JPH04329357A - Immunological measuring method - Google Patents

Abstract

PURPOSE:To prevent a disturbance caused by a nonspecific reaction by adding at least one kind of urea, thiocyanate and hydrochloric acid guanidine into a reagent at the optimum concentration in the immune nephelometry. CONSTITUTION:A sample is added to a reagent, the turbidity of an immune composite body is measured and compared with the turbidity measured in advance to obtain antigenic concentration in the immune nephelometry, and at least one kind of urea, thiocyanate and hydrochloric acid guanidine is added into the reagent at the optimum concentration to prevent a nonspecific reaction. An antigen measurable by the immune nephelometry is used, and sodium thiocyanate is used for the thiocyanate, for example. The optimum concentration differs according to the type of antigens and the type of chemicals, in the measuring system of CRP, for example, the optimum concentration is set to 0.25-0.75mol/L for urea, 0.09-0.31mol/L for sodium thiocyanate, 0.09-0.48mol/L for hydrochloric acid guanidine, and a nonspecific reaction can be suppressed by adding them.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the measurement of antigenic substances in living organisms by immunological measurement methods in order to assist in clinical diagnosis.

0002

BACKGROUND OF THE INVENTION The need to measure antigenic substances in biological samples in clinical tests is increasing year by year. Nowadays, even small facilities cannot process this number of samples without using automatic analyzers that can measure a large number of samples in a short time.

Conventionally, immunoassay methods such as SRID method, agglutination reaction method, immunoturbidimetry, immunonephelometry, enzyme immunoassay, and RIA have been widely used as clinical testing methods. Among these methods, the latex agglutination method and the immunoturbidimetry are methods that can be easily used with a general-purpose automatic analyzer equipped with a colorimeter in the detection system. The latex agglutination method has become more popular in recent years because its measurement sensitivity is higher than that of the immunoturbidimetric method. However, it is well known that the latex agglutination reaction method requires highly purified antibodies and latex with a uniform particle size with little spontaneous aggregation, resulting in high reagent costs. In this respect, immunoturbidimetry can use antibodies with a low degree of purification and does not require any other particularly expensive materials, so it is easier to lower the cost of reagents than the former method. Therefore, when the concentration of antigenic substances contained in a biological sample is high, it is economically more advantageous to use the immunoturbidimetric method than the latex agglutination method.

Although immunoturbidimetry is inexpensive, it is a method that measures optical transmittance (absorbance) by utilizing the diffuse reflection of light by immune complex particles generated by antigen-antibody reactions. , sample turbidity due to lipids, rheumatoid factor (RF), complement, and other reactions whose causes are still unknown, may interfere with slight turbidity. Lipid turbidity is solved by adding a surfactant to the reagent, for example, in JP-A-64-15656. However, the current situation is that effective measures have not been taken or are insufficient for turbidity caused by RF, complement, or other unknown causes.

[0005] Japanese Patent Publication No. 62-29746 reports that non-specific reactions can be prevented by adding guanidine hydrochloride, iodide salt or thiocyanate to an agglutination reaction reagent using red blood cells as a carrier. Also, Tokuhei 2-409
No. 83 successfully prevents nonspecific reactions such as latex agglutination reactions by adding guanidine hydrochloride, thiocyanate, or a chaotropic agent to particles coated with proteins as reagents. However, these patent application publications do not discuss immunoturbidimetry, which involves adding a sample containing an antigen to a reagent in which antibodies have been dissolved and measuring the turbidity of the resulting immune complex to determine the antigen concentration in the sample. It has not been done.

[0006]

[Problems to be Solved by the Invention] As explained in the previous section, the problem to be solved by the present invention is to add a sample containing an antigen to a reagent in which an antibody has been dissolved, and to determine the turbidity of the immune complex formed after a certain period of time. In immunoturbidimetry, which determines the antigen concentration in a sample by measuring turbidity and comparing it with the turbidity previously measured in a sample with a known concentration, it is used to determine the antigen concentration in a sample. The purpose is to prevent non-specific interference.

