JPS60500714A - monoclonal antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] monoclonal antibody Technical field The present invention relates to monoclonal antibodies. More specifically, the present invention provides human Monoclonal antibody against α-type interferon, 1M monoclonal antibody Monoclonal production method, suitable for immunoassay of human α-type interferon How to use antibodies, immunological essence of standard products containing human α-type interferon! ! I! ! 1,7! How to use monoclonal antibodies in production and human α-type interferon Concerning a method for assaying antibodies against cancer.

Background technology Interferon is a substance that has antiviral activity and inhibits cell growth. is the genetic name given to the Kumbaku substance of the group (Steward W, E, ( 1979), “Interferon System”, Nubringer, Vienna and Biology of Interferon Cytesum”, Ernbia North Holland ・Biochemical Press, Amsterdam). Interferon has three Can be classified. Immediately, α-type interferon ([FN-α or white blood cell i interferon), β-type interferon (IFN-β or fibroblast interferon), feron) and γ-type interferon (I FN-γ or immune interferon) ).

(Steward W, E, et al. <1980) “Nomenclature of Interferon” The present invention relates to α-type interferon, especially human Monoclonal antibody against human α-type interferon (Hu-IFN-α) Regarding 1'.

At the same time, British patent application CB 20838358 (this is a national (also published in International Publication W0 81102899).

This monoclonal antibody is produced by the Coller-Milstein cell fusion process. (Kohler and Milstein, Nature). -256, 495-497). G B 2083836 Asserted in A A more specific embodiment of this invention is a cell line called NK2. It is a secreted monoclonal antibody. In addition, for applications in this concurrent layer, N Also discloses immunological purification process using monoclonal antibodies from K2 has been done.

Currently, there are currently no models for human α-interferon derived from cell lines such as NK2. Noclonal antibodies were passed through an immunoadsorption column consisting of antibodies immobilized on a solid phase. Do not completely eliminate interferon activity in preparations containing interferon. has been found. This may make it difficult to use standard materials, which may be objectionable under some circumstances. It becomes necessary to separate the This clear result has attracted the attention of many researchers. (Muers E. et al., Infection, pp. 207-212). these The results showed that monoclonal antibodies against human α-type interferon were This suggests that it is not bonded to all of the component parts of the product. A monoclonal antibody was used to purify all the constituent parts of α-type interferon. To use affinity chromatography, another antibody that binds to all components is It is necessary to search for a body, and +r has two or more complementary specificities. This suggests that the above antibodies need to be used in combination.

The present invention provides a method that is complementary to human α-type interferon occurring within NK2 cells. The objective is to provide monoclonal antibodies with specificity. Hu- IFN-α is derived from a number of different molecular assemblies known as subtypes. It is configured.

There are 8 to 12 confirmed subtypes of Hu-IFN-α, but their relative Further elucidation of the proportion is awaited. There are approximately 165 subtypes each. They have a long length of amino acid residues and have many of the same characteristics. To date, Hu IF N-α (20 different genes have been confirmed, so if you shift the combined PIl) It is assumed that there are up to 20 different subtypes of heat input H++-IFN-α. For historical reasons, the naming of one lipid type of Hu-IFN-α is possible. Laws are not officially recognized; one type of law is the name law, which is based on the Alphahet style; An alternative is the -262 nomenclature system that uses numbers.

The relationship between these nomenclatures is as follows.

A B CD E F GHl, JR α2 α8 α10 αl α4 α3 α5 α6 α7 In this Mei 8th Book III In this case, add 1・)・+1+ to normal alpha, 71□ir, naming 7+.

Kinen-san said that the seven clonal antibodies present in NK2 cells were produced by AJJ Butaibu. Found to selectively bind to human interferon (Hu-IFN-α2) did. Surprisingly, wood-brothers also interact with Heather type humans. Human interferon (+-(u-+FN- A monoclonal antibody that binds selectively by α1) is monoclonal) In the operation of body rJl phasic chromatography, N I<2 is derived from cell threads Found to act secretly and effectively as a complementary antibody to interferon. did.

Disclosure of invention In a first aspect of the invention, the α-type interferon of Hekabu-type human (. D-subtype human α-type interferon rJ4 A monochrome antibody against human α-type interferon, which has a greater binding capacity. provides natural antibodies. This monoclonal antibody is a human α-type interferon. subtype D human interferon than any other subtype of human interferon. It is preferable that the binder has a greater binding capacity than the other. This monoclonal antibody is Y Preferably produced from a cell line designated OK5/19. .

As used herein, the term "binding capacity" refers to human alpha-type interface. In the relative degree of affinity of monoclonal antibodies for specific subtypes of be. This binding ability is simply mo.

It not only shows the characteristics of monoclonal antibodies, but also describes monoclonal antibodies and their anti-monoclonal antibodies. It also represents the affinity of the immunochemical bond formed between corresponding antigenic determinants.

As used herein, the term "complementary binding capacity" refers to According to an embodiment, it refers to a binding capacity that complements that of a monoclonal antibody. complementary conclusion An example of an antibody that has the ability to synthesize is a monoclonal antibody that comes from NK2 cells. It is.

The method for the intracellular preparation of NK2 cells is described in published UK patent application CB2083836A. (Internationally released version W O' 80/02899!!6) It is explained in detail.

