopts adenovirus vector rift valley fever candidate vaccine and recombinant Gn antigen immune rhesus, high binding activity monoclonal antibody variable region gene is obtained from peripheral blood by utilizing flow separation-single cell PCR technology screening, and two human IgG1 subclasses recombinant monoclonal antibodies A1 and C1 are obtained through genetic engineering technology.

The invention firstly provides the monoclonal antibody of the anti-crack valley fever virus Gn protein, which is monoclonal antibody A1 and/or monoclonal antibody C1,

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody A1 are shown as the amino acid sequences of 26 th to 33 th, 51 th to 58 th and 97 th to 115 th of the sequence shown in SEQ ID NO. 1, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as the amino acid sequences of 27 th to 37 th, 55 th to 57 th and 94 th to 102 th of the sequence shown in SEQ ID NO. 2;

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody C1 are shown as the amino acid sequences of SEQ ID NO. 3 at positions 26-33, 51-58 and 97-101, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as the amino acid sequences of SEQ ID NO. 4 at positions 27-38, 56-58 and 95-103.

Wherein, the monoclonal antibody A1 and/or the monoclonal antibody C1 are expressed as one or the combination of the two of the monoclonal antibody A1 and the monoclonal antibody C1.

Preferably, the amino acid sequence of the heavy chain of the monoclonal antibody A1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain is shown as SEQ ID NO. 2.

Preferably, the amino acid sequence of the heavy chain of the monoclonal antibody C1 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 4.

The invention also provides a coding gene of the monoclonal antibody for encoding the anti-crack valley fever virus Gn protein, the sequence of the gene for encoding the heavy chain of the monoclonal antibody A1 is shown as SEQ ID NO. 5, and the sequence of the gene for encoding the light chain of the monoclonal antibody A1 is shown as SEQ ID NO. 6;

the gene sequence of the heavy chain of the monoclonal antibody C1 is shown as SEQ ID NO. 7, and the gene sequence of the light chain of the monoclonal antibody C1 is shown as SEQ ID NO. 8.

The invention also provides application of the monoclonal antibody of the anti-split valley fever virus Gn protein in preparation of a detection kit for detecting the split valley fever virus Gn antigen.

The invention also provides a detection kit for detecting the rift valley fever virus Gn antigen, which comprises the monoclonal antibody of the anti-rift valley fever virus Gn protein. When the monoclonal antibody is one of monoclonal antibody A1 or monoclonal antibody C1, the monoclonal antibody can be directly detected by using detection methods such as Western Blotting, and the like, or can be additionally matched with a secondary antibody specifically combined with an antibody constant region for detection by using detection methods such as ELISA, and the like. When the mab is a combination of mab A1 and mab C1, it can be detected using a double antibody sandwich ELISA detection method.

Preferably, the detection kit is a double antibody sandwich ELISA detection kit, wherein the monoclonal antibodies are monoclonal antibody A1 and monoclonal antibody C1, one of the monoclonal antibody A1 and the monoclonal antibody C1 is used as a capture antibody, the other is used as a detection antibody, and the detection antibody is marked.

The capture antibody is used for binding to an antigen to capture the antigen to be detected, and the detection antibody is used for binding to the antigen again after capturing the antigen by using the capture antigen and detecting by means of color development or fluorescence. The monoclonal antibody A1 can be used as a capture antibody, the monoclonal antibody C1 can be used as a detection antibody, or the monoclonal antibody A1 can be used as a detection antibody in reverse, and the monoclonal antibody C1 can be used as a capture antibody.

Preferably, the label used for labeling the monoclonal antibody in the detection antibody is peroxidase, phosphatase or luciferase.

More preferably, the peroxidase is horseradish peroxidase.

The invention also provides application of the detection kit in detection of the rift valley fever virus Gn antigen for non-diagnosis purposes.

The invention also provides a double-antibody sandwich ELISA detection method for detecting the rift valley fever virus Gn antigen without diagnosis, which comprises the following steps of:

s1, coating an ELISA plate by using a capture antibody;

s2, sealing by using a sealing liquid;

S3, discarding the sealing liquid, cleaning by using a buffer solution, then adding a sample to be detected, and incubating;

s4, cleaning by using a buffer solution, then adding a detection antibody, and incubating;

S5, developing and detecting.

