JPH07500190A - Technology to prevent false reactions during immune testing - 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] Technology to prevent false reactions during immune testing Background of the invention Diagnosis of certain diseases caused by pathogens such as bacteria and viruses depends on the body's immune system. It is based in part on detecting changes. Such changes are caused by bacteria, It detects antibodies produced by the body in response to parasite proteins, sugar proteins, and polysaccharide components. It is shown by

A false positive result is due to the presence of C1q, a component of C11 complex, in all serum samples. It may be caused by doing. Also, false positive results occur in many sera. It is also caused by the presence of rheumatoid factor (RF).

False negative results may be caused by the presence of undissociated C11 conjugates.

C1 complement is composed of three components known as C1q, C1r, and C1s; These three components are combined as a calcium-dependent complex. glycine buffer When diluted with (pH 8,2) and/or treated with EDTA-Na, C1 was It dissociates into the three components (C1q, C1r, C15). In the conventional technology, C1q formation Minutes are heat unstable substances and were thought to be deactivated by heating at 56°C for 30 minutes. . The present inventors determined that C1 and C1q were determined by heating serum samples at 56°C for 30 minutes. It was discovered that although it was attenuated, it was not deactivated.

C1q in human serum binds to antigen-antibody complexes and immunoglobulins such as IgG and IgM. can be matched. A typical concentration of C1q is 70 mcg,/ml. Also that It binds to assembled gamma globulin and causes gamma globulin in solution and agar systems. Precipitate. C1q contains bacteria, bacterial lipopolysaccharide, dextran, heparin, polyimide Nosinic acid, carrageenan, DNA, C-reactive protein, fibronectin, mitcon Doria, platelets, lymphocytes, monocytes, null cells, monosodium urate crystals, cellular bone lattice filament, which binds to a hybrid monoclonal mouse antibody.

Heating serum at 56°C for 30 minutes is not an effective method for inactivating C1q. It was shown that The present invention reduces the temperature to 50% in the presence of organic or inorganic ions. C1 and C1q are deactivated only when heated to 9-64°C for 3-30 minutes. discovered. The higher the temperature, the less heating time is required.

Heating at 56°C for 30 minutes also reversibly attenuates complement, but serum samples at 59-6 Heating at 4°C for approximately 3-30 minutes prevents complement interference in immunoassays. It turns out.

A practical problem arising from interference with C1q is that the presence of the antibody is prevented or the antigen is also present. or increase or decrease in the quantification of the antibody response will result in a change in the sensitivity or accuracy of the assay. sometimes giving false negative results.

None previously known as inhibitors of C1q-IgG or C1-1gM reactions. Compounds such as salts expose C1q by using latex photometry It was surprising and significant that it was found to be an activator of C1. That was something out of the ordinary.

Rheumatoid factor can be found in certain carriers such as latex, sheep cells, bentonite, and charcoal powder. It is recognized as an interfering agent in all immunoassays that use the body. RF falsely promotes immune complex formation. Complement prevents the formation of large insoluble lattice bodies to solubilize antigen-antibody complexes or enhance the shape of immune complexes by It is known that there is. C1q is present in all human and animal serum. ing. Both of these compete with each other and both are important in all immunoassays. resulting in false positive reactions.

Therefore, the main object of the present invention is to improve the complement system C1 and C1q - or more. Provides an improved method to eliminate false positive or negative results caused by ingredients It is about providing.

The main object of the present invention is also to reduce C1q in body fluids tested in immunoassays. and RF.

These and other objects of the invention will be apparent from reading the appended claims. It will be.

Outline of the invention The present invention provides a method for preventing the interference effects of C1q in body fluids.

In its broadest sense, the present invention provides the ability to extract 01 and C1 from body fluids in immunoassays. Including a method for preventing the interference effect of q. The method includes the following steps.

(a) a step of adding an effective amount of a compound to a diluted sample of body fluid, the step of adding an effective amount of a compound to a diluted sample of a body fluid; the mixture of body fluid and compound is heated to a temperature of 59-64°C for a sufficient period of time. in an immunoassay without affecting the reactivity of the antigen or antibody in the body fluid. A step capable of preventing interference of 01 or C1q in the body fluid. (b) bringing the diluted sample containing the compound and the body fluid to a temperature of about 59°C to about 64°C; Heat for sufficient time to remove 01 or CIQ interference in immunoassays. process.

When you want to deactivate C1q and RF in a specific sample in body fluids, the following steps are recommended. including suitable methods.