Binding ability of monoclonal antibodies to human α-type interferon subtypes The force (e.g., a fixed monochrome against human alpha-interfernon) by immobilizing subtype preparations on a solid support (with null antibodies). It is L7 that is determined. Monoclaves labeled with radioisotopes under test ° - The primary antibody is then incubated with the immobilized pure subtype, and after washing, the immobilized Measure the specific radioactivity of the support. Nonspecific binding of labeled monoclonal antibodies Radioactivity Bank Grand Rehe 5 n-60~500714 (3) After making the numerical adjustment for The radioactivity bound (in number of samples) is determined by the monoclonal antibody against the subtype. is the degree of binding ability of against specific subtypes of human α-type interferon. Another preferred technique to measure the binding ability of monoclonal antibodies to neutralize Tests can be used. In this neutralization test, human α-type interferon remains in the test preparation after mixing preparations consisting of subtypes and monoclonal antibodies. inhibits viral RNA synthesis caused by unneutralized interferon. Assess the binding ability by measuring the degree of harm.

In a second aspect of the invention, monoclonal antibodies according to the first aspect of the invention We provide compositions consisting of a combination of monoclonal antibodies with complementary binding abilities. Ru. Monoclonal antibodies with complementary binding ability are derived from NK2 cells. It is preferable that

The advantage of such compositions is that two monoclonal antibodies with complementary binding capabilities The combination of these two types of α-type interferon subtypes is highly effective in most human alpha interferon subtypes. It has a strong binding ability.

In a third aspect of the present invention, immunopurification of a preparation containing human α-type interferon is performed. provides a processing method for In this treatment method, based on the first aspect of the present invention, The monoclonal antibody or composition according to the second aspect of the invention can be prepared on a solid support. identified and an immunopurified medium is formed, which then contains human α-type interferon. The preparation comes into contact with this medium.

Antibodies can also be immobilized, for example on particulate solid supports. each branch The carrier particles are the seven particles according to the first aspect of the present invention. I can do it. A mixture of these particles can be used to make an immunopurification column. I can do it.

In a fourth aspect of the present invention, immunopurification of a preparation containing human α-type interferon is provided. provide a method of management. In this treatment method, the specimen is first subjected to the first aspect of the invention. An immunopurification column consisting of an immobilized monoclonal antibody based on Immunopurification consisting of immobilized monoclonal antibodies with complementary binding capacity column or in the reverse order. A model with complementary binding ability Preferably, the noclonal antibody is derived from NK2 cells. this These columns may be separate or integrated.

Fifth of the present invention! In embodiment 3, a human antibody comprising an antibody according to the first aspect of the invention is produced. The present invention provides an immunoassay method for α-type interferon.

This assay uses a preparation for assaying human α-type interferon. a first antibody against human alpha interferon bound to a solid support; using a second antibody against human α-type interferon labeled with is preferable.

In addition, in this assay method, one of the first and second antibodies is used according to the first aspect of the present invention. It is a monoclonal antibody based on human α-type interferon. Polyclonal antibodies or monoclonal antibodies with complementary binding ability Preferably, it is a body. This assay method uses a standard, a first antibody, and a second antibody in order. contacting human alpha interferon and the first antibody. measuring the amount of the second antibody bound to the solid phase.

Preferably, the preparation is contacted with the first antibody in a first step and then the second antibody is added. Yes. The first antibody is a polyclonal antibody (e.g., sheep anti-α type antibody). and the second antibody is a monoclonal antibody according to the first aspect of the invention. Preferably, it is a body. The label may be a radioisotope label. Preferably, enzymes, chromophores, fluorophores, chemiluminescent chemical groups or may be anything that can emit a detectable signal. Human alpha type I The polyclonal antibody against interferon is a sheep anti-α interferon. It is preferable that Monoclonal antibodies with complementary binding ability can bind to NK2 cells. Preferably, it comes from within the cells.

In a sixth aspect of the present invention, the first antibody has immunity against human α-type interferon. Provides epidemiological assays. In this assay, human α-type interferon Standard for testing the first antibody against human α-type interferon A solid support to which a second antibody is bound, which binds to human α-type interferon. a second antibody that has a label attached to it and is capable of binding to the first antibody; Use No. 3 antibodies. This assay involves bringing a solid support into contact with the standard. The first antibody is attached to the solid phase by the second antibody. contacting the solid support with a third antibody (which causes the third antibody to bind to the third antibody). The first antibody is attached to human α-type interferon and the second antibody in the same phase. and measuring the amount of the third antibody bound to the solid phase. The process consists of the following steps: The second antibody is a sheep anti-interferon and the third antibody is a sheep anti-interferon. The antibody is a radioisotope-labeled sheep antibody directed against the first antibody. It is preferable that Using the assay for antibodies according to the first aspect of the invention In this case, the second antibody may be an antibody with complementary binding ability, but preferably NK2 Using a monoclonal antibody against human α-type interferon that occurs within cells, use

8 In a seventh aspect of the present invention, a monoclonal antibody according to the first 93 aspects of the present invention is used. Methods are provided for producing hybridoma cell lines capable of secretion.