Preferably, the double antibody sandwich ELISA detection method comprises the following steps:

(1) Coating, namely diluting the monoclonal antibody A1 into 2 mug/mL by using coating buffer solution, coating the monoclonal antibody A1 on an ELISA plate at 100 mug/hole and standing at 4 ℃ overnight;

(2) Blocking, namely, washing the plate 4 times by using PBST, adding 5% skimmed milk powder prepared by PBS, 100 mu L/hole, and blocking at 37 ℃ for 2 h;

(3) Adding samples, namely discarding sealing liquid, washing a plate for 4 times by using PBST, adding samples to be detected, and incubating at 37 ℃ for 60 min;

(4) Adding enzyme-labeled antibody, namely washing the plate with PBST for 4 times, adding horseradish peroxidase-labeled monoclonal antibody C1 (0.5 mug/mL) diluted by 1% BSA, 100 mug/hole, and reacting at 37 ℃ for 60 min;

(5) Color development, namely, washing a plate for 4 times by using PBST, adding single-component TMB color development liquid, 100 mu L/hole, and developing color at 37 ℃ in a dark place for 10 min;

(6) Termination by adding 2M H ₂SO₄ to terminate the reaction, 50. Mu.L/well;

(7) Reading the OD _450-630nm by using an enzyme-labeled instrument.

And the result judgment standard is that the ratio of the OD _450-630nm (S) of the sample to be tested to the OD _450-630nm (N) of the negative control is used as a positive judgment standard, when the S/N is more than or equal to 2.1, the sample to be tested is judged to be positive, namely the sample to be tested contains the rift valley fever virus, otherwise, the sample to be tested is judged to be negative, namely the sample to be tested does not contain the rift valley fever virus.

The capture antibody A1 and the detection antibody C1 are selected from a group of monoclonal antibodies of anti-rift valley fever virus Gn protein, can specifically bind to rift valley fever virus, have no cross reaction with other bunyavirus viruses such as fever with thrombocytopenia syndrome virus, have high sensitivity to rift valley fever virus and are favorable for early diagnosis of rift valley fever virus. Meanwhile, the monoclonal antibody in the method has definite sequence, can be obtained through large-scale fermentation, does not need animal feeding and ascites extraction processes, and has low cost.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a double-antibody sandwich ELISA detection kit for detecting a rift valley fever virus Gn antigen, which can specifically detect the rift valley fever virus Gn antigen, has no cross reaction with other bunyavirus such as fever with thrombocytopenia syndrome virus Gn antigen, has definite monoclonal antibody sequence, can be prepared by large-scale culture of the monoclonal antibody, has high sensitivity and has detection limit 781 pg/mL when the double-antibody sandwich ELISA detection kit is used for carrying out double-antibody sandwich ELISA detection.

Drawings

Figure 1 shows the result of SDS-PAGE of truncated RVFV-Gn proteins.

FIG. 2 shows the result of SDS-PAGE of RVFV-Gn specific binding to antibodies A1 and C1.

Figure 3 is a graph of ELISA results (EC ₅₀ assay) for specific binding activity of A1, C1 against RVFV-Gn.

FIG. 4 is a graph showing the results of ELISA for detecting RVFV-Gn protein by the A1-C1 antibody.

FIG. 5 is an ELISA binding diagram of two strains of monoclonal antibodies to recombinant rift valley fever virus Gn antigen (RVFV-Gn) and fever with thrombocytopenia syndrome virus Gn antigen (SFTSV-Gn), respectively.

FIG. 6 shows the detection profile of the invention at the optimal concentration of both antibodies.

Detailed Description

Example 1 acquisition of truncated RVFV-Gn protein

The head region (154-469 aa) of the Gn protein is intercepted in the study to carry out eukaryotic expression and purification to lay a foundation for subsequent rhesus monkey strengthening immunity, gn protein specific memory B cell sorting and Gn protein binding antibody screening.

Open Reading Frame (MORF) corresponding to M gene of rift valley fever virus MP-12 strain (GenBank: DQ 380208.1) is synthesized by Shanghai biological engineering Co., ltd, and primer is designed by using this as template to amplify the gene of truncated Gn protein, and tPA signal peptide (ATGGACGCCATGAAGCGGGGCCTCTGCTGTGTTCTGCTGCTCTGCGGCGCCGTGTTCGTGAGTAACTCG) and Strep II label (TTACTTTTCGAACTGCGGGTGGCTCCAGGCAGATCCTCCAGATCCTCCTCCAGATCCTCCTCCCTTTTCGAACTGCGGGTGGCTCCAAGCGGA) are added to N end and C end of truncated Gn protein respectively by PCR. Restriction sites EcoRI and NotI and pCAGGs vector homology arms are added at both ends of the upstream and downstream primers, respectively, and then the target gene is ligated to the double digested (EcoRI and NotI) linear pCAGGs vector by homologous recombination. Recombinant plasmid containing truncated Gn protein is obtained, and transfected into an Expi293F cell, and the truncated Gn protein is purified by using an affinity chromatography column (STREP TRAP XT), and the expression and purity of the truncated Gn protein are identified by SDS-PAGE, and the purified Gn protein is shown as figure 1, and the size is about 40 kDa.