(a) an effective amount of a compound and an effective amount of a compound of formula R-3H for a diluted sample of body fluid; A step of adding the compound, wherein the mixture of the body fluid and the compound is 59 to 64 When heated for a sufficient time to deactivate C1 or C1q to a temperature of It deactivates CI or C1q without affecting the reactivity of antigens in body fluids. In this compound, R is filled in order for the compound of formula R-5H to deactivate RF. When the mixture is heated to a temperature of 59-62°C for a period of time, the antigen in the body fluid reacts with the mixture. Processes with organic components that do not affect responsiveness (b) The product of step (a) is deactivated by C1q and RF to a temperature of 59-62°C. The process of heating for a sufficient period of time to

In a method useful for the practice of the invention, the 0.25 ml of sample has a pH of 7.8 to B.2 and an ionic strength of 0.025 to (1, ( 1125 in glycine buffer. Next, neutral salt solution (2M NaCl or Add 20ul of 2M KCl or Q, 1M EDTA-Na) to the diluted body fluid. Incubate at 63°C for 13 minutes. If the sample is being calibrated for RF, the preferred temperature is 30 minutes at 60°C.

The present invention also provides that samples of body fluids, i.e., serum, synovial fluid, pleural fluid, or ascites, are includes a screening method to determine whether it contains RF. This method is C 1q and RF reactivity towards latex particles and C1 as defined herein A series based on the use of heat in the presence of suitable compounds to deactivate q. process. Also, a script that proves the existence of C1q using the ophthalony method A method was also devised.

The present invention further provides for making the latex test RF specific to reduce oxidation by C1q. human serum and the body by the use of appropriate compounds and heat used to prevent Includes detection and semi-quantitative methods of RF in liquids.

A technique has also been devised to determine the amount of purified C1q using latex particles.

The present invention also allows automatic latex agglutination testing without interference from C1q or RF. Brief description of new equipment drawings for dynamic implementation Figure 1 is a graph showing the plot from the photometer's X-Y recorder for a negative turbidimetric test. baseline (negative curve) and positive nephelometric test positive curve (slope > 0) ). The dashed line marked “Tangential to maximum value” is “Tangential to maximum slope”. Measure the maximum slope indicated by the overlapping triangles on the right labeled "Measure". This is a line drawn by a tangent meter to

Figure 2 shows the method of the present invention for determining whether a body fluid is RF positive or contains C1q. It is a flowchart showing a series of steps of a screening method.

FIG. 3 is a schematic diagram of a device according to the invention.

FIG. 4 is a graph showing the results of Example 12B.

Detailed description of the invention The term ``body fluids'' refers to the brain as cerebrospinal fluid, synovial fluid, ascites, pleural fluid, blood or the like, urine or and serum. Interference between 01 and C1q in immunoassays when testing body fluids The effect can be prevented by the methods described herein. preferred test The extraction method is latex aggregation technology using photometry.

The photometer used for latex agglutination is model 500 (used for blood aggregation measurement). Chrono-1og Corp.). This appliance is not equipped with a temperature control device. The heater block is controlled to 37°C. Also, this device has a stirring speed of 1 Equipped with a 200 rpm stirring motor.

The reaction between the latex antigen and antibody mixture is performed in a single tube using known methods. I was disappointed. A curve is constructed in which the change in % transmission with respect to time is It is a measure of the degree of change in particle size between particles. The increase in transmission is due to AgAg Larger and fewer latex particles up to 5-6μ in diameter collide during the reaction. This is due to latex particles of 0.8μ size forming. That is the largest size When reaching the particle size, the curve of the S-shaped floc becomes asymptotic. The point of flocculence when changing rapidly is indicated by the slope of the transfer 96 curve. . This slope is measured with a tangential needle within minutes after the latex suspension is added. Ru. When using a negative fluid that does not produce an antigen-antibody reaction, the transfer vs. time is linear. Become.

The maximum slope value is detected within 3 minutes. To obtain quantitative information, serum was diluted. Each aliquot is then subjected to a photometer. Series for different dilutions of serum samples The curve of is then obtained and the slope calculated. a species that contains a certain amount of antigen or antibody A standard curve was constructed by plotting the maximum slope response of each dilution. It will be done. The curve for the unknown sample is compared to this standard curve and the quantitative evaluation is based on this standard curve. It is also useful for comparison.

In Figure 1, a substantially straight convex baseline or negative curve is horizontal. Indicated by a dashed line and a positive "curve". The broken line is the X-Y plotter is the tangent at the point of the S-shaped curve where the slope recorded by is the maximum.

The maximum slope is the area where the rate of change in floc particle size is greatest.

Dilutions of body fluids can be calculated from standard curves without having to test unknown body fluids in several dilutions. It is possible to predict the outcome for any of the solutions.

Preferred compounds for use in the practice of this invention are neutral salts of the Hofmeister permutation, organic Contains salts or diamino compounds. Any of these compounds has a pH of 7.0~g, 5, pH 8.2, ionic strength 0.0125-0.025 u, concentration 40 m M added to a 1:10 dilution of a body fluid sample in ~200mM glycine buffer; and the sample is heated to a temperature of 59-64°C for a sufficient period of time, e.g. 3-30 minutes. , interference with CI or C1q without affecting antigen or antibody reactivity in body fluids. Any device that can prevent Wear.

Below is a list of salts that have been shown to be promoters of latex flocculation tests.