This method involves the steps of immunizing an animal with human α-type interferon, and The stage in which the system produces lymphocytes in response to human α-type interferon, and the tile cell and myeloma cells to form a colony of hybridoma cells; Based on the first aspect of the invention by screening colonies of ibritoma cells The process consists of searching for cells that secrete monoclonal antibodies. This method In the testing step, according to the sixth aspect of the invention (using an assay method, the third antibody is An immunopurification solution consisting of immobilized antibodies with complementary binding capacity is prepared before use in the assay. Antibodies with complementary binding ability are antibodies derived from the NK2 cell line. Preferably it is a clonal antibody. The third antibody is radioactive against the first antibody. Preferably it is a sheep antibody labeled with a sex isotope. The second antibody is U7 is preferably a monoclonal antibody derived from the NK2 cell line.

Brief description of the drawing Figure 1. Figure 2 is a graph showing the results of testing the binding ability of lon. Shows the results of IRMA assay conducted on one group of interferon using human body. Bar graph, Figure 3 is for group 1 interferon using YOK5/19 A bar graph showing the results of testing the binding ability of anti-interferon, Figure 4 is a monochrome About a group of interferons using local and polyclonal antibodies It is a bar graph showing the results of the IRMA test performed.

BEST MODE FOR CARRYING OUT THE INVENTION Preparation of monoclonal antibodies 9'　Special opening U6U-500714 (4) Monoclonal antibody After immunization with α-type interferon and fusing the tile cells and myeloma cells, the desired were prepared by selecting clones secreting antibodies of the same type.

The antigen used for immunization was human α-type interferon (Hu) prepared from white blood cells. -IFN-α(Le) (“P-IF”) (cantylation, 1981), This is what I got from Dr. Kanchiru (Hatsuchi 1912078-A, 38X 10’U/nj! , total protein 28 mg/mA). The antigen is 2 x 106 U/m7 in phosphate buffered salts (PBS)! diluted to , divided into 0.5 m12 (fi) portions and stored at -20°. .

Melt aliquots about every other week and add LOU type young with 1 to 100 mg of adjuvant. Adult rats were injected into multiple subcutaneous areas of the neck and back. Blood for each song to be injected with immunity Samples were taken from the tail. Approximately 19 months later, rats were given birth with white IM bulbs.1. and N Purified by immunoadsorption l'+7 tography using a K2-Sepharose 4B column. Booster 2x1.06U of Hu-IFN-α (“NK2-IFN”) Injected. This interferon can be used as an adjunct to incomplete LJin1. After emulsification in a medium, intramuscular and subcutaneous injections were given. final booster injection After 3 weeks, interferon (P-IF) in I-' BS was administered intravenously (tail vein). This was done by administering an intrapulmonary injection (2X 106 U).

0 Table 1 Immunization schedule To continue the immunization procedure, the rat's tail is 62 balls 1+11 L, +111? After collecting samples of -f approximately every week, α of blood'/H was measured using a plaque reduction assay. The ability to moderate the antiviral activity of 5v interferon was tested. This test Then, 111 layers were added to 1iii, in which an amount of vesicular stomatitis virus was added to balance the titer. When cultured with 10 cells of superior human cells, 10 ) Compared to the virus plaques in the control, the number has decreased to about 20% of that in the evening. Ta. Before adding interferon to ii'4j1i cells, a sample of rat blood was prepared. l￥ (The final dilution rate is) / fi (If you incubate with 1 camphor, several Recovery due to ill pusheek was observed in the game (see Table 2, 116).

On the 11th day of the 604th day, the rats were sterilized and the tiles were removed aseptically. Mark 1Ill-like method (see Galfre and Milschquin, 1981), ]0° IIQ 'Cells were derived from rat hyperlitoma line (Y132/3.0Δ+X20). fused with cells of the non-secreting variant 6X]0' (Gulfre 1 Inn and Fito, 1!17!1). The remaining hepatocytes were kept in liquid nitrogen with low YAA content. It existed.

After fusion, the cells are 2411? 6 plates containing J wells (Linpro) hand! 18　2　m, one bite dispersed in the culture solution of e. -11f! 1 pitch per week for J3 ,.

1'1st ton: 'l　+- was observed in 23 out of 48 (solid).

Table 2 Here, -1 serum has no effect. +- some of the plaque numbers recovered +++-80% of plaque number recovered Three weeks after fusion, culture medium's 2 ln to detect anti-interferon antibodies. Tested. This assay/method is based on an immunoradiometric assay (Secher 1981). - A modification of the 41-step method for measuring feron concentration. sheep resistance The antibody was coated onto a plastic substrate. The substrate is in the form of test tubes or beads or wells of microtiter trays. Ru. The inventor used exclusively a microtitration routine with 96 wells. 10 μp sheep anti-interferon (purified 1gG)) Raction; P B S10.1% NaN:I, 5m M ethylenediamine Add 10-25 μg/mρ in tetraacetic acid per well and incubate for 16 hours at 4"C. Ru. Next, after removing the antibody solution, the wells were treated with “blocking medium” (1% (V/ V) Normal human blood, 0.5% bovine blood 177 albumen (BSA), 0 ．． PBS containing 1% N a N ) and then at room temperature for at least 1 h. (or overnight at 4°C). ■ After removing the 1F culture medium, add 05% BSA to the wells. and 0.1% NaN3.