Example 2 acquisition of monoclonal antibodies that bind to RVFV-Gn protein

The method for obtaining the RVFV-Gn combined monoclonal antibody comprises the steps of firstly constructing recombinant human adenovirus 4 expressing RVFV-Gn protein, cloning an open reading frame (MORF) of a split valley fever virus MP-12 strain M gene (GenBank: DQ 380208.1) synthesized after codon optimization to a plasmid pUC57 to obtain pUC57-opMORF, adding restriction enzyme sites Swa I and pAd4 carrier homology arms at two ends of a target gene by using the pUC57 as a template, and then carrying out homologous recombination on plasmid pAd4 subjected to restriction enzyme Swa I single enzyme and the target gene to obtain recombinant human adenovirus 4 expressing split valley fever virus Gn protein.

The truncated Gn protein obtained by purification is matched to immunize the rhesus monkey twice, and the immunization program is that 1X 10 ⁸ IFUs recombinant human adenovirus 4 expressing the rift valley fever virus Gn protein is immunized with the rhesus monkey by intramuscular injection in the first immunization, and the rhesus monkey is immunized again in the same manner and the same dosage on the 28 th day after immunization. Finally, rhesus monkeys were boosted by intramuscular injection after 0.25 mg Gn protein was thoroughly mixed with 0.25 mg aluminum adjuvant (purchased from Invivogen, vac-alu-50) on days 56 and 182 after priming.

Blood is collected from the vein 210 days after the first time, antibody titer in serum is detected by ELISA method, peripheral blood is collected, gn specific B cells are sorted by a flow cytometer, antibody genes are cloned by single cell PCR technology, a linear expression frame is constructed, 293T cells are co-transfected by light and heavy chains, and after 48 hours of culture, supernatant is collected. Then detecting whether the supernatant contains a binding antibody aiming at Gn Protein or not through ELISA, cloning the full-length genes of the light and heavy chains of the screened positive antibodies into eukaryotic expression plasmids pCDNA3.4 through a homologous recombination mode, transfecting suspension cells Expi293F for large-scale expression, and purifying through a Protein A affinity column (HiTrapTM Protein A HP). Finally obtaining 2 monoclonal antibodies A1 and C1 with better binding activity against Gn protein. The expression and purity were identified by SDS-PAGE and the results are shown in FIG. 2.

The amino acid sequence of the heavy chain of the monoclonal antibody A1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain is shown as SEQ ID NO. 2. The gene sequence of the heavy chain of the monoclonal antibody A1 is shown as SEQ ID NO. 5, and the gene sequence of the light chain of the monoclonal antibody A1 is shown as SEQ ID NO. 6. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody A1 are shown as 26-33, 51-58 and 97-115 amino acid sequences of the sequence shown in SEQ ID NO.1, the amino acid sequences of FR1, FR2, FR3 and FR4 regions are shown as 1-25,34-50,59-96,116-126 amino acid sequences of the sequence shown in SEQ ID NO.1, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are shown as 27-37, 55-57 and 94-102 amino acid sequences of the sequence shown in SEQ ID NO. 2, and the amino acid sequences of FR1, FR2, FR3 and FR4 regions are shown as 1-26,38-54,58-93 and 103-112 amino acid sequences of the sequence shown in SEQ ID NO. 2;

The amino acid sequence of the heavy chain of the monoclonal antibody C1 is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 4. The gene sequence of the heavy chain of the monoclonal antibody C1 is shown as SEQ ID NO. 7, and the gene sequence of the light chain of the monoclonal antibody C1 is shown as SEQ ID NO. 8. The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody C1 are shown as 26-33, 51-58 and 97-101 amino acid sequences of the sequence shown in SEQ ID NO:3, the amino acid sequences of FR1, FR2, FR3 and FR4 regions are shown as 1-25,34-50,59-96 and 102-112 amino acid sequences of the sequence shown in SEQ ID NO:3, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are shown as 27-38, 56-58 and 95-103 amino acid sequences of the sequence shown in SEQ ID NO:4, and the amino acid sequences of FR1, FR2, FR3 and FR4 regions are shown as 1-26,39-55,59-94 and 104-113 amino acid sequences of the sequence shown in SEQ ID NO: 4.