Besides exposing C1q, these salts neutralize the charged groups of C1q, leaving the C1q complex It helps to dissociate AgAg from immunoglobulin and AgAg complexes.

sodium bromide sodium chloride sodium iodide sodium citrate sodium cyanate Sodium perchlorate sodium acetate organic ions Sodium ethylenediamine Ethylenediaminetetraacetic acid tetrasodium propionate Sodium butyrate Neutral salts of other Hofmeister permutations are also heated at a temperature of 59-64°C for a sufficient period of time. Any substance that deactivates C1 or C1q can be used.

Neutral salts are strong electrolytes that serve as sources of ionic strength. The charge of a neutral salt is the charge Change macromolecule conformation by weakening attraction or repulsion reactions within or between charges. tion. Therefore, reactions with charged groups, peptide bonds, amino-carbon By reaction with dipolar groups such as ruboxyl, hydroxyl, primary and tertiary amides , it is the free energy between the folded (association) and cleaved (solution M) forms of the macromolecule. neutral salts have a marked effect on the association and dissociation equilibria. . Since EDTANa4 is used here for its action as a neutral salt, and not used for its chelating or complexing properties. You should be careful.

Although the exact mechanism of the reaction is unknown, these neutral salts used in the present invention Unmasks the C1 complex and the C1q component in serum. It is thought to function as a activator. The same neutral salt can be used with IgG and IgM. used to dissociate C1q from the complex and AgAg complex, and heating is used to deactivate the C1q component.

Inorganic and organic salts introduce structural variations in proteins. These are macromolecules Modify the environment, thus controlling temperature, pH, competitive hydrogen bonding, and hydrophobic binding effectors. through which it behaves in aqueous solution. Neutral salts have a main charge depending on the pH of the solution. Defined as a strong electrolyte that is significantly soluble in water without loading.

It has been observed that gamma globulin aggregates when serum is heated above 53°C.

The present invention brings about a change in the shape of immunoglobulin and irreversibly coagulates C] and q. Binds C1q without causing aggregation to the extent that it binds to collected immunoglobulin provides a novel method based on heating and the use of inorganic or organic anions to do.

This eliminates false positive (non-specific) results due to C1q in immunoassays. It can be prevented.

The latex fixation test is positive in 70-80% of rheumatoid patients and in others. It is also positive for 0-25% of sick patients. Seropositive patients with RA Approximately 25% of people have titers between 1:160 and 1:640. Singer and Brotz suggest that a titer of 80 or higher should be considered positive. About RF According to the Center for Infectious Diseases, many research The laboratory reports a titer of 80 or higher as significant. False positive test is serum It was thought that this may be due to the presence of 01 or C1q in the protein. This is the RF serum. The low titer is the reason for the undefinable false positive result and is also probably due to R This may be the reason why only 80% of A patients have a positive test result.

Prevention of C1q interference results in meaningful potency for RF testing that correlates with treatment symptomatology. It is possible to grant

For the detection and quantification of rheumatoid factors in latex photometric techniques, the above Prepare samples by adding compounds to diluted body fluids and heating at 60°C for approximately 30 minutes. It is preferable to do so. However, the shorter time at the higher temperature, i.e. 63°C. In other words, it may be 3 minutes. coated or uncoated specimens made afterwards. Test the sample for the presence of RF by contacting it with latex particles do.

Aggregation of latex particles is quickly detected visually or by spectrophotometry be able to. The absorbance of a sample increases in direct proportion to the number of RFs in the sample. RF After standardizing the spectrophotometer using a standard sample, the sample is placed in the spectrophotometer and If a flocculent curve is observed within minutes, the sample is positive for RF. this The method provides physicians with the ability to detect C1 or C1q, which can produce false positive results. Fast, sensitive and reproducible test results from samples treated to remove There are benefits to give.

Moreover, the fact that interference by C1 or C1q was prevented suggests that automatic spectrophotometry Allows the meter to be used with microprocessor-controlled heaters and by subjectively reading the slide or tube test. Prevents errors from occurring and provides a permanent printed record. This is for administrative authorities Provide quality assurance and records for proficiency testing. The c1 component and RF are of IgG. It is extremely important to deactivate the c1 component because it binds to the same site. You should understand that. This is because RF must bind to form aggregated 1gG. To reduce the possibility of false negative results caused by 01 binding to an inadvertent site, It is to be removed. This possibility reduces the sensitivity of the test.

If quantitative test data is required, sequentially analyze samples that have been treated qualitatively. Dilute. A series of standard curves in the photometer are prepared with known amounts of purified RF or C1q present. Do not use standardized or equine factor serum and coated latex particles It can be prepared by A test sample is used to determine the amount of RF present in the sample. The curve for the price is compared to the standard curve.

gamma globules when body fluids are heated for 30 minutes at 59-62 °C in the presence of these compounds. Phosphorus aggregation was prevented. Adding a small effective amount of R-5H compound at the same time denatured both C1q and RF, thus inactivating both C1q and RF.

Deactivates RF and C1q in serum and other body fluids (cerebrospinal fluid, pleural fluid, synovial fluid) The following technologies are recommended for this purpose.

