JPS58500037A - Interferon assay method - 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] Name of the invention: Interferon certification ceremony ÷ 6 Technical field The present invention relates to the field of immunoassays. More specifically, the present invention Anti-interferon therapy, including the use of monoclonal antibodies against terferon Concerning body excess immunoassay.

Background technology Interferons are a group of similar proteins that exist in the mammalian body. . interferon.

1 (through cellular metabolic processes including the synthesis of both NA and proteins, at least A protein factor that has a non-virus-specific antiviral effect in cells of be. Interferons are classified into types based on antigen specificity, and one name is rupha, beta, gamma (these were formerly named leukocytes, fibroblasts, (immune) interferon). This antiviral effect In addition, interferons have profound effects as mediators of other cellular events or immune functions. There is a connection. Research on interferon has hitherto been plagued by problems with its testing methods. Things didn't go smoothly depending on the situation. The only widely used assay is for tissue-cultured cells. Factors responsible for virus growth using cells with and without interferon (e.g. viral RNA synthesis or host cell death). this Although their complex bioassays are sensitive, they are laborious and Significantly affected by qualitative variability. In particular, the interfaces present in the test sample Factors other than ferons often influence viral growth. Therefore, it is easy A simple, indirect interferon assay is being considered. Such a test method is It will have wide applications in at least three areas of interferon research: cormorant.

1) Monitoring of interferon research production, bulk production and purification.

2) Quantification of interferon administration in research and clinical applications.

3) Quantification of interferon in biological secretions.

The purpose of the present invention is to provide an antibody excess immunoassay method for interferon that satisfies this requirement. It provides:

When the human body encounters an antigen, it responds by producing antibody molecules from lymphocytes. Reacts to antigens.

Antibodies consist of what is known as a determinant on an antigen. It has the property of selectively binding to specific sites and rendering antigens nontoxic. antigen and anti Although the nature of the interaction with the body is not fully understood, antibodies are It is clear that physical affinities exist for specific determinants of quality. Ru. The reaction between an antibody and a determinant on the antigen for which it has specificity generally causes adducts called “immune complexes” are formed. Formation of this type of A very wide variety of tests of quality is possible. These five assays are collectively referred to as immunological This is known as the verification method.

Immunological assays are broadly classified into the following two types.

(1) Excess analyte; labeled antigen (analyte) This term is a technical term, and in this text it has the meaning ``thing to be analyzed''.) . In this type of assay, antibodies that have specificity for the analyte are Nalyte and a solution containing a known amount of labeled antigen. This person In this method, an antagonistic equilibrium is established, and an unknown amount of analyte is replaced by a known amount of labeled An immune complex with an antibody is formed by competing with the antigen. Between labeled antigen and antibody The amount of analyte can be derived by counting the number of immune complexes produced. Wear. In this type of assay, the ultimate sensitivity of the assay lies in the relative stability of the immune complexes produced. It has a drawback because it is limited by its constantness.

(2) Antibody excess; labeled antibody. In this type of assay, the analyte being quantified is cells are incubated with an excess of labeled antibody molecules. Therefore, the quantification of the analyte The results are collinear, and the maximum sensitivity is theoretically at least one analyte molecule. It is. A modified version of this method prevents too much antibody from dissolving in the solid medium. to allow the analyte to form an immune complex there. Then analyze Incubate excess labeled antibody against the second determinant on the plate with solid medium. Ru.

This type of assay, commonly referred to as a ``sandwich assay'', is an immunoassay. It brings great benefits to the statutory law.

The present invention relates to the second type of assay described above, namely the antibody 11 immunoassay. Of particular relevance, it applies inter alia to the assay of interferons.

According to the present invention, reagents for performing an immunoassay for interferon include - two antibodies against Feron, at least one of which is a monoclonal antibody; One consists of labeled antibodies.

Conventional techniques for producing antibodies against interferon require that animals be given antibodies. Very small amounts of pure interferon are available for immunization to stimulate production. There was a problem because of this. This low antigenicity and the small amount obtained , can only produce antiserum of moderate purity. field of molecular biology Research in is currently providing an alternative source of antibodies.

Between lymphoid cells and myeloma cells obtained from mammals (e.g. mice and rats) It is possible to create hybrid cells with the ability to replicate in vitro by fusion of I know (Cola and Milstein, Nature 256°495-59 7　＞. These hybrid cells are said to secrete antibodies with predetermined specificity. have characteristics. This specificity is due to the lymphocytes produced by the lymphocytes involved in the fusion. This is what the generated antibodies have. Hybrid cells can be cloned and grown in stable culture. When grown in culture, antibodies can be produced against specific determinants in the culture supernatant. I can do it. Antibodies produced by this method are known in the art as monoclonal antibodies. It is known as an anti-inflammatory antibody.