Washed twice with PBS. Solution containing Hu-IFN-cx (100μr, blocking culture medium A voltage of about 4000 V/m7 was added to each well except for the control well. Add 100 μr of blocking medium to control wells. 2 hours at 20-25° After incubation, interferon was removed and 100 lte of the solution to be tested was added to the wafer. Add to the bottle. (Generally speaking, this F4 fluid is the supernatant of the cell culture fluid). Furthermore After incubation (approximately 2 hours at 20-25°), the test solution was removed and the wells were soaked with 0.5% B. Wash twice with PBS containing SA and 0.1% NaN3. completely remove the test solution, and Care must be taken to clean as quickly as possible.

The next step is carried out by incubation with labeled anti-cat Ig antibodies.

100 μl of 1241-labeled sheep anti-rat immunoglobulin antibody (5x 105~lO°"cpm/m7!, 1μci/l+g, 0.5% BSA and purified sheep antibody affinity in PBS containing 0.1% NaNa), respectively. After adding to each well, culture at 20-25° for about 2 hours. Next, join Remove unlabeled antibodies and fill wells with 0.5% BSA and 0.1% NaN+ Wash two or more times with PBS containing

The radioactivity remaining in each well is equal to 1 g of bound sheep anti-rat. This is also the cross-linking between interferon and sheep anti-interferon. It is also the percentage of anti-interface Icon antibodies bound to the solid phase by bridges.

This radioactivity was removed by cutting out the bottom of the wells with a hot wire and transferring each well to a test tube. Alternatively, it can be determined by measuring with a ζ gamma counter.

An alternative to this assay uses plastic rather than sheep anti-interferon. using a monoclonal antibody (eg, NK2) that binds to NK2. in this case, Raheno-C sheep anti-cat Ig antibody is an antibody that binds to NK2 before use. must be passed through a NK2-Sepharose column to remove This test method Now, we have developed an antibody that recognizes a different antigenic determinant than that identified by NK2. Only the body produces clear signals, but binds to IFN species that lack NK2 determinants. Antibodies have an advantage when they do not produce a clear signal.

Using this alternative assay (using NK2-coated plastic), fusion After 3 weeks, remove the supernatant from all cultures containing actively growing cells using an anti-interrupter. - tested to confirm the presence of Feron antibodies. The results (Table 3) show that the NK2 antibody At the same time, it produces anti-interferon antibodies that can bind to α-type interferon. It was suggested that there was only one resulting culture solution (YOK 5/19). α type A Since the interferon molecule is a monomer, this means that the YOK5/19 antibody and NK2 This suggests that each antibody recognizes a different antigenic site. This test was repeated on the culture supernatant 4 to 5 weeks after fusion, but the activity was Stable Table 3 cpIll related to binding (x1O-2) Trial fee + IFN - IFN from immunized rats. Serum (1/1000) 61 8 YOK5/19 culture supernatant 16 6 21 other culture supernatants 5-9 5-8 Cloning and production of monoclonal antibodies Live cells were diluted sequentially, and at each dilution level (96 wells x O, 2 ml) Transferred to a microtitration tray. Cell culture supernatant allows cell growth to still be observed Trays inoculated at the highest possible dilution were removed at 10-14 days and assayed. . Grow cells from the most positive culture medium (YOK5/19 (31)) and For storage, freeze in liquid nitrogen and place about 5 x 107 cells on the front side. FI AOxLOU rat belly primed with pristane Injected into the membranous cavity (Gulfre and Milstein, 1 (181), ascites tumor develops. After 2 weeks of survival, the cells were removed from the peritoneum and returned to the culture medium. cells grow well , continued to produce anti-interferon antibodies. These cells (, Y OK 5/ 19f31 (A s)) was again diluted and fractionated as described above, and the sheep Anti-interferon and unpurified Hu-I FN-α (Le) or cloned Based on the binding assay method using IFN-α1 (Table 4), YOK 5/1 9(3) (As) (3,80> was selected.

Next, use agarose instead of agar and remove the mouse peritoneum before the addition of agarose. Cells from this culture were grown as described above, except that the cells were attached to Vetri dishes. Sea urchin was successfully cloned on a semi-solid support (Gulfre and Milstein , 1981). This is a subcutaneous injection into the peritoneal cavity of a freshly killed Baβb/C mouse. Depending on the vessel.

This was achieved by washing with 8 mj of cell culture medium (2%).Then, the cell suspension was The cells were divided into 0-20 x 9cIn Petri dishes and cultured for 4-24 hours. After this cultivation, The culture solution was removed and dissolved agarose (0.5% in the culture solution) was added.

Table 4 )1u-IFN-α(Le) Hu-IFN-α1(Le) *Non-specific binding The combination (100-200) is a spear.

Clones were picked from Petri dishes containing approximately 20 clones.

Of the 13 clones tested in the anti-interferon assay, 10 were positive. It was sex.

Selected clone YOK5/19f31 (As) (3,80), 31 Re-cloning was performed and the next Niku o-7YOK 5/19 (31 (As) (3,8 0).

31.9 was isolated. This clone was renamed YOK 5/19.31.9 However, the antibody produced thereby was simply referred to as YOK5/19 antibody.