EXAMPLE 3 determination of monoclonal antibody A1, C1 binding Activity

1. The purified RVFV-Gn protein coated ELISA plate (2. Mu.g/mL, 100. Mu.L/well) was taken overnight at 4℃and after 4 times of PBST washing, 2h was blocked with 5% nonfat milk powder at 37 ℃;

2. after PBST is washed for 4 times, monoclonal antibodies A1 and C1 aiming at Gn protein antigen with initial concentration of 9 mug/mL are respectively added, and the mixture is diluted in a gradient way according to the proportion of 1:3, and incubated at 37 ℃ for 1 h;

3. After 4 PBST washes, HRP-labeled goat anti-human IgG antibody (Sigma, AP 113P) was added at a dilution ratio of 1:5000,100 μl/well, incubated at 37 ℃ for 1 h;

4. After washing with PBST 4 times, 100. Mu.L of a single-component TMB developing solution was added, the color developed at 37℃was 10min, 50. Mu.L of ELISA stop solution was added, and finally the OD _450-630nm value was read with an ELISA reader.

The binding curves of mAb A1, C1 and RVFV-Gn were obtained as shown in FIG. 3 with the concentration of mAb as X axis and OD _450-630nm as Y axis.

Table 1 ELISA binding EC of monoclonal antibodies A1, C1 to RVFV-Gn antigen ₅₀

Table 1 shows EC ₅₀ values for mab A1, C1.

Example 4 sensitivity test of antibodies to A1-C1 detection

To establish a double-antibody sandwich ELISA method for detecting Gn antigen, detection sensitivity experiments need to be carried out on the screened antibody pairs, A1 monoclonal antibody is used as a capture antibody, and C1 monoclonal antibody is used as a detection antibody (the detection monoclonal antibody is obtained by adopting an HRP direct labeling method).

The experimental method comprises the following steps:

1. the capture antibody A1 was coated and 2. Mu.g/mL was applied to a 96-well ELISA plate and two duplicate wells were set. Incubate overnight at 4 ℃. The plates were washed 4 times for the next day with 5% nonfat dry milk at 37 ℃ for 2 hours.

2. After blocking was completed, plates were washed 4 times with PBST, RVFV-Gn protein (concentration gradient 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.562 ng/mL, 0.781 ng/mL, 0 ng/mL) was added as primary antibody, 100. Mu.L/well, and incubated at 37℃for 1 hour.

3. After completion of the primary antibody incubation, the plates were washed 4 times with PBST, HRP-labeled C1 was added as the secondary antibody (concentration of 2. Mu.g/mL), and incubated at 37℃for 1 hour.

4. After completion of the secondary antibody incubation, the plate was washed 4 times with PBST, 100. Mu.L of a single-component TMB developing solution was added, the color development was performed at 37℃for 10 minutes, 50. Mu.L of a stop solution was added, and the absorbance at OD _450-630nm was measured to determine the binding activity.

Table 2 shows the OD _450-630nm of the antibody pair A1-C1 for RVFV-Gn detection. And taking the ratio of the OD _450-630nm value S of the sample to be detected to the OD _450-630nm value N of the negative control as a standard for judging the detection positive, judging that the sample to be detected contains RVFV-Gn when the S/N is more than or equal to 2.1, otherwise judging that the sample to be detected is negative, namely, the sample to be detected does not contain RVFV-Gn.

TABLE 2 detection of OD _450-630nm value of RVFV-Gn by antibody pair A1-C1

The binding curves for A1-C1 and RVFV-Gn obtained with RVFV-Gn concentration on the X-axis and OD _450-630nm on the Y-axis are shown in FIG. 4 (the X-axis scale interval is shown in Log10 growth, so the data for RVFV-Gn concentration of 0 is not shown).

Example 5 specificity of antibodies for detection

And (3) carrying out a binding test on the two monoclonal antibodies A1 and C1 and the recombinant rift valley fever virus Gn antigen and the fever with thrombocytopenia syndrome virus (SEVERE FEVER WITH thrombocytopenia syndrome virus, SFTSV) Gn antigen respectively, and judging whether the A1 and the C1 have detection specificity by detecting OD _450-630nm values.