The advantage of this technique is that antigenic impurities present on the antigen or in the immunizing material with which the mammal was immunized specific, uncontaminated by antibodies raised to other determinants on any of the above The aim is to provide a source of antibodies that can Other advantages of this technique include screening Antigens that cannot be used in pure form for assays such as interferon It is possible to use even antigens that exist in low concentrations in immune substances such as be. In addition to the convenient source of antibodies that cell fusion technology provides, monoclonal The unique determinant specificity of antibodies has been exploited in various ways in the field of immunoassays. ing. In particular, monoclonal antibodies bind to only one determinant. be. In immunoassays, commonly known as polyclonal antibodies, Previously used antibodies do not have this specificity and are not polyclonal. Assays using antibody antibodies tended to be inaccurate due to lack of specificity.

In one embodiment of the invention, both antibodies to interferon are monoclonal antibodies. It is.

A particularly preferred monoclonal antibody against interferon is l-lu-IFN. α-specific monoclonal antibody NK2, whose isolation and properties were described by D.S. DC, Park Niyol Nature 285, pp. 446-450 (1980 ) is explained in the paper Ken.

As mentioned above, both antibodies against interferon are monoclonal antibodies. can be done. However, conveniently one of the antibodies may be e.g. 2, and the other one was produced by conventional techniques. It is an antibody. Antibodies produced by such conventional methods can be used in humans, sheep, horses, It can be produced from mice, goats, guinea pigs, chickens, rats, etc. those antibodies is usually purified as much as possible and then by known methods to increase specificity, e.g. For example, it is modified by blocking.

Assays can be performed in liquid phase or using solid supports. If liquid phase For assays, it is a homogeneous assay that does not require separation of reactants, or a homogeneous assay that does not require separation of reactants. This is a unique test that requires Separation is performed by immunoprecipitation, bound antigen-antibody such as absorption or solubility by charcoal with free antigen and antibody rather than complexes. This can be done by phase separation based on different physical properties.

The assay according to the invention is preferably performed on a solid to which the unlabeled antibody is initially attached. This is done using a shaped support. This test should be conducted using the following two or three methods. Can be done. (a) an interface in which the product will react with the labeled antibody; - Reaction of Feron antigen with antibody bound to solid phase in excess (bl-labeled antibody and The antibody bound to the solid phase reacts with both antigens, (the antibody bound to the solid phase with excess C1 and This is the reaction of the labeled antibody with the antigen that will react.

The solid support can be fixed or unfixed. fixed support Examples of bodies are plates, test tubes, trays, wells. unfixed supports Examples are beads, particles, powders. Typical materials that can be used as supports include, for example, Polystyrene, polyvinyl chloride, polyethylene, polyacrylamide, nylon synthetic polymers such as resins, e.g. cellulose, polysaccharides, sepharose, Gallose, natural polymers like dextran, silica, glass, collagens are structural proteins such as polynucleotides, cells such as red blood cells, 5taphylococcus aureus )including. Attachment of antibodies to solid supports can be achieved directly by absorption, adsorption, or covalent bonding. This can be done manually or with a binder.

Labeled monoclonal antibodies, whether isotopically labeled or It may be labeled with something other than an isotope. Preferably the isotope For this reason, this assay is different from the immunoradiometric assay (IMRA). Become. Labeling can be done directly or indirectly (combined), with appropriate labels and There are 125.1, 1311, 32p, 14C, and 3H. labeling techniques Techniques include chloramine-T oxidation method, conjugation labeling method (Polton and Hunter, 1973b, Bio!l:hem, J 133゜ 529), lactoperoxyguse and iodogen: y (iodogen) operation including.

Non-isotopic labels that can be used in our assay include enzymes, which Meko's assay is an enzyme immunoassay (EIA) or an enzyme-linked immunosorbent assay (EL). ISA). Suitable enzyme markers include β-galactosidase, peroxidase oxidase, alkaline phosphatase, glucose oxidase, etc. The test method is It can also be a fluorescent immunoassay (FIA), with examples of markers such as Fluorophores such as cein, rhodamine, chelated rare earth elements There is a phore. Assays can be performed using bioluminescent markers or e.g. Luminescent immunoassays that include chemiluminescent markers such as IA).

Other methods of non-isotopic labeling are all well known in the art. Known heavy metals, coenzymes, latex, free radicals, cells by use of particle counting Is there a sign operation?

The assay operation is suitably carried out at a temperature ranging from 4C to 37C, preferably at room temperature. cormorant. When culturing is carried out twice in a row, the first culture is carried out for about 4 hours, and the second culture is carried out at 8 o'clock. It will be carried out for between 16 hours, for example - overnight.

Brief description of the drawing Figure 1 shows the standard curve for quantifying interferon using an immunoassay. be.

FIG. 2 is a standard curve for low concentrations of interferon.

Figure 3 is a graph showing the interferon assay results in the presence of human serum. It's rough.

BEST MODE FOR CARRYING OUT THE INVENTION As one representative example, an immunoradiometric assay (IRMA) will be described. child In this assay, described below, a sheep anti-interferon antibody is used in a solid phase. Attach to polystyrene and immobilize the interferon in the sample on the solid phase. Do it like this. This bound interferon then becomes 1251-NK2 (Model Add noclonal anti-Hu-IFNα) and count the total number bound to the solid phase. It is detected by

This antibody NK2 was produced by D, S, Sechartori, C, Park (Hature 285). 446-450 (1980)) and International Special Prepared as described in patent application no. PCT/GI381100067.

Three forms of polystyrene were used as solid phase.

(1) 3ml test tube (LP3, Luckham, Ltd., Bun gers Hi II.

U, ni,) (2+” micro titration tray with 96 depressions (M241Gibco Europe, Ltd, Uxbridge, U, Ni, ) (31 6,5tnrn beads (Northumbria Biologicals Li d.

In the first case, combine the entire test tube Trap measurements were made to measure 125■-NK2. When using a tray, each depression The bottom of the tube was cut out using a hot wire and transformed into a clean tube for measurement. in the third case , tray with 20 or 60 beads (93-0402, A, b) botLaboratories, Basingstoke, U. ), and then transferred to test tubes for measurement. The culture volume used was 1ml or 01ml ((hollow) and 0.2ml (beads). Although all samples were tested satisfactorily, it was not convenient to assay a large number of samples. Therefore, beads were more suitable.

NK2 antibodies were obtained by ammonium sulfate from the serum and ascites fluid of mice with NK2 tumors. Chloramine = T Label by method and then desalt by applying to Sephadex G-50 (line) column. did. The labeled IgG is approximately 201/μmole, or 125 μmol per mole of IgG. It had a specific activity of about 1 atom.

For each assay, standard curves were prepared using interferon refaremes standard Mf ( Drawn using C69/19 or laboratory standard.

In one such assay (see Figure 1), IgG was combined with sheep anti-Hu-IFNα Ammonium sulfate precipitation and DE from anti-dish supernatant (450,000 neutralization units/ml) AE-cellulose purified by ion-exchange chromatography followed by sheep Ig Q (PBS (phosphate buffer), 5mM EDTA, 20% in aN3 to 01% Polystyrene peas were cultured for 16 hours at 40 μg/ml). It was applied to. Hundreds of Bees 7: K fl cloth L, PBS, 105'6 horse plate 0.1516 was immersed in a culture solution (JIS culture solution) consisting of aN3, and incubated at 4C for 1- (Preserved in S culture medium. Assay tray (with 20 or 60 depressions) , Abbott) with H8 culture solution and culture at 4C. Dilute and then add a pair of samples (200 μC) to the antibody-coated beads. .

After 4 hours at 4C, each bead was combined with a dispenser/aspirator. Pentawash”.

Abotl Laboratories) was used to wash with H8 culture medium of mi. It was. After removing the remaining culture solution, 125I-NK2 (purified 1gQ, 40, 000 cpm) and cultured at 4C for 16 hours. Beads as mentioned above I washed it and then took it to the gamma counter. The dashed lines indicate the inter-applied beads. Background cpm range when cultured in H8 culture medium without feron Show the world. The total number bound at 8,000 U/ml is the maximum, and the in No further increase was observed when the terferon concentration was increased to 106 U/ml. It was. Low levels of non-specific binding (<1% input a number) This is an important feature of the statutory law.

In the second experiment (see Figure 2), we found that at low interferon concentrations The total number was shown to be proportional to interferon concentration. interferon sample is prepared by diluting the reference research standard in l-18 culture medium. 23,000 cpm of 125I-NK2 was added to each bead. Assays were performed as described for FIG. 1, except for the following. The dotted line is the background It shows the cpm boundary and is the average of 8 values (s, e, = ±5 cpm). 5 It can be seen that concentrations of 0 U/mL or higher can be easily quantified. Standard test conditions ( υIJ (used when drawing Figure 1) was used during the purification of interferon. The obtained sample was adjusted to make it easier to quantify.

Such samples have values of 103-107 U/mg.

When a solution containing 103 U/ml is successively diluted, the titration curve obtained is a standard curve. It matched the line. For the same sample, the results of the above immunoassay method and the known Comparing the results with the antiviral assay results, it appears that there may be some experimental error, but the results are similar. We are doing so.

According to the immunoassay method of the present invention, the assay has great advantages over conventional biological assay methods. become. Polystyrene coated with antibodies can be used in 4C, so materials can be assayed immediately, and results are available within 24 hours from the start of the assay. It will be done. Standard curves can be compared to the very large inherent variability of biological assays. In comparison, there is almost no variation from test to test. Input cpm is 47,000 2,0 OOU/mA in separate assays ranging from 53,000 CI) m ’ solution was measured four times.

The value was 3063±345 cpm. used to apply to polystyrene The IgG solution can be reused without any apparent decrease in the sensitivity of the assay. Therefore, only a small amount of sheep antibody is required. The quality of sheep antibodies depends on the success of the assay. is not a decisive factor, and as mentioned above, other antibodies against Hu-IFNα It can also be substituted in exactly the same way. This assay tests the specificity of monoclonal antibody NK2. This is the product of a hybrid myeloma cell line in culture. Therefore, it can be produced in large quantities without any change in quality. Finally, this The cost of the assay is low (in particular, all plastic products except beads are recyclable). It is suitable for automation, and one person can process hundreds of samples per day. can also be tested.

In traditional assays for interferon in serum or other biological secretions, Due to the limitations of other non-interferon substances that affect virus replication, Dilute the sample until the effect no longer masks the action of interferon. There was a need. PBS, 1096 horse dish serum, o, l, 96 NaN3 or Miki Affinity chromatography with NKz-Sepharose in normal human serum Diluting a sample of interferon purified by A titration curve is obtained, which indicates that the immunoassay method of the present invention solves this problem. It shows that there is. In this experiment, Inter7niro/(a) in H8 culture medium (b) undiluted normal human serum, not beads. 1 except that a 3 ml test tube was used as the polystyrene support. Samples were assayed as described above. The solid line represents the coupling c in case (a) VC. Indicates pm.

The dashed line shows the results in case (b).

lower amounts when quantifying serum interferon by biological assays. However, interferon in the serum of patients treated with interferon It is sufficient to detect. However, even with this sensitivity, normal humans Insufficient to quantify interferon concentration (if any) in serum .

- If the conditions are adjusted as described above to maintain a constant interferon concentration, The immunoassay method of the present invention can reliably quantify approximately 50 U/mJ.

The sensitivity of this assay is such that interferon can be detected using a small NK2-affinity column. It can be further increased by pre-concentrating, purifying and storing.

Engraving of drawings (no changes to content) [I = y-7zo>] (RRU/ml) FIG, 1 FIG.3 Commissioner of the Patent Office Wakasugi Kazuo Shun - 1.Display of the incident PCT/GB 81100239 2. Name of the invention Interferon assay method 3. Sparrow with correction Relationship to the incident: Patent applicant 4. Director, Shinjuku District, Tokyo, Shinjuku 1-8-1 (Hirado Building) 6. Due to amendments increasing number of inventions 2

Claims (1)

[Claims] 1. At least one of the two is a monoclonal antibody and one is labeled Interferon, which is an antibody, is made up of two antibodies against interferon. Reagent kit for immunoassay. 2. Both antibodies are monoclonal antibodies, and one is a labeled antibody. The kit according to item 1. 3 The labeled antibody is a monoclonal antibody specific to human α-type interferon. The kit according to claim 1 or 2, which is a body. 4. The kit according to claim 3, wherein the monoclonal antibody is NK2. . 5. The monoclonal antibody in the patent claim is radioactively labeled NK2. The kit according to scope item 4. 6. The unlabeled antibody is bound to a solid support. The kit according to any one of Items 5 to 5. 7. The sample to be assayed is a mixture of interferon in the sample and the protein bound to the solid support. placed in contact with a solid support that forms an immune complex with the interferon antibody. The labeled monoclonal antibody is attached to interferon bound to a solid support. A solid support that forms an immune complex between the labeled monoclonal antibody and attached to a solid support that will be placed in contact and the immune complexes will be quantified. Interferon antibody and labeled monoclonal against interferon An immunoassay method for interferon consisting of an antibody. 8. The labeled monoclonal antibody reacts with the sample to be assayed, and the reaction The product is reacted with an excess of antibody bound to the solid support, and the resulting immune complexes are determined. The interferon antibody attached to the solid support and the interferon that will be quantified are interferon, which consists of a labeled monoclonal antibody directed against interferon. immunoassay method. 9. The labeled monoclonal antibody and the antibody bound to the solid support are assayed. A solid sample is reacted with the sample and the resulting immune complexes are quantified. Interferon antibody attached to the support and labeled against interferon An immunoassay for interferon consisting of a monoclonal antibody. 10. The labeled monoclonal antibody is radiolabeled NK2. The immunoassay method according to any one of claims 7 to 9.