[0007] In immunoassays, how to prevent non-specific reactions is an important issue. If a non-specific reactive substance such as rheumatoid factor or complement can be identified, the animal species of the antibody may be changed or a portion of the antibody such as F(ab')2 fragment (Japanese Patent Publication No. 63-63863) may be used. methods are being tried. However, other reactions whose causes are unknown are often not resolved by the above methods. In order to prevent such reactions whose causes are unknown, methods of increasing the concentration of salts in reagents have been well studied. Commonly used salts include sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate. However, although this method can reduce nonspecific reactions by increasing the concentration of salts above a physiological concentration, it is also known to interfere with antigen-antibody reactions and significantly reduce measurement sensitivity.

[0008] The agglutination reaction discussed in Japanese Patent Publication No. 62-29746 and Japanese Patent Publication No. 2-40983 utilizes the reaction and binding between antibodies immobilized on a carrier and antigens in a sample for measurement. . Although the immunoturbidimetry and the agglutination reaction method using a carrier, which are the subject of the present invention, are essentially the same immunological reaction (edited by Ohara, Science of Immunology 1, published by Buneido Shoten, 1977), However, in an agglutination reaction method using a carrier, particularly in a passive agglutination reaction method using an antibody bound to a carrier as a reagent, the antibody exists as a solid. For this reason, the reagent blind values are high and it is difficult to improve reproducibility, and the reagent nozzles of automatic analyzers commonly used in clinical tests are clogged (Japanese Patent Laid-Open No. 15656/1983).
In particular, it has been experienced that the latex carrier melts into the styrene cells of the reaction tank and destroys the cells. Furthermore, while the agglutination reaction using a carrier is a reaction between a solid antibody and an antigen in a solution, the immunonephelometry, which is the subject of the present invention, involves antibody molecules dissolved in a solution. Therefore, it is not considered that the technique achieved by the agglutination method using a carrier can be directly applied to the immunoturbidimetry that is the subject of the present invention. Even if one comes up with the idea of adding urea, guanidine hydrochloride, or thiocyanate (hereinafter abbreviated as these drugs) to an agglutination reaction using a carrier, no one has thought of adding these drugs to immunoturbidimetry. There wasn't. This is because these drugs are known to act as a type of chaotropic ion that interferes with the formation of molecular associations, including hydrogen bonds and ionic bonds.

[0009] In the antigen-antibody reaction or the reaction in which immune complexes gather to form a precipitate, the associative forces of molecules including hydrogen bonds play a large role. Therefore, it is well known among those skilled in the art that antigen-antibody reactions and the formation of precipitates are hindered in solutions containing these drugs. Taking advantage of this property, affinity chromatography traps antigens and antibodies in a sample with antibodies or antigens immobilized on a suitable carrier, and after washing unbound components, 3 mol/L sodium thiocyanate or 8 mol/L urea solution is used. We routinely use methods to elute antigens and antibodies through

[0010] Generally, these drugs are recognized as substances that interfere with immunological reactions. However, as a result of intensive research, the present inventor found that by limiting the concentration of these drugs and using them at the optimum concentration, non-specific reactions could be prevented without substantially reducing the measurement sensitivity of immunonephelometry. Completed.

[0011]

[Means for Solving the Problems] In order to solve the above problems, the present invention adds a sample containing an antigen to a reagent in which an antibody is dissolved, and measures the turbidity of the immune complex formed after a certain period of time.
In immunoturbidimetry, which determines the antigen concentration in a sample by comparing it with the turbidity previously measured with a sample of known concentration, at least one of urea, thiocyanate, and guanidine hydrochloride is added to the reagent to induce a nonspecific reaction. Provided is an immunoassay method characterized in that the compound is added at an optimal concentration for preventing the disease.

