TWI418786B - Monoclonal antibody against hemagglutinin of avian influenza viruses subtype h6n1 and methods of making and using same - Google Patents

Description

Monoclonal antibody against avian influenza H6N1 subtype virus hemagglutination protein and preparation and application thereof

The present invention relates to a monoclonal antibody against avian influenza virus hemagglutination protein and a preparation method and application thereof, in particular to a monoclonal antibody against avian influenza H6N1 subtype virus hemagglutination protein, and a fusion tumor of the monoclonal antibody. a cell strain, and a test kit and method for applying the monoclonal antibody.

Avian influenza (AI) is caused by an influenza A virus infection of the Orthomyxoviridae family. Type A influenza virus has two kinds of glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA), and the antigenicity of agglutinating proteins and neuraminidase The difference can be divided into many immunological subtypes. There are currently 16 kinds of hemagglutination protein antigen subtypes H1~H16 (Fouchier et al., J Virol 79(5):2814-22, 2005) and Nine neuraminidase antigen subtypes N1 to N9 (Webster and Laver, Virology 69(2): 511-22, 1976).

In addition, according to the pathogenicity of poultry influenza virus, it can be divided into high pathogenicity and low pathogenicity. The highly pathogenic high-pathogenic avian influenza virus (HPAIV) is mainly H5 and H7 subtypes. It can cause systemic diseases with high mortality in chickens and may even cause death in humans (Lamb and Krug, Fields Virology 1487-1531, 2001; OIE, 2008). The remaining pathogenicity is called Low pathogenic avian influenza virus (LPAIV), which is widely found in various poultry. According to the current field investigation, the H6N1 subtype avian influenza virus belonging to the low pathogenic poultry influenza virus (LPAIV) is prevalent in Taiwanese native poultry (Lin Shizhen, Avian Influenza Information of the Animal Health Laboratory of the Executive Yuan Agricultural Committee, 2004; Happy et al., Taiwan Provincial Animal Husbandry and Veterinary Society reported 59:45-55, 1992; Zheng Mingzhu et al., Animal Health Laboratory Research Report 42:43-50, 2007). H6N1 has almost become one of the endemic poultry epidemics in Asia in the past few decades. Taiwan first erupted the H6N1 subtype poultry influenza virus in 1972, which has a very high mortality rate for poultry. In 1998, there were plans to monitor the waterfowl and poultry epidemics in Taiwan. In recent serum monitoring, more than 50% of laying hens and chickens were found to be H6N1 seropositive (Yang Pingzheng et al., Taiwan Veterinary Medicine 32:24-29, 2006), and more and more H6 subtype strains have been isolated.

Although H6N1 is low in pathogenicity, when the virus forms latent infections in poultry, it may cause weightlessness, dysplasia, decreased egg production and easy infection of other diseases, resulting in economic losses for poultry farmers, and more, If the virus persists in a population for a long time, it may mutate into a highly pathogenic poultry influenza virus (HPAIV) or form a new avian influenza virus, infecting a non-resisting population, which in turn causes a global pandemic. The establishment of a convenient, accurate and rapid mass production method for the identification of H6 subtype poultry influenza virus detection cannot be ignored.

Today's poultry influenza virus detection method comprises direct detection of viral proteins or nucleic acids by reverse transcription polymerase chain reaction and immediate quantitative reverse transcription polymerase chain reaction, and separation of antigens by virus detection by comparing influenza virus samples to HA The ability to initially assess the titer of the virus and the use of specific antibodies to distinguish between viral serotypes such as hemagglutination inhibition test (HI teat), neuraminidase inhibition test (NI test) and enzyme-linked immunosorbent assay Method such as enzyme-linked immunosorbent assay (ELISA). Among them, the method of detecting avian influenza virus by antibody has the advantages of simplicity and rapidity, however, the accuracy and sensitivity of the method depend on whether the antibody recognizes an important epitope and its good specificity, and therefore, diversity (diversity) and specific antibodies play an important role in this method.

According to the study, infective avian influenza viruses in nature, most of the HA subunits contain 3-5 glycosylation sites (Schulze, J Infect Dis 176: S24-8, 1997), these active HAs are large. Some of the surface is covered with glycans, and the number, size and type of sugars depend on the difference in the host, and the position is determined by the nucleic acid sequence of the viral gene. Furthermore, since the influenza virus is an RNA virus, these glycosylation sites are frequently changed with mutations, and the resulting diversity is presumed to be a strategy for the virus to counteract host neutralizing antibodies and various antiviral mechanisms. In addition, huge sugars will mask part of the HA surface, affecting its physiological and immunological properties, which may make the host's immune system unable to recognize the virus. Therefore, studies have pointed out that the effects of HA surface sugar must be considered in the development of influenza vaccine. (Wang et al., PNAS 106 (43): 18137-42, 2009). In addition to the development of vaccines, the location of the antibodies identified by the individual antibodies is also affected by the saccharide. Therefore, it is necessary to screen for the location of the saccharide-masking or to directly identify the monoclonal antibodies of the specific saccharide.