The experimental method comprises the following steps:

1. The SFTSV-Gn protein (Gn antigen of SFTSV, purchased from ACROBiosystems, GNN-S52H 3) was coated on an ELISA plate (2. Mu.g/mL, 100. Mu.L/well) at 4℃overnight, and after 4 times of PBST washing, 2H was blocked with 5% nonfat milk powder at 37 ℃;

2. After PBST is washed for 4 times, monoclonal antibodies A1 and C1 aiming at RVFV-Gn protein antigen with initial concentration of 200 ng/mL are respectively added, and the mixture is diluted in a gradient mode according to the proportion of 1:2, and incubated at 37 ℃ for 1 h;

4. After washing with PBST 4 times, 100. Mu.L of a single-component TMB developing solution was added, the color developed at 37℃was 10 min, and then 50. Mu.L of ELISA stop solution was added, and finally the OD _450-630nm value was read by an ELISA reader.

The binding experiments of the two monoclonal antibodies A1 and C1 and RVFV-Gn antigen are the same as those of the example 3, the result of the example 3 is integrated with the result of the example, and the binding curves of the monoclonal antibodies A1 and C1 and RVFV-Gn and SFTSV-Gn respectively are obtained by taking the concentration of the monoclonal antibodies as the X axis and the OD _450-630nm as the Y axis as shown in figure 5.

FIG. 5 shows binding curves of monoclonal antibodies A1 and C1 to RVFV-Gn antigen and SFTSV-Gn antigen, respectively, and shows that the monoclonal antibodies A1 and C1 can specifically bind to RVFV-Gn antigen, do not bind to SFTSV-Gn antigen, and have detection specificity.

Example 6 optimization of a double antibody sandwich ELISA detection scheme

The blank hole OD _450-630nm value of the antibody for detecting the RVFV-Gn by the A1-C1 is larger, which is related to the concentration of the added HRP-marked antibody, and the adding amount of the monoclonal antibody in the detection process needs to be optimized for establishing a double-antibody sandwich ELISA method for detecting the RVFV-Gn antigen.

The specific optimization scheme is that the addition amount of the HRP-labeled antibody C1 is reduced.

Experimental operation:

1. Antibody A1 was coated and two replicate wells were set up in 96-well ELISA plates at a concentration of 2. Mu.g/mL. Incubate overnight at 4 ℃. The plates were washed 4 times for the next day with 5% nonfat dry milk at 37 ℃ for 2 hours.

2. After blocking was completed, plates were washed 4 times with PBST, RVFV-Gn protein was added, diluted with 1% BSA, 100. Mu.L/well, and incubated at 37℃for 1 hour. The concentration of the first hole after RVFV-Gn protein dilution is 50 ng/mL, and the concentration is diluted in a 2-time gradient way, respectively 50 ng/mL、25 ng/mL、12.5 ng/mL、6.25 ng/mL、3.125 ng/mL、1.562 ng/mL、0.781 ng/mL、0.390 ng/mL、0.195 ng/mL、97.5 pg/mL、48.75 pg/mL、0 ng/mL.

3. After incubation was completed, the plates were washed 4 times with PBST, HRP-labeled antibody C1 was added as secondary antibody, 100. Mu.L/well, and incubated at 37℃for 1 hour. The secondary antibodies had concentrations of 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, and 0.125. Mu.g/mL, respectively.

4. After the secondary antibody incubation was completed, the plate was washed 4 times with PBST, a single-component TMB developing solution was added at 100. Mu.L/well, color development at 37℃was 10min, and a 50. Mu.L stop solution was added, and the absorbance at OD _450-630nm was measured.

Table 3 shows that the OD _450-630nm values of RVFV-Gn detected with different concentrations of HRP-C1, the OD _450-630nm values of blank wells (without RVFV-Gn protein added) gradually decreased with decreasing HRP-labeled antibody concentration, and blank well OD _450-630nm values <0.10 were generally required.

As can be seen from Table 3, the sensitivity of detecting Gn protein was 0.39 ng/mL when the HRP-C1 concentration was 1. Mu.g/mL and 0.5. Mu.g/mL, and the sensitivity of detecting Gn protein was 0.781 ng/mL when the HRP-C1 concentration was 0.25. Mu.g/mL and 0.125. Mu.g/mL. Therefore, HRP-labeled antibody concentration under the conditions that the blank OD _450-630nm value is smaller than 0.10 and the detection sensitivity is 0.39 ng/mL is selected as a kit, namely the coated antibody A1 is 2 mug/mL, and the detection antibody HRP-C1 is 0.5 mug/mL.

TABLE 3 detection of OD _450-630nm values for RVFV-Gn at different HRP-C1 concentrations

FIG. 6 is a graph showing the results of calculation using ELISA CALC with RVFV-Gn concentration of X and OD _450-630nm of Y, at a detection antibody HRP-C1 of 0.5 μg/mL, and fitting model using logistic curve (four parameters).

Example 7 sensitivity of double antibody sandwich ELISA detection of RVFV-Gn in serum

The invention aims to detect whether human serum contains rift valley fever virus or not, so that the detection sensitivity of RVFV-Gn in human serum by the double-antibody sandwich ELISA is required to be measured.

Particular embodiments are the detection of the extent of the double antibody sandwich ELISA in this case by dilution of RVFV-Gn proteins with laboratory-preserved single serum (serum of healthy volunteers).

TABLE 4 double antibody sandwich ELISA detection of OD _450-630nm value of RVFV-Gn in serum

Table 4 shows the OD _450-630nm of the RVFV-Gn in the serum detected by the double-antibody sandwich ELISA, and from Table 4, the detection sensitivity of the double-antibody sandwich ELISA is lower than that of 1% BSA, but the detection sensitivity can still reach 781: 781 pg/mL.

Claims

1. A monoclonal antibody against Rift Valley fever virus Gn protein, characterized in that it is monoclonal antibody A1 and/or monoclonal antibody C1,

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of monoclonal antibody A1 are shown in the amino acid sequences at positions 26-33, 51-58 and 97-115 of SEQ ID NO:1; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in the amino acid sequences at positions 27-37, 55-57 and 94-102 of SEQ ID NO:2;

The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of monoclonal antibody C1 are shown in the amino acid sequences at positions 26-33, 51-58, and 97-101 of SEQ ID NO:3; the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are shown in the amino acid sequences at positions 27-38, 56-58, and 95-103 of SEQ ID NO:4.

2. The monoclonal antibody against Rift Valley fever virus Gn protein according to claim 1, characterized in that the amino acid sequence of the heavy chain of monoclonal antibody A1 is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 2;

The amino acid sequence of the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain is shown in SEQ ID NO:4.

3. The gene encoding the monoclonal antibody against the Rift Valley fever virus Gn protein according to claim 2, characterized in that the gene sequence encoding the heavy chain of monoclonal antibody A1 is as shown in SEQ ID NO: 5, and the gene sequence encoding the light chain of monoclonal antibody A1 is as shown in SEQ ID NO: 6;

The gene sequence encoding the heavy chain of monoclonal antibody C1 is shown in SEQ ID NO:7, and the gene sequence encoding the light chain of monoclonal antibody C1 is shown in SEQ ID NO:8.

4. Use of the monoclonal antibody against Rift Valley fever virus Gn protein according to any one of claims 1 to 3 in the preparation of a detection kit for detecting Rift Valley fever virus Gn antigen.

5. A detection kit for detecting Rift Valley fever virus Gn antigen, characterized in that it comprises the monoclonal antibody against Rift Valley fever virus Gn protein according to any one of claims 1 to 3.

6. The detection kit according to claim 5, characterized in that the detection kit is a double antibody sandwich ELISA detection kit, wherein the monoclonal antibodies are monoclonal antibody A1 and monoclonal antibody C1, one of monoclonal antibody A1 and monoclonal antibody C1 is used as a capture antibody, and the other is used as a detection antibody, and the detection antibody is labeled.

7. The detection kit according to claim 6, characterized in that the marker used to label the monoclonal antibody in the detection antibody is peroxidase, phosphatase or luciferase.

8. The detection kit according to claim 7, characterized in that the peroxidase is horseradish peroxidase.

9. Use of the detection kit according to any one of claims 6 to 8 for non-diagnostic purposes in detecting Rift Valley fever virus Gn antigen.

10. A double antibody sandwich ELISA detection method for detecting Rift Valley fever virus Gn antigen for non-diagnostic purposes, characterized in that the detection kit according to any one of claims 6 to 8 is used, and the double antibody sandwich ELISA detection method comprises the following steps:

S1, coating the ELISA plate with capture antibody;

S2, blocking with blocking solution;

S3, discard the blocking solution, wash with buffer, then add the sample to be tested and incubate;

S4, wash with buffer, then add detection antibody and incubate;

S5, color development and detection.