In the present invention, samples include human or animal blood, serum, plasma, urine, spinal fluid, saliva, sweat, ascites, amniotic fluid, and extracts of human or animal cells or organs.

[0013] Any antigen can be used as long as it can be measured by immunoturbidimetry; examples include immunoglobulin (Ig) G, IgM, IgA, complement C3, complement C4, Examples include C-reactive protein (CRP), transferrin, α1-acid glycoprotein, and haptoglobulin.

[0014] The antibody may be any antibody specific to the antigen contained in the sample.

[0015] The fixed period of time may be any period of time during which the turbidity of an immune complex produced by adding a sample containing an antigen to a reagent in which an antibody has been dissolved is within a measurable range. Further, the turbidity may be measured once or multiple times during the immune reaction.

As the thiocyanate, thiocyanates such as sodium thiocyanate, potassium thiocyanate, calcium thiocyanate, and ammonium thiocyanate can be used.

The optimum concentration for preventing non-specific reactions will be explained below. Table 1 shows the basic reagents for immunoturbidimetry shown in Example 1 below, plus an optimal amount of anti-human CRP rabbit antibody, A for urea, B for sodium thiocyanate (Na thiocyanate), and C for guanidine hydrochloride. This is data obtained by measuring the CRP value in a human serum sample by immunoturbidimetry using the same reagent.

[0018]

[Table 1]

[0019] For A, the value measured using the SRID method, which is said to be almost unaffected by non-specific reactions, was 9 ± 3 m.
g/L, but in immunoturbidimetry without adding urea, it is 12
mg/L, and an average error of 3 mg/L was confirmed. In B, the measured value by the SRID method was 19±3 mg/L, whereas it was measured to be 24 mg/L without sodium thiocyanate, confirming an average error of 5 mg/L. SR in C
The value measured by the ID method was 39 ± 4 mg/L, but without the addition of guanidine hydrochloride it was 57 mg/L, with an average of 1
A correct error of 8 mg/L was confirmed.

Table 2 shows data obtained when the C3 and C4 values in serum samples were measured by replacing the antibody in the CRP measurement reagent with an antibody against complement component C3 or C4.

[0021]

[Table 2]

In D, urea was added as an additive, and in E, sodium thiocyanate was added. In the SRID method, C3 is 94
0±30 mg/L, and C4 was determined to be 270±30 mg/L. However, in immunoturbidimetry without the addition of these drugs, the concentrations were 980-1000 mg/L and 300-310 mg/L, respectively.
It can be seen that the value is slightly high at mg/L. However, it can be seen that in any measurement system, when a large amount of these drugs is added, the measured value decreases.

[0023] As shown from the above data, the optimal concentrations of these drugs vary depending on the type of antigen to be measured and the type of these drugs. The optimum amount of urea in the CRP measurement system is approximately 0.25 to 0.75 mol/L, preferably 0.3 to 0.58 mol/L. Also, sodium thiocyanate is approximately 0.09 to 0.
31 mol/L, preferably 0.12 to 0.22 mo
It is within the range of 1/L. Furthermore, guanidine hydrochloride is 0.09
to 0.48 mol/L, preferably 0.10 to 0.4
It is 0 mol/L. In the C3 and C4 measurement system,
The optimal amount of urea is approximately 0.05 to 0.33 mol/
L, preferably 0.08 to 0.25 mol/L. In addition, for sodium thiocyanate, it is approximately 0.04
to 0.09 mol/L, preferably 0.06 mol/L
It is near L. However, the optimum concentration of the drug of the present invention is not particularly limited by the values shown here, and in fact, the optimum concentration is determined depending on the type of drug, the antigen substance to be measured, and the type of antibody against it. It is something that Furthermore, the effects will be explained using examples.

[0024]

[Examples] The present invention will be explained below according to Examples.
The present invention is not limited in any way by these Examples.