In summary, due to the large number of poultry influenza virus subtypes, it is possible to quickly and effectively distinguish the differences between different subtypes as the key to epidemic prevention. Taiwanese poultry influenza H6N1 serosubtype is prevalent in Taiwanese native poultry. And it will cause serious economic losses. However, there is no specific single antibody against the avian influenza H6N1 subtype virus or a diagnostic kit for detecting the virus. Therefore, in order to reduce the livestock diseases caused by avian influenza, As well as preventing the future of new viruses threatening human health, the development of specific monoclonal antibodies against the avian influenza H6N1 subtype virus is a top priority.

In order to quickly and accurately detect the presence of the avian influenza H6N1 subtype virus and effectively distinguish the difference between the H6N1 subtype virus and various other avian influenza subtype viruses, one of the objects of the present invention is to provide a fusion tumor cell line, which can Producing a monoclonal antibody against a hemagglutination protein of avian influenza H6N1 subtype virus, and the monoclonal antibody does not cross the hemagglutinating protein of at least one other avian influenza virus subtype other than the H6N1 subtype virus (for example, H5N2 subtype) The reaction, further, the fusion tumor cell line was named EB2, and the registration number of the Republic of China Food Industry Development Institute was BCRC 960409.

Another object of the present invention is to provide a monoclonal antibody having antigen-binding properties, which is produced by a fusion tumor cell strain deposited with the Republic of China Food Industry Development Institute and having the accession number BCRC 960409, wherein the antigen binding property is In order to identify and bind to the agglutination protein of the avian influenza H6N1 subtype virus, and not to cross-react with the agglutinin protein of the avian influenza H5N2 subtype virus; the monoclonal resistance system belongs to the IgG _2b subtype, and the The fusion tumor cell line EB2 was prepared.

A further object of the present invention is to provide a kit for detecting the presence of avian influenza H6N1 subtype virus in a sample, which comprises at least the above-mentioned monoclonal antibody, and the kit is a blocking ELISA. Kits or Western immunoblotting kits.

It is still another object of the present invention to provide a method for detecting the presence of avian influenza H6N1 subtype virus in a sample, the steps comprising: (1) contacting a sample with the above-mentioned monoclonal antibody; and (2) borrowing an immune response The presence of the avian influenza H6N1 subtype virus in the sample is detected, wherein the immune response is blocking ELISA or Western immunoblotting.

The monoclonal antibody produced by the fusion tumor cell line EB2 of the present invention is highly specific to the avian influenza H6N1 subtype virus, and does not cross-react with other subtypes of avian influenza virus, that is, the monoclonal antibody of the present invention against avian influenza The H6N1 subtype virus has good discriminating ability, and the kit, or method for testing the poultry epidemic cold developed by the monoclonal antibody with rapid, high sensitivity and specificity can accurately detect and further control the H6N1 subtype virus. The purpose of proliferation.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

In order to rapidly and accurately detect the avian influenza H6N1 subtype virus, the present invention immunizes a mouse with a virus particle of a H6N1 subtype virus strain isolated from Taiwan, and prepares a fusion tumor cell line, and is initially purified by a virus particle. The hemagglutination protein (HA) of intact carbohydrates is screened for monoclonal antibody to enhance the binding of the monoclonal antibody to the H6N1 subtype avian influenza virus, and the obtained monoclonal antibody binding site is prevented from being masked by sugar, in order to obtain the right The avian influenza H6N1 subtype virus strain is highly specific and can be practically applied to the wild-type detection of monoclonal antibodies; and the specificity analysis result of the purified antibody shows that the monoclonal antibody prepared by the present invention does not affect at least one other subtype. The avian influenza virus strain such as H5N2 cross-reacts, so the fusion tumor cell strain and the monoclonal antibody of the present invention can be applied to the development of diagnostic reagents or kits, and can be used for detection of H6N1 subtype virus in clinical samples, which is beneficial to the detection. Effective monitoring of avian influenza.

