CN115353562A - Monoclonal antibody and application thereof in preparation of kit for disease diagnosis - Google Patents

Monoclonal antibody and application thereof in preparation of kit for disease diagnosis Download PDF

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CN115353562A
CN115353562A CN202210747456.9A CN202210747456A CN115353562A CN 115353562 A CN115353562 A CN 115353562A CN 202210747456 A CN202210747456 A CN 202210747456A CN 115353562 A CN115353562 A CN 115353562A
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张倡埼
朱高茂
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Beijing Reyan Biotechnology Co ltd
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Abstract

The invention relates to a monoclonal antibody and application thereof in preparing a kit for disease diagnosis. The invention develops a specific monoclonal antibody aiming at the H5N1HA protein, and the antibody can be combined with the HA protein with high affinity and HAs strict specificity. After the monoclonal antibody is prepared into the ELISA detection kit, the target can be detected with an extremely low detection lower limit, and the application prospect is good.

Description

Monoclonal antibody and application thereof in preparation of kit for disease diagnosis
Technical Field
The application relates to the field of biology, and relates to a monoclonal antibody and application thereof in preparing a kit for disease diagnosis.
Background
Avian influenza (Avianinfluenza, AIorbirdflu) is a short term for avian influenza, and refers to an animal infectious disease caused by avian influenza virus, which often occurs in birds and sometimes in lower mammals, and so far, only sporadic cases occur in humans. Avian Influenza Virus (AIV), which is classified into high-, medium-, and low- Π non-pathogenic according to its pathogenicity to chickens, is not human. Highly pathogenic are some strains in H5 and H7 subtype viruses, and the medium pathogenic mainly refers to H9N2 and H6N8 subtype strains. Other strains were all low Π non-pathogenic. The avian influenza virus not only brings catastrophic damage to poultry and animal husbandry, but also poses serious threat to public health. Therefore, the harm of avian influenza has attracted general attention from countries around the world.
Human infection with avian influenza is a human disease caused by avian influenza virus. Avian influenza virus belongs to influenza A virus, and is divided into three grades of high, medium and low/non-pathogenicity according to the difference of pathogenicity of avian influenza virus to chickens and turkeys. Because of the characteristics of hemagglutinin structure of avian influenza virus, generally, poultry is infected, and when the virus is subjected to gene reassortment in the replication process, the structure is changed, the ability of infecting people is obtained, and the possibility that people are infected with avian influenza diseases is caused. To date, avian influenza virus subtypes capable of directly infecting humans have been found: H5N1, H7N2, H7N3, H7N7, H9N2 and H7N9 subtypes. Among them, highly pathogenic H5N1 subtypes and new avian influenza H7N9 subtypes found for the first time in 2013 on human bodies are particularly attracting attention, not only causing human casualties, but also creating poultry breeding. Particularly, since 2003, human-to-human avian influenza events have been reported in domestic and foreign media, and since 1997, some thought that highly pathogenic avian H5N1 influenza virus would be worried about causing pandemics of influenza worldwide, and people faced with the serious roll of spanish influenza tragedy in 1918.
Avian influenza is of many types. The lower animals examined up to now comprise more than 30 species of pig, horse, cattle, mule, donkey, dog, cat, rabbit, deer, sheep, goat, experimental mouse, bison, mink, tree, tiger, monkey, snake, pangolin, chicken, pheasant, pigeon, goose, domestic duck, bisque, seagull, black-face lutet, oil tank, spale, small, red-foot snipe, spotted goose, and the like. The avian influenza viruses that have been isolated include H2N3, H3N1, H3N3, H3N6, H3N7, H3N8, H3N9, H4N2, H4N5, H4N6, H4N7, H4N8, H4N9, H5N1, H5N4, H5N8, H6N2, H6N4, H6N5, H6N8, H7N7, H9N2, H9N9, H10N3, H10N5, H10N9, H11N2, H11N6, H11N9 and H12N9, and the majority of the 9N subtypes and 16H subtypes of influenza A viruses that have been found so far have been isolated.