To obtain serum and ascites containing YOK5/19 antibodies, extract from the culture medium as described above. Cells (1-5 x 10' per rat) were added to F1 hybrid rats of the AOXLOU strain. injected intraperitoneally or subcutaneously (Gulfre and Milstein, 1981). Next one No significant differences were observed when using a humming loan.

YOK 5/19 (31 (As) (3,80), 8. YOK 5/19f 31 (As) (3,80), 22. yOK 5/19+31 (8s) ( 3,80), 31. Y OK 5/19f31 (As) (3,80) , 31.9 10-20 days after injection, rats were sacrificed and ascites and/or blood were removed. I started out. After the blood was allowed to clot, the serum was removed and stored at -2o. ascites After separating the cells by centrifugation, the supernatant was removed and stored at -20°. stomach Newborn cells from suspensions prepared from watery cells or solid subcutaneous tumors were injected into rats and passed through the cell line. Cellulose acetate samples of serum and ascites YOK5/1 in animals was subjected to electrophoresis (Microzone, Beckman). The production of 9 antibodies was detected.

To purify the YOK5/19 antibody, the following experimental design was adopted.

Add 100 μl of retained serum and ascites to 100 pI) of saturated ammonium sulfate solution. was added with stirring at 4°C. Precipitate 10. OOOrpm (MSE21)X2 After collecting by centrifugation for 0 min, add 10 mM sodium phosphate buffer, pH 7.5. 44 mA). This solution was mixed with 10 mM sodium phosphate, pH 7. , 5 (5x 2 liters) and centrifuged 16 Separation (10, OOOrpm x 20 minutes), DE 52 (7,5c m　X　3.85c+n.

denatured and insoluble molecules that are in equilibrium in the same buffer. protein was removed. The column was then heated with 420mρ of 10mM N-phosphate. Elute with thorium buffer pH 7.4, then concentrate stepwise from 10 to 100 mM. Sodium phosphate solution with increased strength, pl + 7.4 (800 mβ + 800 n It eluted with u). The fractions were collected (12,3 mβ) and the absorbance of this column eluate was (280 nm). The first peak eluted from the solution at the first concentration The fraction was identified as pure YOK5/19 by cellulose acetate electrophoresis; The mixture was collected, dialyzed against distilled water, and then freeze-dried. In such experiments The protein yield was 313 mg.

YOK5/ for purification of interferon by immunoadsorption chromatography Binding of +9 antibody to Sepharose 4B.

YOK5/19 antibody purified as described above was purified as previously described (Sethi Ger and Haack, 1980), Omg per ml of swollen Sepharose. CNBr-activated Sepharose, IB (Pharmacia) with protein content. Combined. Measuring the protein concentration in the solution before and after reaction with Sepharose Judging by this, 95% or more of the bonds were formed.

Radioisotope labeling of YOK5/1.9 antibody Y purified as described above The OK5/19 antibody was used as previously described for NK2 (Secher, 1981). Labeling was carried out with the radioactive isotope 124■ using chloramine-T. Approximately 2 4YOK5/ with a specific activity of 0μci/μg (3Ci/p mole) 19 Hu-IFN-tx neutralization ability YOK 5/19.8 and YOK 5/19.2 The serum of No. 2 was tested using an antiviral assay (Atherton and Haak, +975); According to the results of 6 tests examining the ability to neutralize the activity of Hu-IFN-α, 2500 0.7 when tested against tJ Hu-IFN-α(Ly) (Namalva) and 25 U of Hu-IFN-α was neutralized with 1.8. child These results indicate that at high antibody concentrations, YOK5/19 is a major constituent of Namalva TFN. It has been shown that it can neutralize the antiviral activity of minutes.

YOK5/19 and NK2 anti-interface using various Hu-IFN-α preparation methods. Elon's peculiarity In the selection and cloning of YOK5/19, this assay applied NK2. A plastic well was used. This well allows YOK5/19 to The antigenic sites identified are different from those identified by NK2, but at least Some Hu-IFN-α molecules have both antigenic sites and have both monoclonal sites. It was found that local antibodies can bind simultaneously. Different types of Hu-I When using FN-α, the specificity of the two antibodies was Using feron coated wells and comparing the cpm of binding. I investigated further. The IF'N group consists of crude leukocytes IFN (Hu-IFN-(X (1, e)), crude leukocyte IFN is transferred to an NK2-Sepharose column. When NK2 is removed, the eluate is interferon (“N K2 eluate''), cloned Hu-IFN-α (Pzabquib) and cloned A, B, C, D, F of Hu-IFN-α.

1, J, and K were identified. The cpm for binding when IFN is not added to the assay is , one value of non-specific binding (usually 200-400 cp) subtracted from other numbers. m) was clarified.

Tables 5 and 6 show the results of different such experiments and also demonstrate the reproducibility of the assay method. It is something.

According to the experimental results, NK2 distinguishes between A, B, C, and D, but not F. On the other hand, YOK5/19 clearly shows that it is most active against lFN-D. Ru.

Table 5 IFN Values subtract the hackground cpm obtained when IFN was not added to the assay. The cpm (xlO-2) related to the bond (solid phase deposit) after subtraction is shown.

Y OK 5/19 (11 is the secondary culture of the original YOK5/16. YO K4. [, C6 is a culture from a different fusion that was terminated.