Example 1 Preparation of monoclonal antibodies against avian influenza H6N1 subtype virus hemagglutination protein (HA) 1. Source and preparation of antigen

The antigen used in the embodiment of the present invention is Taiwan H6N1 subtype avian influenza virus strain A/Chicken/Taiwan/2838V/00, GenBank number is EF681878, and the specific pathogen free (SPF) chicken embryo is inoculated 9-11 days old. Eggs, and cultured in a 37 ° C incubator for 3 to 4 days, collecting the allantoic fluid of the embryonic egg of the dead chicken, and detecting and determining the virus containing the avian influenza virus, which is initially purified and concentrated for subsequent immunization use.

Among them, the avian influenza virus is purified and concentrated by centrifuging the collected allantoic fluid at 3,000 rpm for 15 minutes at 4 ° C, taking the supernatant, adding 1% BEI, and placing the virus at 37 ° C overnight to inactivate the virus. . After ultracentrifugation at 70,000 g for 3 hours at 4 ° C, the supernatant was removed, and the precipitate was reconstituted with NET buffer of 1/100 volume. The solution was slowly added to the sucrose gradient (sucrose gradient was 10%, 20 %, 30%, 40% and 50%), after ultracentrifugation at 50,000 g for 3 hours at 4 ° C, the visible virus layer was aspirated with a needle, and washed with 4 mL of NET buffer, and then at 50,000 at 4 ° C. g ultracentrifugation for 3 hours, depending on the amount of precipitate, 200 ~ 500mL of NET buffer back to dissolve, stored at -20 ° C. In addition, virus quantification can be determined by any method for determining protein content, here using Bio-Rad's microassay system (protein analysis kit), using Bradford's protein assay, and bovine serum albumin as the standard protein. .

2. Preparation of fusion tumor cell strain (1) Immunization of mice

The antigen of the immunized mouse is an avian influenza H6N1 subtype virus particle which has been treated with formaldehyde and has no infectious activity. An emulsion containing the antigen was prepared by mixing an equal amount of the synthetic adjuvant TiterMax with the purified and concentrated H6N1 subtype virus, and the emulsion containing 100 μg of the antigen was intraperitoneally injected (Intra-peritoneal, ip) at 6 weeks of age. BALB/c female mice (purchased from the Experimental Animal Center of National Taiwan University of Medicine) were injected with an antigen-containing emulsion at a concentration of 50 μg every 2 weeks for a total of 4 injections. The antiserum was collected from the tail vein of the mouse every other week after the immunization of the mouse, and the antibody against the avian influenza virus was detected by Western immunoblotting using the antibody in the antiserum as the primary antibody. potency. A strong antibody titer reaction was measured up to 6 weeks later, and 50 μg of the antigen without adjuvant was subjected to the last immunization in the same manner, and cell fusion was performed 5 days later.

(2) Preparation of fusion tumor cells

On the day of fusion, the spleens of the mice were taken out, and splenocytes were isolated to make all spleen cells and a T80 (about 2 × 10 ⁷ cells) of mouse bone marrow cancer cells (Sp2/0-Ag14, ATCC CRL- 1581) Convergence. The cell mixture was collected by centrifugation, and 0.7 ml of polyethylene glycol 1500 (polyethyleneglycol 1500, PEG 1500) at 37 ° C was added to the cell mixture, and gently mixed for 1 minute to fuse the cells.

Next, 10 ml of 37 ° C DMEM medium (Dulbecco's modified Eagle's medium, available from Biochrom AG, FG 0435) was slowly added to dilute PEG 1500 over 4 minutes, and PEG 1500 was removed by centrifugation. The cells collected after centrifugation were gently resuspended in 30 ml of DMEMX medium (HAT-DMEMX, supplemented with 15% fetal bovine serum) containing hypoxanthine-aminopterin-thymidine (HAT). And placed in a 37 ° C CO ₂ incubator for 1-2 hours, and then the fused cells were evenly distributed into a 96-well microtiter plate. The day of cell fusion was designated as day 0.

On the first day after the completion of the fusion, add 2 drops of HAT-DMEMX at 37 ° C in each well of a 96-well microplate, and then, on the 6th, 11th and 14th day after the completion of the fusion, two drops of 37 °C HAT-DMEMX was added to each well of the culture dish. The two-week culture system is a preliminary screening, and the successfully merged fusion tumor cells can continue to divide and grow, and the cells aggregated can be observed. On the contrary, the fusion cells that have not been successfully fused die.

3. Screening and identification of fusion tumor cell lines

Two weeks after the initial screening, avian influenza H6N1 subtype virus (A/chicken/Taiwan/2838V/00) and avian influenza H5N2 subtype virus (A/Duck/Taiwan/3233/04, this virus is based on the former example 1 The source of the antigen of point 1 was prepared by the method described above. Western immunoblotting was simultaneously performed to screen for fusion tumor cells.

Considering the specific location of the HA of the H6N1 subtype avian influenza virus particle obtained from chicken embryo eggs, there are some large glycans, which will mask part of the protein surface, if using non-sugar-containing protein or The synthesized short-chain peptide is used for immunization and subsequent antibody screening. Although a monoclonal antibody with potency can also be obtained, if the antibody is applied to the natural H6N1 subtype avian influenza virus particle detection, it may identify the binding position of the protein. It will be obscured by sugar and cannot be actually used for testing. In order to avoid this problem, the present invention uses a virus particle obtained by obtaining a saccharide from a chicken embryo egg to preliminarily purify HA (which is prepared by the method described in the above "Source and Preparation of Antigen") to screen individual antibodies, although prepared. The procedure is more complicated, but once the method is applied to the monoclonal antibody with potency, the original characteristics of the antibody can avoid the interference of the sugar, and the avian influenza virus with the sugar on the surface of the virus in nature is carried out. Detection.

Among the many fusion tumor cells producing antibodies with effective valency, only the fusion tumor cells which were positive for H6N1 subtype virus and negative for H5N2 virus were selected and then monocultured by limiting dilution. According to this method, a single antibody fusion tumor cell line with the strongest titer to produce avian influenza H6N1 subtype hemagglutination protein was selected and named as EB2, and deposited in the food industry development on March 12, 1999, in the Republic of China. Institute (address No. 331, Food Road, Hsinchu City), its registration number is BCRC 960409.

The EB2 fusion tumor cell line is rapidly and stablely split (see Figure A) and tested for potency after multiple passages (>20), which can be diluted by more than 24,000 times (see Figure B).

4. Subtype analysis, production and purification of individual antibodies (1) Subtype analysis of individual antibodies

An appropriate amount of freeze-dried monoclonal antibody powder produced by the fusion tumor cell line EB2 was dissolved in PBS buffer, and the isotype of the individual antibody was performed using the ImmunoPure monoclonal antibody isotyping kit (Pierce Chemical, Rockford, IL). analysis. The results are shown in the second figure. As can be seen from the figure, the monoclonal antibody system produced by the fusion tumor cell line EB2 belongs to the IgG _2b subtype, and its light chain is a kappa chain.

(2) Large-scale preparation of monoclonal antibodies

In the embodiment of the present invention, a monoclonal antibody is produced by injecting a fusion tumor cell strain into a mouse to induce the production of ascites.

BALB/c mice over eight weeks were injected with 0.5 mL of pristine in each mouse to stimulate the peritoneal cavity of the mice. After 5-7 days, the fusion cell line EB2 with an effective price of 10 ⁶ or more was centrifuged to remove the supernatant, and then suspended in 0.5 ml of DMEM at 37 ° C, and then injected into the abdominal cavity of the mouse with a syringe. After one to two weeks, the mouse's abdomen swelled until the ground was pulled, and the mouse peritoneal fluid was taken. After centrifugation, the supernatant was taken. The supernatant contained the monoclonal antibody produced by the fusion tumor cell line EB2.

(3) Purification of monoclonal antibodies

The obtained mouse ascites was diluted 3 times with PBS buffer or the supernatant of the EB2 fusion tumor cell line was directly collected, subjected to 40% saturated ammonium sulfate precipitation, and the collected precipitate was dissolved in 50 mM sodium hydrogencarbonate buffer ( pH 8.3) and dialyzed overnight at 4 °C. The precipitate produced during dialysis was removed by centrifugation, and the supernatant was subjected to DEAE-Sephacel column chromatography. The DEAE-Sephacel column was first washed with 50 mM sodium bicarbonate buffer (pH 8.3), and after loading the sample, a linear gradient of 50 mM sodium bicarbonate buffer (pH 8.3) containing 25 to 250 mM sodium chloride. ), the fraction containing the monoclonal antibody is eluted at a concentration of 200 mM to 300 mM of sodium chloride.

Example 2 Specificity analysis of monoclonal antibodies produced by fusion cell line EB2

The H6N1 and H5N2 subtype avian influenza viruses were taken, and 1 ug/well was used for protein electrophoresis using 15% SDS-PAGE film, and then the antigenic proteins were transferred to the PVDF membrane by electrophoretic transfer method, and then The PVDF membrane was separately transferred to a solution containing no antibody (this is used as a control group), a primary antibody solution containing H6N1 or H5N2 multi-strain antibody (the multi-stranded anti-system is diluted 2,000-fold) (this is used as a control group), and contains a fusion tumor cell. The monoclonal antibody produced by the strain EB2 (the monoclonal antibody was 2,000-fold diluted) was used as a primary antibody solution (this was used as an experimental group).

After the antibody antigen binding reaction and the step of rinsing the unbound antibody, a commercially available secondary antibody that recognizes the primary antibody, such as a goat conjugated peroxidase-goat-anti-mouse (horse radish peroxidase-goat-anti-mouse, is used. HRP-GAM, this secondary anti-system is diluted 5,000 times and then uniquely identified. Since the secondary antibody has been linked to peroxidase (HRP), it can emit luminescence through the catalytic matrix reaction to carry out specific antibodies. Specificity analysis. The result is shown in the third figure.

Please refer to the third figure. The results of the figure show that the monoclonal antibody produced by the fusion tumor cell line EB2 is only transferred to H6N1 compared to the complex antibody immunological band presented on the transfer membrane of H6N1 in the control group. An apparent band is present at a size of about 55 KDa, which is the relative position of the known HA sub-body HA1, and its identity is confirmed by LC/MS/MS sequencing. As for the transfer film of H5N2 avian influenza virus, there is no band, and it can be seen that the monoclonal antibody system produced by the fusion cell line EB2 can specifically recognize and bind to the H6N1 subtype avian influenza virus (A/chicken/Taiwan/ 2838V/00) HA protein, but has no binding ability to H5N2 avian influenza virus. Therefore, the monoclonal antibody system produced by the fusion tumor cell line EB2 can be applied to the detection method of avian influenza H6N1 subtype virus, for example, western immune dot blotting. Method or enzyme-linked immunosorbent assay (ELISA), biochip, and strip test, which are required to be detected by antibodies, and can also be used to detect a diagnostic kit for the presence of avian influenza H6N1 subtype virus in a sample. Among them, the enzyme-linked immunosorbent assay (ELISA) system includes a blocking ELISA (bELISA), etc. In addition, the test strip portion can be used without special equipment, and can achieve the effect of local detection and immediate prevention. It is quite helpful for the prevention and treatment of avian influenza diseases.

The first A is a schematic diagram of an EB2 fusion tumor cell line of the present invention.

The first B is a graph showing the results of the titer of the EB2 fusion tumor cell line of the present invention after repeated passages.

The second graph is a graph showing the results of subtype analysis of individual antibodies of the present invention.

The third panel is a graph showing the results of specificity analysis of individual antibodies of the present invention.

Claims (9)

A monoclonal antibody produced by a fusion cell strain deposited in the Republic of China Food Industry Development Institute and registered under the number BCRC 960409, wherein the antigen binding property of the monoclonal antibody comprises a blood cell for the avian influenza H6N1 subtype virus The agglutinated protein has a specific binding and does not bind to a hemagglutinating protein of the avian influenza subtype virus other than the avian influenza H6N1 subtype virus. The monoclonal antibody according to the first aspect of the patent application is a hemagglutinin protein which does not bind to the avian influenza H5N2 subtype virus. The monoclonal antibody according to the first aspect of the patent application belongs to the IgG _2b subtype and the light chain of the monoclonal antibody is a kappa chain. The monoclonal antibody according to the first aspect of the patent application is screened using a preliminary purification protein of avian influenza H6N1 subtype virus particles having a saccharide. A fusion tumor cell line is a monoclonal antibody that specifically binds to a hemagglutination protein of avian influenza H6N1 subtype virus, wherein the fusion tumor cell line is deposited in the Republic of China Food Industry Development Institute and the accession number is BCRC 960409. A kit for detecting the presence of avian influenza H6N1 subtype virus in a specimen, comprising at least the monoclonal antibody as described in claim 1 of the patent application. The kit described in claim 6 is a blocking ELISA kit, a Western immunoblotting kit, a biochip kit or a test strip (strip) ) Set. A method for detecting the presence of avian influenza H6N1 subtype virus in a sample, the steps comprising: (1) contacting a sample with a monoclonal antibody as described in claim 1; and (2) borrowing an immune response The presence of the avian influenza H6N1 subtype virus in the specimen was detected. The method of claim 8, wherein the immune response is a blocking ELISA or a Western immunoblotting.