Example 1 A solution having the following composition was prepared as a basic reagent for immunoturbidimetry. Basic reagent for immunoturbidimetry polyethylene glycol (PEG) #
6000 3.5% pH8
．． 0 Tris-HCl buffer
10mmol/L Sodium chloride
0.9%

A reagent in which urea was added to the basic reagent for immunoturbidimetry at a concentration of 0.42 mol/L and a reagent without urea were used as the first reagent, and then anti-human with Becker units of 4 mg/mL was added. A second reagent solution was prepared by adding CRP rabbit antibody solution to the basic reagent for immunoturbidimetry at a concentration of 6 v/v%. Add 14 samples of serum or CRP standard solution to 260 μL of the first test solution.
After mixing μL, the wavelength was 340 nm after 4 minutes and 30 seconds at 37℃.
The absorbance A1 was measured at 340 nm, and the absorbance A2 at 340 nm was measured again 5 minutes after adding 130 μL of the second test solution at 37°C. The CRP concentration of the sample serum is (A2-0.678A
1) was converted from the turbidity of the standard solution as the true turbidity. A general-purpose automatic analyzer was used for measurement operations. SRI using CRP concentration of 5 mg/L as the cut-off value for 55 patient serum samples.
The measurement sensitivity (detection rate of true positive) and specificity (detection rate of true negative) of this immunoturbidimetric method were calculated using the measured value of method D as the true value. The results are shown in Table 3.

[0027]

[Table 3]

[0028] The measurement sensitivity of immunoturbidimetry is the same at 100% with and without the addition of urea, but the specificity increases by 11.4 points to 82.9% and 94.3% with the addition of urea. did.

Example 2 0.50 mo of urea was added to the basic reagent for immunoturbidimetry in Example 1.
The reagent added to l/L and the reagent without urea were used as the first reagent, and then 4 mg of Becker unit was added.
A second reagent solution was prepared by adding 12 v/v% of anti-human CRP rabbit antibody solution to the basic reagent for immunoturbidimetry. Add sample serum or CRP to 260 μL of the first test solution.
After mixing 12 μL of the standard solution, absorbance A1 was measured at a wavelength of 340 nm after 4 minutes and 30 seconds at 37° C., and absorbance A2 at 340 nm was measured again 5 minutes after adding 65 μL of the second test solution at 37° C. The CRP concentration of the sample serum is (A2-0.
807A1) as the true turbidity, which was calculated from the turbidity of the standard solution. A general-purpose automatic analyzer was used for measurement operations. The measurement sensitivity and specificity of this immune nephelometric method were calculated for 55 patients' serum samples, with a CRP concentration of 5 mg/L as the cutoff value and the measured value of the SRID method as the true value. The results are shown in Table 4.

[0030]

[Table 4]

[0031] The measurement sensitivity of immunoturbidimetry is the same at 100% with and without the addition of urea, but the specificity increases by 8.6 points with the addition of urea to 85.7% and 94.3%. did.

[0032]

[Effect of the invention] By adding a sample containing an antigen to a reagent containing dissolved antibodies, measuring the turbidity of the immune complex formed after a certain period of time, and comparing it with the turbidity previously measured with a sample of known concentration. In immunoturbidimetry to determine the antigen concentration in a sample, adding at least one of urea, thiocyanate, and guanidine hydrochloride to the reagent at an optimal concentration suppresses nonspecific reactions and improves measurement sensitivity ( This made it possible to significantly increase the specificity (the degree of detection of true negatives) without decreasing the degree of detection of true positives.

Claims (1)

[Claims] Claim 1: By adding a sample containing an antigen to a reagent containing dissolved antibodies, measuring the turbidity of the immune complex formed after a certain period of time, and comparing it with the turbidity previously measured with a sample of known concentration. In immunoturbidimetry for determining antigen concentration in a sample, at least one of urea, thiocyanate, and guanidine hydrochloride is added to the reagent at an optimal concentration to prevent nonspecific reactions. Immunological measurement method.