Table 6 IFN 8 B (: [] F I J K 1lu-IFN-αl Crude IFN-αNK 2-[1iN NK2-/8 liquid YOK 5/19.　31.9 is also a lodge: Hu-IFN-A from Roche was tested using the same assay method. Ta. The results suggest that under this test condition, no discrimination was made between .

"Anti-interferon assay used to measure antibody concentration" Duplicate anti-interferon assay The terferon binding assay tests the yoK5/1.9 antibody in different samples. It was also used to measure relative concentrations. Use serial dilutions for each sample. For each dilution, add appropriate IFN (e.g. IFN-α1 or crude IFN). The cpm of binding (solid phase deposits) in this assay method used was measured.

The results of such an experiment are shown in FIG. From this experimental result, the force 1i11i (maximum The half dilution of cpm for large binding is between 1/10' and 1/105. , is more than 100 times the concentration in 21N of YOK5/19 cells before cloning. I understand that. C. In order to obtain the cpnt value related to tag land coupling, use the α-type interface. - A control experiment was conducted in the absence of feron.

yo　K　5/　19.31.9 antibody to immunoglobulin class, and radioactive As mentioned above, SI) S-polyacrylamide gel LJ (Gulfre and Milstein, 1) ．． 98]) 6 According to autoradiography, one (γ) H chain and one L chain This clearly shows the existence of Y OK 5/19.31.9 from a single cell. This proves that the antibody is an IgG antibody. It is something that

A similar conclusion is that the S Also obtained from DS polyacrylamide gel electrophoresis. O)・Radiograph The only visible Han 1 had the mobility of ■1 chain (γ) and I-chain.

Immunoradiometric assay using YOK5/19 The previously described immunoradiometric assay for measuring IFN-α Some similar methods of infectious radiation assay (IRMA) (Secher, 1981) are used. devised and tested.

In these methods, 1241-labeled YOK5, prepared as described above, /19 antibody or YOK5/19 antibody coated plastic heat (or other solid support) was used. This method is performed as follows. First, as mentioned above YOK5 purified by ammonium sulfate precipitation and ion exchange chromatography /19 antibody in PBS containing 0.05% 1laN3 and 5mM EDTA. (After diluting to lμg/mff, 200 polystyrene beads were placed in this solution. Crushed at 4°C for 16 hours. Next, wash the beads and add 0.1% NaN3 and 0.5% B. Stored in PBS containing SA. Beads and tracer coated with YOK5/19 IRMA using YOK 5/19 labeled with radioactive isotope as Leave it, TFNi! ! 1 degree is 105U/mj! Until then, there was no noticeable binding of radioactivity. I couldn't see it. This means that one molecule of interferon produces 5/19 YOKs. This shows that only one antigenic site exists. Similar observation is made in NK2 This is consistent with the fact that the IFN-α molecule is a monomer. IRMA No of IFN-α using clonal antibody, solid phase antibody labeled Antibody (*) Assayability *This labeled antibody is labeled with a radioactive isotope. It is an antibody that has been tested.

Table 7 lists the monochromes tested as possible IRMs against Hu-IFN-α. The combination of null antibodies is listed. Using sheep anti-IFN-α in solution (Hawkins and Sechar, 1983).

Not distinguished by radioisotope-labeled antibodies in some assays The same problem of inhibition due to competition for the solid phase by other IFN-α species may occur. It was. This is true for assays when polyclonal sheep antibodies are coated on beads. (No. of Table 7.

1 and 2), and to a lesser extent, it was also seen in test No. 6.

on a solid support with radioisotope labeled NK2 as a tracer. The combination of YOK5/19 did not show any signs of this inhibition, with 50.0OOU/ m E shows 3900 cpm related to coupling for an input of 65,000 cpm Ta. IFN-α with both NK2 and YOK 5/19 antigenic determinants Only species can be distinguished by assay using two monoclonal antibodies. stop To further investigate this specificity, we again used a panel of IFNs (all approximately 5 0000. /　mj! >. The results of this comparison are shown in FIG. (A.

I3. C, D, F, I, and J contain the individual subtypes of human α-type interferon. Type standard, Cr is interferon crude standard, Ef is NK2 immunopurification column. This is the eluate from. NK is interferon purified with NK2 immunopurification column. αl is a standard α1 type human interferon, b/g is Hasoku Ground 1. ゛, that is, a control. ) In Figure 2a, this assay involves the use of solid-phase NK2 antibody and radioisotope. A topically labeled YOK5/19 antibody was used. Figure 2 shows this test. are solid-phase YOK5/19 antibody, radioisotope-labeled NK2 antibody, and IF. B, F and α1 of N-α (all appear not to be identified in the combined test) )It was used. All other species tested were identified by at least one assay. solid The antibody in the phase is produced at a concentration much higher than the effective concentration of the radioisotope-labeled antibody. Due to the fact that they have effective concentrations, different relative reactivities are probably This is probably due to the difference in binding strength between the two monoclonal antibodies.