A plurality of people think that the highly pathogenic avian H5N1 virus breaks through the barrier of a human host, and the H5N1 strain separated from a patient in 1997 is formed by gene reassortment of an A pi goose pi 1 pi 96 (H5N 1) strain and other avian H5N1 strains, thereby breaking through the barrier of the human host. Clearly this inference lacks scientific basis. It has been mentioned previously that highly pathogenic avian H5N1 was first introduced in 1959 in scotland, and that highly pathogenic H5N1 strains were also isolated in turkeys in england in 1990. Outbreaks of H5N1 influenza occurred in 1997 in 3 rd 3 chicken farms in hong kong kingdom with virus genetic characteristics very similar to those isolated from patients, and in hong kong china, in 1979, H5N3 strain was isolated from ducks, and in wild birds and ducks in other countries, H5N1 strain has been isolated already. The H5N1 strain of Guangdong goose in 1996 is similar to the H5N1 strain of hong Kong in 1997, the rest 7 gene segments are obviously different from the H5N1 strain in 1997, and meanwhile, the H strain in 1996 is used for infecting chicks at 4-6 weeks through nasal cavity and oral cavity with large dose, so that the chicks do not get ill, do not expel toxin and do not respond to antibodies. Therefore, the avian H5N1 influenza in the 1997 population is subjectively considered to be related to the H5N1 virus of Guangdong goose in 1996, and is unrelated to the rest, which lacks scientific basis. The fact that the H5N1 strain has not evolved from the hong Kong H5N1 strain in 1997 since 2003 is clearly indicated. Forcing some to change the view that the antigenicity and genetic properties of highly pathogenic H5N1 strains differ regionally. Others believe that, to date, highly pathogenic avian H5N1 strains have not breached the barrier of human hosts. First analyzed from an epidemiological perspective, the worldwide diagnosis of cases has fallen short of 200 since 5 months 1997 if the human host barrier has been breached. Secondly, analysis is carried out on the aspect of pathogenic molecular biology, 8 segments of genomes of H5N1 strains separated from patients II dead to date all belong to avian influenza viruses and do not contain any human influenza virus segments; their receptor specificity is avian influenza virus; the HA protein molecules of the above-mentioned materials have a series of basic amino acids. Therefore, the possibility of the attacked person is considered to be some genetic abnormality or immunocompromised person.
At present, the main prevention of the avian influenza is the prevention. Detection is the best way to prevent. The traditional immunodiagnosis detection technology is to perform separation culture of viruses, perform typing through a hemagglutination test, a hemagglutination inhibition test and an agar diffusion test, and determine the strength of a strain by means of experimental animals. The virus separation adopts a chick embryo allantois amplification method and an ultracentrifugation purification method, the hemagglutination of the separated virus is detected by using an HI antigen test, and the subtype is determined by using 16 prepared standard HA antiserums to perform an HI antigen inhibition test. Agar diffusion assays are commonly used to detect influenza a common antigen Nucleoprotein (NP) or Matrix Protein (MP), and commonly used methods are the two-way double diffusion assay (IDD), the immuno-single-radiation diffusion assay (SRD) and the countercurrent immunoelectrophoresis. The method is simple, convenient and quick, and can be used for both qualitative and quantitative determination. At present, accurate McAb diagnosis technology is successfully developed for carrying out fluorescent antibody labeling and enzyme-linked immunosorbent assay (ELISA), the sensitivity is high, standardized operation can be realized, and the result is easy to analyze. If the competitive ELISA method is adopted to detect the HA antibody of the avian influenza, the sensitivity reaches 96.2 percent, and the agar immunodiffusion method (AGID) is used to detect the HA antibody, and the sensitivity reaches 90 percent. However, the current ELISA detection methods for detecting H5 are not sufficient. The available alternatives are also not abundant. Moreover, there are not many studies on whether the corresponding vaccine was inoculated to detect the production of the corresponding antibody.