Body fluids were diluted with low ionic strength glycine buffer 0.02μ (ionic strength) pH 8.2 at 1/2 Dilute to 10. Dissolve 0.1M EDTA-Tetra Na in 0.25ml serum diluent. 10 μm to 20 μl of solution and 2 μl of freshly prepared 5M mercaptoethanol solution. Immediately after the addition of 1 ml, the body fluid is heated at 60° C. for 30 minutes.

These methods are limited to testing antigens in body fluids. antibody test is not suggested. Another limitation of this method is that the antigen to be detected and quantified is SS-bound. This means that it must not contain. The viral capsular antigen is mercaptoe. In some cases that are sensitive to the denaturing effects of tanol it is tested should not do.

By this method, the effects of C1q and RF can be reduced by dissociating the AgAg complex. If the antigen is captured by AgAg1 aggregates, it can also be released. Ru.

Antibodies can be denatured to release exposed antigen.

This method is suitable for the detection of all bacteria, viruses, and parasites that do not contain SS bonds. It can be used for testing.

Preferred compounds for use in the practice of this invention are neutral salts of the Hofmeister permutation, organic acids, Contains salts or diamino compounds. If any of these compounds have a pH of 8.2, Concentration 4 (added to 1/8 diluted body fluid sample in 1mM to 200mM glycine buffer) , the sample is heated at a temperature of 59-62°C for a sufficient period of time, e.g. 30 minutes, to remove body fluids. C1 or C1q can be inactivated without affecting the reactivity of the antigen or antibody in the If it can be used, it can be used in implementing the present invention.

To deactivate RF, use mercaptoethanol, cystine, dithioerythritide. Contains ruthioglycolic acid, thioalcohol, lower alkyl (C) mercaptan, etc. It is necessary to use a specific compound of formula R-5H.

Other suitable compounds that deactivate RF using the methods described herein may also be used. The amount of R-SH compound produced is not critical and is preferably between 25mM and 50mM. Preferably about 30mM (in final dilution) can be used. The sample is approx. Heat at a temperature of 59-62° C. for a sufficient time to deactivate C1q and RF. General 30 minutes is usually effective.

Description of the preferred embodiment The following examples are presented to illustrate the invention. These define the scope of the attached claims. It should not be construed as limiting.

In Examples 1 to 12 detailed below, the following two techniques further enhance the application of the present invention. used to generate experimental data for illustration.

A, Latex photometric rate response as representative of a specific immunoassay technique.

B, Ophthalony analysis to detect antigen-antibody reactions by precipitation techniques.

Immunoprecipitation is the simplest and most direct way to demonstrate antigen-antibody reactions in the laboratory. The method can be applied to all other immunoassays.

Since the invention was developed using latex photometry, this method will first be described.

Reagents nigericin saline buffer.

Add 2.5ml of IN NaOH to 975ml of 0.1M glycine and dilute with distilled water for 10 minutes. 00ml and adjust the pH to 8.2.

Then add 10 g of sodium chloride to each 1000 ml buffer.

Glycine buffer No. 2 Dilute 100 ml of glycine saline buffer with 700 ml of distilled water, Make a 1:8 dilution of

2. Latex particles: Bang Laboratory, Indianapolis, Indiana Ceragen Diagnostinox, polystyrene latex 10% solid particles, Size: 0.777μ diameter.

3. Glycine saline buffer No. 1 human gamma globulin) P [ENTEX 0.5g% solution.

4. Glycine buffer No., latex 1096 particle size 0.7 in 110ml Prepare a latex particle suspension by adding 77μ diameter and 25μm diameter.

5. Latex human gamma globulin suspension.

Glycine saline buffer No. 110ml with 25μl of latex 10% and human Add 50 μl of gamma globulin.

6. EDTA tetrasodium 0.1M In the general method of the present invention, the test sample The serum or body fluid to be prepared is diluted 1=10 with glycine buffer No. 2.

0.1 M EDT A tetrasodium dihydrate in 0.25 ml of diluted serum Add 0 μl and incubate for 3-30 minutes at 59-64 °C in a temperature-controlled bath. . IgG-coated or uncoated latex particles are then added. In addition, the presence of flocs or agglomerations of latex particles. Screening to determine if is RF positive or if it contains C1q Describe the programming method.

Dilute the serum to a 1:10 dilution with glycine buffer NO12. The diluted sample is Place in chronolog photometer and stir continuously. This sample is not coated. Add 0.25 ml of latex particle suspension. Photometer record J1 shows a straight line. If so, it indicates that there is no detectable C1q or RF present. S-shaped curve If obtained, it indicates that the sample contains C1q or rheumatoid factor. .

If a straight line is still visible in the sample treated with latex particles after 3 minutes, the blood Add neutral salt (110 μl of 2M NaC) to the supernatant and latex mixture.

Next, if you get a flocculent curve on your photometer, this is the masked C1q or Indicates the presence of RF or a mixture of C1q and RF. A fluffy curve should not be obtained. If so, prepare aliquots of serum, buffer and 2M NaCl as described above.

Heat this mixture at 56°C for 30 minutes and add the uncoated latex. Ru. A flocculent curve should be observed, indicating the presence of C1q and RF. shows.

Even when a flocculent curve is obtained, fresh serum, buffer, and 2M NaCl Prepare aliquots and heat at 63°C for 3 minutes. and uncoated la Add Tex. When a flocculent curve is observed, it indicates the presence of RF. confirm. When the flocculent curve is not observed, it means that the serum is not sensitive to RF. It is assumed that the sample contains C1q by inference.

This example demonstrates a screening method for RF detection when RF is present in body fluids. This method also detects the presence of C1q in samples used in immunoassays. This example is used to verify that interference caused by The C1q component of the serum aggregates by heating at 63°C for 3 min in the presence of salt. This indicates that the enzyme is deactivated.

A commercial preparation of C1q-deficient human serum was tested with a latex IgG preparation in a photometer. will be tested. Glycine buffer No. 2, pH 8 for latex particle suspension When 0.25 ml of serum diluted to 1.10 with 2 was added, latex particles coagulated. No collection occurs. C], commercially available C]Q preparation by purifying 0.25 ml of q-loss serum Serum shows strong positive latex agglutination when combined with Next, the diluted serum was diluted with 2MNa. Heated at 63° C. for 3 minutes in the presence of C115 μm. Latex coated with IgG Agglutination was detected when the sample was tested at 59-64°C. When not treated for a long time, when C1 or negative serum was added, IgG was purified in different ways. It has been shown to aggregate with complement components and rheumatoid factor.

1, C1, glycine buffer No., 20 μ2 in 20.25 ml, C1 + EDT A-Na, 0.1M EDTA-Na 10 μl added 3, C1q, 10μ! 4, C1r, 10μm 5, C1s, 10μm 6.01 inhibitor, 10μm 7. Purified RF, 10μm 8. Purified RF+C110μm 9. Purified RF negative serum 25μI CI Diamedx 0.04m g/ml, C1q Siga+a O, 5 mg/ml, C1r human 250 u g 1 Calbiochea+ , C1s Ca1biochea+ 500 μg/ml, RF purified 1 mg/m l, C inhibitor 0.5/mg Ca1bloches This table shows CI (1), C1r(4), C15(5) and C1 inhibitors aggregate latex IgG particles Indicates that it will not be allowed. When C1 is treated with EDTA-Na, it resolves the C1q component. released, which causes the latex IgG particles to aggregate. Purified RF preparation (7) is latte When C1 is added to purified RF (8), it aggregates c Inhibits RF agglutination resistance of negative serum containing l.

Implementation example 3 This example shows that latex particles coated or uncoated with HgG are Indicates that it is used for presence detection.

Uncoated latex particles or latex particles coated with human gamma globulin Dlaaedlx CorpSSigmal, ab, Behrlng, Purified C1q from At1antic Lab (each concentration 0.5 mg CIQ/m l). All of these preparations are coated or coated with gamma globulin. Latex particles that were not mixed were agglomerated.

A diluted solution of C1q was prepared from C1q manufactured by Cytotech.

These dilutions use uncoated latex particles and are quantified using the techniques described above. did. The results are as follows.

0.5 1.2 0.025 0. [lO O, (11250,30 0,006250・18 0.003125 0.09 0.00+5625 0.045 0.0007B12 0.027 0.000390B 0.014 Implementation example 4 This example shows various concentrations of neutral salts for normal serum heated at 60°C for 30 minutes. Show the impact of

Add various amounts of EDTA tetrasodium solution to 0.25 ml of negative serum. and tested by the 11 light method using latex IgG.

0.1M slope slope EDTA Tetra Na Add μl, do not heat, heat at 60℃ 0 0.08 0 10 0.958 0 20 1.12 0 30 0.815 0 40 0.788 0 50 0.11i47 0 60 0.647 0 70 0.660 0 80 0.660 0 too　o,eeo　.

Normal serum heated at 60°C for 30 minutes in the presence of neutral salts (EDTA-TetraNa) showed a positive response when tested photometrically using latex IgG, whereas and shows a negative reaction.

Use EDTA Tetra Na at the same concentration as normal serum and use uncoated latex. The subsequent test after heating at 63℃±1℃ for 3 minutes was zero throw for each concentration. This example describes the testing of 100 typical donor sera by latex photometry. I am reporting on this.

heating heating heating heating heating heating 56℃ 56℃ 60℃ 63＠±1℃% 30 minutes 30 minutes 30 Minutes 3 minutes Added salt No added salt Added salt Added salt %%%%%% Non-singular 20 100 100 No agglomeration 80 100 100 *Contrast ″ 100 serum samples obtained from apparently healthy blood donors were first subjected to the slide method (Rheum ate+c, new chassis-H cranberry, wampolc laboratory Standard latex fixed test tube method of Singer and Plotz, ^m, J, Med21:888-892 (1956)) showed the presence of rheumatoid factor. I tested it for its existence. Tests were negative for all sera.

Each serum was diluted 1:10 with glycine buffer No. 2 and treated as follows.

1. Add salt fish without heating 2.Heat at 56℃ for 30 minutes, add 15μl of 0.1M EDT A sodium 3.Heat at 60℃ for 30 minutes, add salt fish 4. Heat at 60℃ for 30 minutes, add 15μl of 0.1M EDTA-1-IJ'7 5.Heat at 63℃±1℃, add 15μm of 0.1M EDTA sodium The table shows that with this method, 80% of unheated serum is negative for C1q; , when heated at 56℃ for 30 minutes with or without addition of salt, serum scarcity c1q We show that it is true. From normal serum that had previously aggregated latex particles at 56°C. C1q of 60 °C for 30 minutes or 63 °C ± 1 °C, only when heated in the presence of salt. It was deactivated in 3 minutes.

Serum should not be processed at temperatures of 63°C ± 1°C without added salts. because, The sample contains high levels of gamma globulin, i.e. greater than 18 mg/ml. This is because high levels of gamma globulin can cause aggregation. .

Example of implementation The example shows the effect of salt and heat on RF-containing serum.

Rheumatoid serum 5o was diluted 1:10 with glycine buffer No. 1, without heating. , 56℃, heating for 30 minutes, addition of EDTA-Na 15μ+, and 60"C1 Tested by heating for 30 minutes and adding EDTA-Na. These sera were tested using the slide method. and latex IgG tube method were all positive.

50% of these serums (diluted to 1.1o with buffer) were latex IgG71 It was negative by FI light method. This suppression is due to inhibition of RF by the 01 component of complement. It was something. With the use of heat and salt, the c1 complement component is dissociated and the RF is released. Reacts with latex IgG. Add 15 μl of 0.1M EDTA-Na When heated at 60℃ for 30 minutes, C1 component 1;! C1q, C1r SC1s l: Dissociates and can prevent aggregation of clq with latex particles. Result is It was as follows.

Without heating Heating 56" Heating 60" Yang and Yin for RF Negative Positive Positive RF desire for IgG-coated latex particles at 63°C ± 1°C for 3 minutes. aggregation of C1q in latex IgG particles as it may reduce To prevent this, a temperature of 60°C for 30 minutes should be used.

Implementation example 7 The present invention also provides an immune system in which it is desirable to inactivate C1 or C1q in a body fluid sample. It includes a novel device that automatically performs the epidemic assay.

This device can be used for:

1. Screening method to confirm that C1q is inactivated 2. Screening method for detection of C1q in body fluids 3. Presence of RF in body fluid samples Screening method to confirm the presence of 4. Method to demonstrate the absence of RF in body fluids after treatment with Mercabutoecool 5. Screening method for the detection of RF 6. Screening of RF titer in body fluids Method 7 for quantifying RF (IgM) in body fluids 8, 01 and C from body fluid samples Bacteria, molds, parasites, and Ligadia using latex flocculation in a device that removes 1q. , how to detect viral diseases FIG. 3 is a schematic diagram of the device of the invention. The basic configuration of the present invention is an irradiation device comprising a light source 1. Unit, filter or prism 2, sample cell for holding samples of body fluids 3. An electronically controlled device adapted to hold the test sample tube in the vicinity of cell 4. It includes a variable electric heater block 4. Electronic control of heater block 4 3 to a temperature of approximately 59-64°C for a period of 3 to 30 minutes or more. Provide heating temperature. The sample cell 3 has several associated reagent supply lines 6. 7.8, these are equipped with automatic metering valves, and these valves are electronically controlled to required amount of dispersed latex particles, chemical solution, and/or antigen or antibody. solution and/or mercaptoethanol solution. As shown in Figure 3, Line 6 is provided to supply dispersed latex particles, line 7 is 2M Na Line 8 is provided to supply a neutral salt solution such as C1, and line 8 is provided to remove RF from the body fluid sample. Supplying mercaptoethanol solution for immunoassays where it is desired to remove It is set up for the purpose of Reference cell 3A with light source IA serves as a control test for compensation. It is set up to hold fees. Heater 4A and lines 6A, 17A and 8A are It is provided for cases where row control is desired. Photocell 12.14 is It is arranged to sense the light transmitted through cells 3 and 3A.

Amplifier 16 is used to amplify the signals from photocells 12-14. The signal is CRT which is sent to the interface 18 and processed by the computer and is not shown in FIG. and/or displayed on a printer. Cells 3 and 3A are preferably of known Equipped with a magnetic stirrer, which keeps the components in the test cell in suspension used for.

This novel device of the invention operates as follows. Serum (glycine buffer No. 2 analyte (diluted 1:10 with When testing for ans, place the analyte in cell 3 and add 1 part of KCI 2M aqueous solution. 0 μl is weighed into cell 3 via line 6. Operate heater block 4 The temperature of the cell was raised to 63° C. and heated for 3 minutes to inactivate C1 and C1q.

After that, the dispersion coated with cryptococcus neoror*ans antibody Weigh 15 μl of latex particles into cell 3 via line 6 while magnetically stirring. Rotate the stirrer at 1000 rpm. The change in absorbance vs. being done in cell 3A Compare with the light and check whether flocculent lumps have formed. Detection of flocculent lumps using Cry This is read as a positive result indicating the presence of Ptococcus neoloraans antigen. I can see it.

The apparatus of the present invention is suitable for performing optical agglutination tests.

The basic configuration of a known photometer such as the log model 570VS may be provided. this The device also includes a variable heater block means and an associated timer means and sample as described above. It may also be modified to include drug delivery means. Adaptations that may be modified to embody the present invention. A suitable photometer is U.S. Pat. No. 4,118,192, which is incorporated herein by reference. Eiken Cheslcal Co. A commercially available photometer such as Karu LA-2000 may be used.

Implementation example 8 This experiment shows the effect of temperature on the Clq molecule, which is present in purified form. It is thermally unstable at 56℃ for 30 minutes, but when combined with C1q, it is thermally stable at that temperature. Thermostable experimental example of anti-C1 when heated for 10 to 30 minutes q Anti-C1n 1* Normal serum, 25μ+ ppt ppt2 zero without heating, normal serum, 56℃, Heat for 30 minutes, ppt ppt2M NaC115μ! Added, 25μm3 normal blood Clear, heated at 63℃ for 3 minutes, no ppt, very weak 2M NaC, 115μI added, 25μI ppLHC1q15mg/ml foμl ppt 1) llt 54 ctq 5B℃, heated for 30 minutes lOμ+ No ppt No ppt 8* C1n IOμl No ppt ppt7IICIQ 1OuI +C1n IOμl ppt ppt8* ctq IOμl + CIn 10μI, ppl ppt56℃, 10 minutes heating 9 * C1q 1Oμl + C1n 1OuI, ppt ppt56℃, 20 heating for minutes 10* CIQ 1 OuI + CIn loμI, ppt ppt56℃, 3 Heat for 0 minutes 11* C1q lOu I +ctn toμI, no ppt ppt* Comparison C1r inhibitor was 10 International Units (Calbloches).

In the present invention shown in Experimental Example No. 3, in the presence of neutral salts, C1q in serum was heated at 63°C for 3 minutes. It is thermally unstable at a temperature of 56℃ for 30 minutes in the presence of a neutral salt. It shows that it is not fixed.

(CA L A S ) Meridian Diagnostic Inc. , tested by latex agglutination test. Latex grains in these kits The offspring are coated with anti-cryptococcal globulin. One of the sera is RF negative and other sera were tested in the Singer and Block latex gamma globulin test. It had a titer of 1.60 for RF. Techniques used in this kit should remove the presence of RF, a non-specific agglutination factor, from the fluid being tested. Ru.

Pronase solution (Meridian Catalog N o, 140050) Add 200 It I and add 5 ml of serum Pronase reagent. The solution was incubated at 6° C. for 15 minutes, and some solutions were boiled for 5 minutes to complete the enzymatic digestion. child These serums use latex particles coated with anti-cryptococcal globulin. It showed aggregation titers of 1:256 and 1:512 by the tube method.

The above technique is compared to the technique of the present invention, including the removal of C1q and RF from the sample. It was done. It also includes two controls. One control is 1 mg C1q (purified C 1q) 25 μl, which is added to 0.25 μl of glycine buffer. Second The control is a 1=50 dilution of RF serum obtained from a patient with active rheumatoid arthritis.

0.25 ml of a 1:10 dilution of serum and two controls with EDTA tetranate. Add 10 μl of a 0.1M mercaptoethanol solution and 2 μm of a 5M mercaptoethanol solution. Ru. Heat serum and controls at 60°C for 30 minutes.

Serum was serially diluted with 0, 1M glycine buffer pH 8, 2 and added to a crypt ml tube. Antibody (Meridian Diagnostic Inc. latex kit) ) was tested. Both serum and control were tested with the same reagents. Both serums are Cohesive forces 1i1fl:256 and 1:512 were shown. This titer is Meri Similar to that obtained by Dian DlagnO8tles' original method. It was something that All controls were negative, and by using the technology of the present invention, We showed that C1q and RF were removed from the serum of this patient.

It was identified as serotype 8. The patient's serum was WeI co■e Diagn.

Latex grains tested positive with 5tjc's ve+cogen antigen detection kit showed the child. In this kit, the latex particles are S, pneu+goni coated with ae rabbit antibody.

Before testing, add 1 volume of 0.1M EDTA sodium (pH 7,4) to the serum sample. It was heated for 5 minutes in rear seat water to which 3 volumes were added. Cool the sample at room temperature and It was clarified by centrifugation before being tested. Use the supernatant from the clot and both blood It was found that the serum was positive.

Two sera obtained from patients with pneumonia were subjected to Singer and Brotzno latex agglutination tests. Detected by As, J, Med, 21 + 80.1956 It was also positive for the presence of RF.

Patient serum was diluted 1.10 with glycine buffer diluted 1:8 with distilled water.

EDT for 0.25 ml of diluted serum and for two 0.25 ml controls. Add AO, 10 μl of 1M solution and 2 lambda of 5M mercaptoethanol solution The serum was then heated at 60°C for 30 minutes. Control was RF-containing blood obtained from serum of RA patients. 1150 dilution of the supernatant and 1 mg of C1q purified product (S!gga Co,) It was a glycine buffer solution (pH 8.2) containing

After treatment, serum and control were added to VelcogenSWelcome Diagnostic A kit was used to test for the presence of S, pnOU+wOnlae.

Two sera were positive for S, pneua+onlae and two controls. The test result was negative.

This example shows that C1,q and RF are removed from serum containing S, pneumoniae. When tested, the serum is positive for S. pneumoniae antigen.

The invention includes kits for performing immunoassays.

Kits of the invention may include any known kit components and further include: (a) When added to a sample of body fluid, the mixture of body fluid and compound has a temperature of 59 to 62 °C. Cl and C1q can be lost to a degree without affecting antigen or antibody reactivity in body fluids. Deactivation of 01 or C1q in body fluids when heated for a sufficient time to activate and (b) a solution containing C1q.

Kits of the invention may include any known kit components and further include: (a) When added to a sample of body fluid, the mixture of body fluid and compound has a temperature of 59 to 64 °C. C1 and C1q without affecting antigen or antibody reactivity in body fluids. Deactivates 01 or C1q in body fluids when heated for a sufficient time to deactivate it. and (b) a solution containing C1q.

Examples of these kits are:

1. Reagent for removing interference of C1q complement component of C1 in fluid or serum (c) Antigen control (d) Antibody control (e) Neutral salt (preferably 2M NaCl), and (r) for antigen-antibody reaction buffer solution 2. For in vitro diagnosis, the kit contains reagents used in each immunoassay. include.

(a) Latex antigen or antibody emulsion (b) Latex antigen control (C) Latex rheumatoid factor control (d) Latex antibody control, and (e) Neutral salt (preferably 2M NaC1)3, RF identification and C1q screen -ning technology (a) Latex particle solution 0.70-0.90 micron diameter (b ) HgG 2% (c) Glycine buffer pH 8, 2 μy o, t (d) RF purified protein (e) RF standard serum (r) Ophthalony board (selective) (g) Purified C1q (h) Neutral salt 2M NaCl or 2M KCI, and (i) 5M mercaptoethanol aqueous solution 4, for C1q interference removal and RF drying For removing debris (a) 0.1M glycine buffer saline pH 8.2 (b) O, 1M EDTA Na solution (c) 5M mercaptoethanol concentrated solution (d) RF Serum A 1:50 dilution of human serum (tested for HIV) as a control) or a purified RF preparation. (e) C1q complement component diluted solution (1 mg C1q purified tongue self-prepared as a control for CIQ) Add 25 μl of the substance to 0.25 ml of glycine saline buffer (pH 8,2). ), and (') Buffer containing 0.1% sodium azide as a preservative Zv≧J Figure 2 FIG.3 o O's Q lie Sio　c:s　o　o　.

) χ admiration Procedural amendment form)

Claims (1)

[Claims] Claim 1 Cl and Clq from body fluids to remove Cl and Clq interference in immunoassays. A method for removing the influence of (a) a step of adding an effective amount of a compound to a diluted sample of body fluid, the step of adding an effective amount of a compound to a diluted sample of a body fluid; the mixture of body fluid and compound is heated to a temperature of 59-64°C for a sufficient period of time. in an immunoassay without affecting the reactivity of the antigen or antibody in the body fluid. A step capable of preventing interference of Cl or Clq in the body fluid. (b) bringing the diluted sample containing the compound and the body fluid to a temperature of about 59°C to about 64°C; Heating for a sufficient period of time to remove Cl or Clq interference in immunoassays process. Claim 2 An improvement in an immunoassay kit comprising: (a) a compound according to claim 1; (b) Solution containing Clq component Claim 3 Photometric device for aggregation detection, including the following configurations: (a) Light source (b) Sample cell holding a sample of body fluid (c) a controlled variable heater block provided near the cell; (d) reagent supply means and for metering a measured amount of reagent into said sample cell; valve means (e) photovoltaic means for detecting light transmitted through the sample cell; (f) amplifier means for amplifying the signal from the photovoltaic means; (g) processing the signal and a microprocessor controlling the valve means; and (h) Display means for recording the detection results of aggregation. Claim 4 A method for quantifying purified Clq, comprising the following steps (a) Sequentially measuring samples of Clq diluting step (b) contacting the sequentially diluted sample from step (a) with latex particles; Touching process (c) Determining the amount of floc in each diluted sample in the photometer (d) Determining the maximum slope of each sample (e) Determining the maximum slope of each sample as the standard The process of comparing with the value.