The importance of antibody concentration (and avidity) is important when using standard anti-interferon antibodies as described above. This was proven using the 2-way test method. In this assay, anti-interferon was tested at 10 μg/ml or is 1 Mg/mj! Purified 2 Y OK 5/191gG. In the case of IFN-α-response, one test cpm ranges from approximately 1400 at IOIJg/ml to 110 at 1Mg/mA. 00, but in the case of IFN-tx-A, the decrease was from about 1500 cpm to The background level was approximately Ocpm. About A, C, and D of Hu-IFN-α were also tested over a wide range of concentrations. The results in Figure 3 are relative to the two tests. This difference in reactivity cannot be based on inaccurate estimates of IFN concentrations. This suggests that. (Figure 3' a is 10 μg/mn, Figure 3 is 1 Mg /m7! ).

Using only one labeled monoclonal antibody along with a polyclonal solid-phase antibody A similar analysis of the antigen specificity of the assay used is shown in FIG. In Figure 4 , the solid-phase antibody is sheep anti-α interferon, and in Figure 4a, the radioactive isotope The plain labeled antibody is YOK5/19 antibody. The antibody tested is the NK2 antibody.

Use of YOK5/19-Sepharose 4B column for IFN-α purification ゛ E. coli that produces Hu-IFN (1' and Hu-IFN- (X) from leukocytes In the immunopurification of Hu-I FN-α from YOK5/1 as described above, 9 purified from serum and ascites of tumor-bearing rats and conjugated to Sepharose 4B. IgG was used.

In the preliminary exam, 45ml! To purify IFN-α from crude leukocyte IFN, Experimental design developed for NK2-Sepharose (Secher and Haak, 198 YOK 5/19-Sepharose 4B small challah according to the same experimental design as 0) (0.6m7!') was used. Packed with approximately 2.2 x 106U of IFN-α After that, 46% of this IFN-α (judged by IRMA) was eluted at pH 2, and one It was collected as a fraction.

Proteins contained in the fractions were precipitated with 1-lichloroacetic acid, and S -According to the results of PAGE analysis, it has a molecular weight of 15,000 to 20,000. One major band and a second, much weaker band with a somewhat higher molecular weight. has become clear. This was confirmed once on the YOK5/19 Sepharose column. This suggests that IFN can be purified simply by passing it through.

According to the specificity data obtained with the anti-interferon assay (see above): YOK5/19 antibody recognizes interferon in NK2-column eluate and YOK 5-Sepharose and NK2-Sepharose have crude IF/Ni1 compound. This suggests that there may be complementarity in the purification of IFN between these two groups. . This means that NK2-Sepharose and YOK5/19-Sepharose can be used consecutively. It was confirmed by using the Crude IFN-α(Le) was converted into NK2-Sepharo When passed through a column, 50-60% of the flu Activity is lost (but can be recovered by elution from the column with p112) Met). Most of the remaining IFN activity was caused by YOK5/19 Sepharose Kara I lost it by passing it through the system. According to the antiviral assay, approximately 10% TFNL is not maintained by the combination of NK2 and YOK 5-cepharose. It suggests that.

In another experiment, crude IFN (1 400mp) YOK 5--fzVyn-1, no 1 mff (7),'+ram passed through. YOK5-IFN was eluted with 0.1M citric acid and then stained with silver. It was analyzed by SD S-PAGE (Shinto et al., 1981). IFN for acidic fraction contained most of the activity. And 1 with mobility indicating Hu-IFN-α The main hunt was on the same gel.

YOK for purifying IFN-α species that cannot be purified with NK2-Sepharose As a further illustration of the capabilities of 5-sepharose, a recombinant human α1 gene product containing The crude bacterial lysate (70 mf) was transferred to a 4.4 ml column of YOK5 Sepharose. I passed it to Mu. (This IFNα1 is present in IRMA or in NK2-Sepharose. some were not identified by NK2). The column was washed with PBS and then zeroed. ．． Washed with 1M ammonium acetate. The bound IFN-α1 is converted into a volatile PI. +2 no loose fi/l! L (4,5% (V/V)) lIc0OI (,0,01M vinegar After elution with ammonium It was assayed by IRMA using YOK5/19 labeled with a radioisotope.

The results showed that approximately 65% of the IFN in the column was recovered in a fraction of 2 x 4 mβ. It showed that. The purity of this IFN is determined by a single molecule with a molecular weight of approximately 19,000. 5DS-PAGE revealing major hunts, l! : Indicated by silver staining.

The purity of purified αI is determined by lyophilizing the most active fraction and converting this protein into By transferring to Sokuman 89o3 sequencer and determining the N-terminal amino acid sequence. confirmed. This sequence is more than 20 times the sequence predicted from the DNA sequence of the α1 gene. There was a complete match for the residues.

Notes on naming In this specification, clone YOK 5/19.31.9 (originally YOK 5 /19 (also known as 31 (Ag) (3,80), 31.9) or similar Clonete YOK 5/19.31. YOK 5/19.22. YOK 5 The antibody produced by YOK5/19.8 cells is called YOK5/19 antibody.

The abbreviation for YOK5 is NK for NK2/13.35.6 It is the same as the abbreviation 2 (Secher and Haak, 1980). YOK5 Sefa The interferon purified with ROS is called YOK5-IFN, which is NK5-IFN. It also follows common practice regarding 2.