Disclosure of Invention
The invention provides a monoclonal antibody specifically binding to H5N 1.
Further, the monoclonal antibody is a monoclonal antibody specific for the HA protein of H5N 1.
Further, the monoclonal antibody is H5-3F5, and the amino acid sequence of the heavy chain variable region is
Figure BDA0003717346080000031
The variable region of the light chain has the amino acid sequence of
Figure BDA0003717346080000032
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2, the activity of the antibody is ensured to be unchanged on the basis of more than 95 percent of homology of the amino acid sequence shown in the sequence table 2.
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2 of the amino acid sequence shown in the sequence table 2 is more than 90 percent of homology, so that the activity of the antibody is ensured to be unchanged.
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2 is more than 85 percent of homology and ensures that the activity of the antibody is not changed.
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2 is more than 80 percent of homology, and the activity of the antibody is ensured to be unchanged.
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2 is more than 75 percent of homology, and ensures that the activity of the antibody is not changed.
Furthermore, the light and heavy chain variable regions of the monoclonal antibody of the invention can adopt conservative substitution, and the conservative substitution between the light and heavy chain variable regions and the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region and the amino acid sequence shown in SEQ ID NO:2 is more than 70 percent of homology, and the activity of the antibody is ensured to be unchanged.
Further, the invention provides application of the monoclonal antibody of the HA protein specific to the H5N1 in preparing a kit for diagnosing the H5N 1.
Further, the invention provides an application of the monoclonal antibody specifically aiming at the HA protein of the H5N1 in preparing a kit for diagnosing the HA protein of the H5N 1.
In some embodiments, ELISA assays can be performed using magnetic beads. The ELISA assay can be performed in, for example, a tube, such as an Eppendorf tube. The beads can be used as solid supports. At least one antibody (e.g., a capture antibody) can be conjugated to the magnetic particles. A biological fluid sample comprising an antigen can be added to a tube comprising at least one antibody conjugated to a magnetic bead. After binding the antigen to the antibody, a magnetic device, such as a magnet, may be used in the separation and/or washing steps. By attracting antibodies with bound antigen (antibody-antigen complex) to one side of the tube, the remaining biological fluid sample can be removed from the tube using a pipette. A wash solution may be added to the tube. The magnet may be removed from the tube and the antibody-antigen complex may be dispersed in the wash solution. Closing the magnet again will attract the antibodies conjugated to the magnetic beads to the side of the tube. Any unbound antigen is present in the wash solution, and the wash solution containing any unbound antigen can be removed using a pipette. In addition, a fluid containing a second antibody may be added to the tube. By removing the magnet, the antibody-antigen complex can be dispersed in the fluid. The second antibody is allowed to bind to the antigen bound to the first antibody. After a certain time, after binding the secondary antibody to the antigen, the magnet can be brought close to the test tube. In a similar manner, as described above, a wash solution may be added to the tube. Any unbound secondary antibody can be removed from the tube. The second antibody may be conjugated to a label, such as biotin, and the label may be detected or measured using the techniques described herein, for example by adding streptavidin-conjugated HRP to the tube, followed by TMB, and spectrophotometry may be used, for example by measuring absorbance at 450 nm.
Further, the present application provides a new assay method, which comprises adding the sample to be tested into the wells of the polystyrene reaction plate, 50 μ l per well, and then placing the diagnostic membrane into the well with the sample application surface facing up to 1 per well. Slightly shaking on a micro-oscillator for 1h; removing the liquid from the wells by blotting with Tween20-TBS for 3 times (1 st for 1min, 2 nd for 3min, and 3 rd for 5 min); diluting the H5-3F5 monoclonal antibody with Tween20-TBS, adding 50 μ l of shaking into each well, and washing the plate for 50 min; diluting enzyme-labeled rabbit anti-mouse IgG with Tween20-TBS at a ratio of 1:500, adding into each well, shaking for 55min, and washing the plate; substrate solution [6.5ml D ] was addedAB (8 mg/10 ml) with 2. Mu.l H 2 O 2 (30％)]Shaking 65 μ l per well for 10min; after the substrate solution was aspirated, the substrate solution was washed with TBS for 2 times and then with distilled water for 2 times, each time for 1-2 min, and then air-dried, followed by interpretation. The following controls were required for each run: positive and negative antigen control TBS control (no antibody) and substrate control (no enzyme conjugate). The control tests can be interpreted when all the control tests have correct results. If the membrane has brown yellow spots, the reaction is positive; negative reactions were, for example, unclear spot coloration and irregular shape or no spots.
The present disclosure also relates to a method of identifying H5N1 in a sample, the method comprising the acts of:
a. incubating the sample with a primary antibody; and
b. chromogen-conjugated secondary antibodies were added to the incubated samples and immunohistochemical analysis was performed to identify H5N1 virus in the samples.
The antibody kit corresponding to the method further comprises an antibody, a coated plate, a strong positive control, a weak positive control, a negative control, an HRP goat anti-mouse secondary antibody, a 20-time concentrated washing solution, a diluent, a substrate solution and a stop solution.
The above 20-fold concentrated washing solution is 0.01mol/LpH7.4 PBST containing 0.05% -0.5% Tween-20, which is 20-fold concentrated; the diluent is PBS containing 1-10% of BSA, 0.05-0.5% of Tween-20pH7.4; the substrate solution is soluble TMB; the stop solution is 1-2mol/L H 2 SO 4 And (3) solution.
The volume or number of each component of the antibody kit is as follows: coating the plate for 2 blocks; 0.3mL of EHD-2 monoclonal antibody; strong positive control 0.6mL; 0.6mL of weak positive control; negative control 0.6mL; 0.3mL of goat anti-mouse secondary antibody marked by HRP; 50mL of 20-time concentrated washing solution; 60mL of diluent; 25mL of substrate solution; 25mL of stop solution; 6 pieces of sealing paper; 1 part of a specification.
Further, the present disclosure relates to a kit for identifying H5N1 in a sample, the kit comprising an anti-H5N 1 monoclonal antibody and optionally a component selected from the group consisting of a secondary antibody, an immunohistochemical analysis reagent, a pharmaceutically acceptable excipient and instructions or any combination thereof. The present disclosure relates to a method of treating H5N1 comprising the act of administering to a subject in need thereof a monoclonal antibody, optionally with a pharmaceutically acceptable excipient. In one embodiment of the present disclosure, the subject is a mammal, including a human.
In one embodiment of the present disclosure, the excipient is a pharmaceutically acceptable excipient selected from granulating agents, binders, lubricants, disintegrants, glidants, anti-adherents, antistatic agents, surfactants, antioxidants, gums, coating agents, coloring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, plant cellulose materials and spheronizing agents or any combination thereof.
Advantageous effects
The invention develops a specific monoclonal antibody aiming at the H5N1HA protein, and the antibody can be combined with the HA protein with high affinity and HAs strict specificity. After the monoclonal antibody is prepared into the ELISA detection kit, the target can be detected with an extremely low detection lower limit, and the application prospect is good.
Drawings
FIG. 1 is a diagram showing the result of epitope identification of binding polypeptides specific to monoclonal antibody
FIG. 2 shows the result of subtype identification of monoclonal antibody
Detailed Description
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Example 1 preparation of monoclonal antibody specific for avian influenza virus H5
6-8 weeks old BALB/c mice, total 5. H5N1HA protein (BIOHJSW, cat # orb-EHJ 075488) is used as immunogen, and is mixed with Freund's complete adjuvant according to the proportion of 1; after 2 weeks, the immunization is carried out according to the dose of 100 mu g/mouse after the immunization is carried out by mixing the three-way antibody with Freund incomplete adjuvant according to the proportion of 1; and detecting the titer of the polyclonal antibody by adopting an indirect ELISA detection method, wherein the titer of a No. 2 mouse reaching 1.
After 5 days of boosting, the mice were killed by removing their necks and sterilized in a beaker containing 75% alcohol for 5min. Cutting off epidermis with aseptic scissors and forceps in a clean bench, cutting off abdominal cavity with a second set of scissors, taking out spleen, placing on 120-mesh aseptic filter screen, cutting up and grinding spleen with scissors, adding GNK lotion for washing, filtering spleen cells into small aseptic beaker individually, and making into single cell suspension; transferring the spleen cell suspension into a centrifuge tube, adding GNK to 40mL, performing centrifugation for 10min at 1200 r/min; discarding the supernatant, performing cell mass elasticity, adding 10mL of GNK respectively, transferring the spleen cell suspension into a mouse SP2/0 cell bottle, adding GNK to 40mL and 1200r/min, centrifuging for 10min, and discarding the supernatant; gently scattering the cell mass, dropwise adding 50% of fusion agent PEG-1500 1mL, dropwise adding, slowly adding and then quickly adding within 1min, standing for 90s, slowly dropwise adding 15mLGNK to terminate fusion, standing in a water bath at 37 ℃ for 5min, supplementing GNK to 40mL, then standing for 1000r/min, and centrifuging for 10min; discard the supernatant, gently break up the cell pellet, gently mix the fused cells with selection medium containing HAT. The suspension cells were dispersed into 96-well cell culture plates at 220. Mu.L/well cell culture medium. Half of the culture medium can be changed after 6 days of culture, the supernatant of the hybridoma cells is detected after 10 days, and positive clone holes are screened. Screening positive cloning wells of the wells with hybridoma cells by indirect ELISA method, setting mouse positive serum and unfused cell wells as control groups, respectively, and screening coated positive screening antigens are H5N1HA protein (BIOHJSW, cat # orb-EHJ 075488) and anti-screening antigen H7N9 HA protein (cat # 40104-V08H, chinesia). The number of positive wells was 14, and the positive rate was 10.5% (see table 1). Transferring the selected cell wells with higher positive values into a 24-well plate containing an HT medium for expanded culture. After 2 days, the titer of the antibody of the cell supernatant is measured by the same method, 4 cell strains with higher titer and rich sharp positive reaction are selected, and strong positive holes detected by indirect ELISA are subcloned by a limiting dilution method, so that H5-1A3, H5-3F5 and H5-4G6 anti-H5 avian influenza monoclonal hybridoma cell strains which are screened out in a co-screening way are obtained.
TABLE 1 screening results for Positive clones
Figure BDA0003717346080000071
Figure BDA0003717346080000081
And (3) specific identification of the three monoclonal antibodies: coating: diluting H5 HA, H7 HA, H1 HA and H3 HA with CBS according to 5 μ g/mL concentration, respectively, spreading on 96-well enzyme-labeled plate at 50 μ L/well, and standing overnight at 4 deg.C; and (3) sealing: washing the plate for 2 times by PBST, completely spin-drying the washing solution, adding 200 mu L of sealing solution into each hole, and sealing for 1h at 37 ℃; adding a primary antibody: removing the confining liquid, washing the plate for 2 times by PBST, thoroughly spin-drying the washing liquid, and sequentially mixing the supernatant of the three monoclonal antibody cells according to the following ratio of 1:200,1:400,1:800-1:25600, 50. Mu.L per well, and, at the same time, mouse 1: taking 500 diluted positive serum as positive control, and adsorbing at 37 deg.C for 1h; adding a secondary antibody: discard primary antibody, wash plate 6 times with PBST, spin-dry wash thoroughly, add 50. Mu.L 1:20000 times diluted goat anti-mouse secondary antibody labeled with HRP reacts for 30min at 37 ℃; color development: discarding the secondary antibody, washing the plate for 6 times by PBST, thoroughly spin-drying the washing solution, developing the color by TMB color developing solution, setting a blank control, timely stopping according to the color development condition, and reading. The results are shown in Table 2.
TABLE 2 result of specific identification (OD 450)
Cloning well H5 HA H7 HA H1 HA H3 HA
H5-1A3 2.553 0.262 0.252 0.331
H5-3F5 2.621 0.237 0.231 0.412
H5-4G6 2.701 0.253 0.324 0.289
The results in table 2 show that all three monoclonal antibodies prepared by the present invention have good specificity, and can specifically bind to HA protein of H5, but not to HA proteins of other subtypes.
Example 2H5-3F5 monoclonal antibody binding site assay
According to the amino acid sequence of H5N1, truncated peptides with different lengths are respectively designed, an ELISA method is adopted for specific identification, each hole is coated with 200ng of the truncated peptide, and HRP-labeled H5-3F5 monoclonal antibody is developed. The blank control is used for zero setting, and the absorbance value of A450nm is measured by an enzyme-linked immunosorbent assay. H5N1HA protein (BIOHJSW, cat # orb-EHJ 075488) and H7N9 HA protein (cat # 40104-V08H, yi Qiao Shen) were used as controls. The positive standard was a P/N value (positive/negative) >2.0 and the negative standard was a P/N value <0.5. The results are shown in FIG. 1.
As can be seen from the results shown in FIG. 1, the H5-3F5 monoclonal antibody specifically binds to the H5N1-14 polypeptide fragment, and therefore, the target site sequence of the antibody can be determined to be ELLVLMENERLDFHDSN.
Example 3 affinity assay for H5-3F5 monoclonal antibodies
Evaluation of binding ability of H5-3F5 antibody to H5N1HA protein surface plasmon resonance analysis was performed using Biacore 8K. The method comprises the following specific steps:
first, an anti-mouse IgG antibody was immobilized on the channel of the CM5 chip in an amino-coupled manner. The fixed amount is controlled around 8,000 response values (RU). The purified H5-3F5 antibody is then bound by means of antibody capture. In addition, HA protein was serially diluted in 2 mM HEPES,150mM NaCl, pH7.4 solution at double ratio. Then, the serially diluted HA proteins were sequentially passed through each channel (one by one from a low concentration). Kinetic curves of the binding of the H5-3F5 antibody to the HA protein were recorded and kinetic constants were calculated using BIA evaluation software 8K software. The results show that the H5-3F5 antibody can bind HA protein with high affinity, HAs a KD value of 4.07E-10 (M), and HAs excellent binding property.
Example 4 identification of preparation sequence of H5-3F5 monoclonal antibody
Selecting healthy BALB/c female mice, inoculating 500 mu L of sterile liquid paraffin to the mice in advance by an intraperitoneal injection method for ascites preparation one week, transferring the screened positive hybridoma H5-3F5 strain to a cell bottle for amplification culture, discarding cell culture solution when the cells are in logarithmic growth phase and in the best state, washing the cells once with sterile PBS, blowing the cells off with PBS, centrifuging at 1000r/min for 10min, and performing resuspension counting, wherein the inoculation amount of the cells in the abdomen of each female mouse is about 5 multiplied by 10 6 And (4) respectively. Observing the abdominal swelling condition of the mouse after one week until the abdominal swelling condition and the skin of the mouse are in a tight state, and collecting ascites. Purifying the ascites of the monoclonal antibody by an n-octanoic acid-ammonium sulfate method, and adopting an indirect ELISA method to treat the ascites titerThe detection is carried out, and the titer is detected to be 1. The concentration of the ascites of the H5-3F5 monoclonal antibody after purification is 4.6mg/mL. And 2 electrophoretic bands of the purified monoclonal antibody are clear through SDS-PAGE detection, which proves that the purified antibody has higher purity.
Separating total RNA from hybridoma cells according to the instruction of a reagent TriZol, carrying out reverse transcription on the total RNA into cDNA according to the instruction of a TIANCcript first strand cDNA synthesis kit, amplifying antibody fragments of VH and VL according to specific primers, respectively cloning the amplified antibody fragments into standard cloning vectors, and sequencing. After the sequencing is subjected to data analysis, the amino acid sequence of the heavy chain variable region of the H5-3F5 monoclonal antibody is shown as SEQ ID No:1, and the light chain variable region amino acid sequence of the H5-3F5 monoclonal antibody is SEQ ID No:2, or a pharmaceutically acceptable salt thereof.
Example 5 identification of subtype of H5-3F5 monoclonal antibody
Analysis of the subtype was carried out by referring to the procedure of the Mouse monoclonal antibody subtype identification kit (kit) of proteintech corporation. And (4) taking out the Mouse monoclonal antibody subtype identification kit, and balancing for 30min at room temperature. (a quantity of 20 XPBST diluted with double distilled water to 1 XPBST, 100 times the concentration of goat anti-mouse IgM + IgG-HRP with 1 XPBST to 1 XPBST IgM + lgG-HRP); H5-3F5 monoclonal antibody is mixed with 1 xPBST according to the ratio of 1:20 dilution, 50 uL/hole into the lath, one sample corresponding to one lath; adding 1 Xgoat anti-mouse IgM + IgG-HRP into the sample hole according to 50L/hole, and gently mixing on a mixer; covering a sealing plate film, and incubating at room temperature for 1h; discarding liquid in the hole, washing the plate for 3 times by using washing liquid, and patting dry; adding the prepared developing solution into the holes (developing solution formula, solution A: solution B =1:100, namely, 10uLA solution and 1mLB solution are mixed evenly and used immediately); developing at room temperature in dark for 10min, adding stop solution at 100 μ L/well; and reading OD450 by a microplate reader, wherein the deepest color or the highest OD450 value is the corresponding subtype.
As can be seen from the results in FIG. 2, the H5-3F5 monoclonal antibody belongs to the IgG2a, kappa light chain type.
Example 6ELISA Rapid detection assay
Respectively adding H5N1AIV, H7N9AIV, newcastle disease virus, chicken infectious bursal disease into polystyrene reaction plate holeVirus, infectious bronchitis virus and normal chick embryo allantoic fluid were each 50. Mu.l and then diagnostic patches were placed into the wells with the sample side up for 1 patch per well. Slightly shaking on a micro-oscillator for 1h; removing the liquid from the wells by blotting with Tween20-TBS for 3 times (1 st for 1min, 2 nd for 3min, and 3 rd for 5 min); diluting the H5-3F5 monoclonal antibody with Tween20-TBS, adding 50 μ l of the diluted monoclonal antibody into each well, shaking for 50min, and washing the plate; diluting enzyme-labeled rabbit anti-mouse IgG with Tween20-TBS at a ratio of 1:500, adding into each well, shaking for 55min, and washing the plate; adding 2. Mu.l H into substrate solution [6.5ml DAB (8 mg/10 ml) 2 O 2 (30％)]Shaking 65 μ l per well for 10min; after the substrate solution was aspirated, the substrate solution was washed with TBS for 2 times and then with distilled water for 2 times, each time for 1-2 min, and then air-dried, followed by interpretation. The following controls were required for each run: positive and negative antigen control TBS control (no antibody) and substrate control (no enzyme conjugate). The control tests can be interpreted when all the tests have correct results. Positive reaction if brown yellow spots appear on the membrane; negative reactions were, for example, unclear spot coloration and irregular shape or no spots.
The result shows that the H5-3F5 monoclonal antibody can only produce spot reaction with H5N1AIV, but does not react with H7N9AIV, newcastle disease virus, chicken infectious bursal disease virus, chicken infectious bronchitis virus and normal chicken embryo allantoic fluid, and the antibody can be used for identifying the H5N1 AIV.
Through Dot-ELISA square matrix test on the optimal antibody coating concentration, the lowest protein content of the rabbit adsorbed by each membrane is 3.2 multiplied by 10 －9 g, above which significant mottling can occur. Meanwhile, the minimum detection concentration of H5N1AIV was 1.9X 10 －9 g, when the amount of the monoclonal antibody-bound AIV on the patch exceeds this amount, a distinct spot color appears, indicating that the present invention has a better lower limit of detection.
Finally, it should be noted that: the skilled person in the art can produce monoclonal antibodies by any existing method or possible method in the future based on the amino acid sequence of the monoclonal antibodies disclosed in the present application, and should be covered by the claims of the present application. Similarly, any form of commercial application of the monoclonal antibody of the present invention, including but not limited to kits, antibody chips, etc., should be covered by the claims of this application.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Beijing Yan Biotechnology Ltd
<120> monoclonal antibody and use thereof in preparation of kit for disease diagnosis
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Claims (5)

1. <xnotran> H5N1 HA , H5-3F5, EVQLVESGGGLVQPGGSLRLSCAASGFSLSEAVSGWVRQAPGKGLEWVGVCNNRIPFYLTWCKLFRFTISKDNSKNTLYLQMNSLRAEDTAVYYCARQRSYCNFQPHPNGWGQGTLVTVSS; </xnotran>
<xnotran> AYQMTQSPSSVSASVGDRVTITCGQNMMWIPAWLWYQQKPGKAPKLLIYETNGEHYGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCMMMNARKSLQATKFGGGTKVEIK. </xnotran>
2. Use of the monoclonal antibody of claim 1 in the preparation of a kit for detecting H5N1HA protein or purifying H5N1HA protein.
3. Use of the monoclonal antibody of claim 1 in the preparation of a kit for the detection of H5N1 or purification of H5N 1.
4. Use according to claim 2 or 3, characterized in that the kit is an ELISA kit.
5. The use according to claim 4, wherein the ELISA kit further comprises a coated plate, a strong positive control, a weak positive control, a negative control, a HRP goat anti-mouse secondary antibody, a washing solution, a dilution solution, a substrate solution and a stop solution.
CN202210747456.9A 2022-06-28 2022-06-28 Monoclonal antibody and application thereof in preparation of kit for disease diagnosis Pending CN115353562A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814623A (en) * 2005-02-06 2006-08-09 厦门大学 H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use
CN101379397A (en) * 2006-01-26 2009-03-04 Hx诊断公司 Monoclonal antibody for H5 subtype fowl-influenza virus hemagglutinin protein or combined active fragment, and uses thereof
CN101580545A (en) * 2008-05-14 2009-11-18 中国科学院上海生命科学研究院 Monoclonal antibody of hemagglutinin protein resisting H5N1 resource and application thereof
CN103254308A (en) * 2007-06-15 2013-08-21 厦门大学 Monoclonal antibody of haemagglutinin protein of H5 subtype of avian influenza virus, or binding activity segment thereof and application of monoclonal antibody or binding activity segment

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1814623A (en) * 2005-02-06 2006-08-09 厦门大学 H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use
CN101379397A (en) * 2006-01-26 2009-03-04 Hx诊断公司 Monoclonal antibody for H5 subtype fowl-influenza virus hemagglutinin protein or combined active fragment, and uses thereof
CN103254308A (en) * 2007-06-15 2013-08-21 厦门大学 Monoclonal antibody of haemagglutinin protein of H5 subtype of avian influenza virus, or binding activity segment thereof and application of monoclonal antibody or binding activity segment
CN101580545A (en) * 2008-05-14 2009-11-18 中国科学院上海生命科学研究院 Monoclonal antibody of hemagglutinin protein resisting H5N1 resource and application thereof

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