References Atherton, N., T. A. and Hark, D. S. (1975) ``Viruses and Porines.'' Induction of interferon by cleotide: Cambutosequantel, Ni, Hilbo Nen, S., and Koishti Feng, ■.

(1981) “Large-scale partial purification of human leukocyte interferon” ``In Engimology'', (MG S, Hest Riki) Vol, 7B, pp 499-505. Academic Press, New York and London.

Garfret, G. and Milshfin, C. (198]) “Monoclonal Antibodies” MEN Law 2 Planning and Operation”, “Merinth in Engimo.

Collie”, (editors J, J Langon, 11. Van Bounakis) Vol.

■, 3-46. Academic Press, New York and London.

Gar7shi,G,,mil'/Yutai7. C, and Rai l-, I3. (1979 ) “Fd portion of rat rat myeloma and mouse IgG Mo2T corner antibody” , Nature, 277, +3], -133.

Hokitus, R, E, and -fzchi+=, l], S, (+983) "Clinical “Immunoassays for Chemistry” (editors Xiong, Mon, Hunter, J.E.T., Collie) 2nd edition, PP582-587゜ Scotto, G, M,, Secha-, D, S, 7u'7-; (, D,, Hate, ,1,.

Cantil, N., and Chirel, D., A. J. (1981) “Interferon. "Toxicity", Br1tish Med, J, 282.1345-1348゜ Setcher, O. S. (1981) “Human leukemia using monoclonal antibodies” “Immunoradiometric assay for bulbar interferon”. Nature.

Opening □, 501~503° Sechar, D, S, and Bar 1. D. (1980) “Human leukocyte interface” "Monoclonal Antibodies for Large-Scale Purification of Ron". Nature Liu 5.446~ 450° Ray, W., Poulikas, T., Shinin v., p., and Hankosoku.

6 R, (19B2) “Protein silver and silver in polyacrylamide gels” International search report

Claims (1)

[Claims] 1. Monoclonal antibodies are more effective than A subtype human α-type interferon. Has greater binding ability to human α-type interferon of the D subtype. A monoclonal antibody against human α-type interferon characterized by body. 2. Monoclonal antibodies are effective against any subtype of human α-type interferon. Rimo D subtype has greater binding ability to human α-type interferon. The monoclonal antibody according to claim 1, characterized in that it has and. 3. Complementary binding ability with the monoclonal antibody sensitive to claim 1 or 2 0 composition consisting of a combination with a powerful monoclonal antibody. 4. Monoclonal antibodies with complementary binding ability can be found in NK2 cells. The composition according to claim 3, which is 5. Monoclonal antibodies or claims based on claim 1 or 2 The composition according to paragraph 3 or 4 is immobilized on a solid support to prepare an immunopurification medium. is formed, and the preparation containing human α-type interferon comes into contact with this medium. A processing method for immunopurification of a preparation containing human α-type interferon . 6. The specimen is a fixed monoclonal product according to claim 1 or 2. Immobilized monochrome with complementary binding capacity to immunopurification columns consisting of antibodies. The column is passed sequentially through an immunopurification column consisting of a null antibody, one first. Processing method for immunopurification of a preparation containing human α-type interferon prepared in . 7. The claim that the antibody with complementary binding ability is derived from NK2 cells. The treatment method described in item 5 or 6. 8. Using the antibody according to claim 1 or 2, human α-type protein consisting of diζ - Immunoassay for Feron. 9. Preparation for assaying human α-type interferon and binding to solid support The first antibody against human α-type interferon and the label are attached. A second antibody against human α-type interferon is used. The immunoassay method according to item 8. In this assay, one of the first and second antibodies One is a monoclonal antibody according to claim 1 or 2, and another antibody is a monoclonal antibody according to claim 1 or 2. The body uses polyclonal antibodies or complementary antibodies against human alpha interferon. It is a monoclonal antibody with binding ability. This assay consists of a standard, a first antibody, and f. f52 antibody sequentially or in combination; and human α-type interface. A step of measuring the amount of the second antibody bound to the solid phase by the first antibody and the first antibody. consists of 10. Standard material for testing the first antibody against human α-type interferon , a solid support to which a second antibody against human α-type interferon is bound. , a second antibody that binds to human α-type interferon, and a label attached. a third antibody capable of binding to the first antibody; Immunoassay for first antibodies against interferon. This assay is based on a solid phase support. The carrier is brought into contact with the standard, thereby causing the first antibody to be attached in phase by the second antibody. The step of binding to human α-type interferon, and the step of attaching the solid phase support to a third the third antibody is brought into contact with the human alpha interferon and the second antibody. The first antibody attached to the solid phase by the antibody 11, human α-type interface Steps of immunizing the animal with Ron and 29 The stage in which the immune system of a human being produces lymphocytes that respond to human α-type interferon. , preparing a sample of lung cells from an animal, and combining the lung cells with myeloma cells. The stage of fusion to form a colony of hybridoma cells; The monoclonal clone according to claim 1 or 2 is obtained by screening colonies of the method according to claim 1 or 2, comprising the step of searching for cells secreting null antibodies. To produce hybridoma cell lines capable of producing the monoclonal antibodies described above. How to do it. In this method, in the screening step, claim 1O Assay described and immobilized antibodies with complementary binding capacity prior to use in the assay. and a third antibody passed through an immunopurification column consisting of: