JPH03197866A - Reagent for immune measurement having ability to make simultaneous decision of multiple items and immune measuring method for simultaneous decision of multiple items - Google Patents

Abstract

PURPOSE:To make simultaneous decision of multiple items by using the reagent contg. prescribed ratios of a non-radioactive material labeled antibody and non-radioactive material labeled antigen which specifically conjugate with respective measuring objects and, if necessary, the same kind of an unlabeled antibody and unlabeled antigen. CONSTITUTION:The measurement is made by the screening reagent or measuring method contg. either one or two or more of both of the non-radioactive material labeled antibody and non-radioactive material labeled antigen which specifically conjugate with the respective measuring objects and, if necessary, either or one or more of both of the unlabeled antibody non-radioactive material labeled antibody of the same kind of the non-radioactive material labeled antibody and the unlabeled antigen of the same kind. A person with which abnormality is not found is decided to be normal in all of the measurement items and is no longer measured. Only the person with which the abnormal value is found is subjected to the measurement of each of the respective items and specification is made as to which of items is abnormal. The number of processing of the specimens is drastically decreased in this way without impairing the purposes to discover the abnormality.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to an immunoassay reagent capable of simultaneous multi-item determination and a multi-item simultaneous determination immunoassay method. More specifically, two or more types of trace components contained in biological components such as blood and urine are measured at the same time, and then a judgment operation is performed, and if there is an abnormality in any of the trace components (-items), it can be detected. The present invention relates to an immunoassay reagent capable of simultaneous multi-item determination and an immunoassay method for simultaneous multi-item determination.

<Prior art> Some trace components contained in blood, urine, etc. are detected or increase in amount during specific diseases, and measuring their amounts is very meaningful for disease diagnosis. It is recognized that

However, there is not a complete one-to-one correspondence between trace components that serve as disease markers and the disease; several types of markers may be present in one disease, especially in a group of diseases such as infectious diseases and tumors. The reality is that it is possible to suspect a disease group based on changes in any marker. For these reasons, measuring a few markers is important in the field of clinical testing to ensure that diseases are not overlooked. However, measuring many markers separately is difficult in daily life. Testing, especially when detecting rare tumors from a large number of specimens in mass screenings, places an enormous burden in terms of labor and cost.Therefore, a simple micro-testing method is proposed as a solution. It is desired to develop immunoassay reagents and immunoassay methods capable of simultaneous multi-item determination in accordance with the above.

Now, with the development of radioimmunoassay, the sensitivity of methods for quantifying biological components using immunoassays has increased dramatically, making it possible to quantify trace components that were previously unmeasurable. In this method, antibodies are immobilized on the surface of a solid phase such as a plastic tube, and a sample containing the analyte to be measured and a radioisotope-labeled antigen are placed in the tube, or the part of the sample that does not react is discarded. After that, add a radioisotope-labeled antigen and react for a certain period of time, then wash the tube and count the amount of radioisotope immobilized on the tube. Since there is a correlation between the amounts, the amount of antigen can be measured.However, since this method uses radioisotopes, there are many restrictions on its disposal, and operations such as separating and washing labeled substances are complicated. There is a drawback.

Therefore, various labels have been devised to replace radioisotopes. One is an enzyme such as peroxidase, galactosidase, or alkaline phosphatase, and the other is a fluorescent substance such as fluorescein isothiocyanate or rhodamine, or a luminescent substance such as luminol or acridinium. Measurement systems similar to radioimmunoassays have been constructed by labeling such substances and are in actual use.

Furthermore, a homogeneous immunoassay method (homogeneous immunoassay method) has also been developed that utilizes the change in the signal of a labeled substance due to an antigen-antibody reaction. For example, as shown in Japanese Patent Publication No. 53-27763, there is a difference between the enzyme activity of an enzyme-labeled antigen in which a low-molecular-weight antigen is bound to an enzyme, and the enzyme activity when an antibody binds to the enzyme-labeled antigen. A method that takes advantage of the fact that there is
This method utilizes the fact that when an enzyme-labeled antibody causes an agglutination reaction with an antigen, changes in enzyme activity can be detected by selecting an appropriate substrate concentration, as disclosed in Japanese Patent No. 22459. This homogeneous immunoassay can be measured simply by adding a reagent to the sample and reacting.
It has the advantage of not requiring complicated operations such as separation and washing of labeled substances.

On the other hand, another type of immunoassay is immunoturbidimetry. In this method, an antibody against the antigen is added to a sample containing the antigen to be measured, and the amount of antigen is determined by measuring the change in light transmittance of the reaction solution due to the resulting aggregate using an absorbance meter.

Although this method also has a simple measurement operation, it cannot be used to measure trace amounts of antigens because it lacks sensitivity. However, recently, the sensitivity has been dramatically increased by using reagents in which antibodies are bound to latex particles, and it has come to be used for measuring trace amounts of antigens. Many trace markers that are considered useful for disease diagnosis have been measured using these various measurement methods.

In principle, all of the above methods measure one type of substance in one measurement; however, in order to effectively utilize a small amount of sample and save labor in measurement operations, some methods include simultaneous measurement of multiple items. Attempts are also being made to develop For example, JP-A-54-1190
As shown in No. 26, enzyme immunoassay is used to label antibodies for two or more substances to be measured with different enzymes, and the antigen-antibody reaction part is performed simultaneously by mixing reagents to determine the enzyme activity. At the time of measurement, the concentration of the target substance to be measured can be determined separately by separating the solid phase and adding each enzyme substrate, or as shown in Japanese Patent Application Laid-open No. 56-2558, one antibody has a radioisotope. After the antigen-antibody reaction is completed, the enzyme activity is first measured, and the amount of radioisotope is then measured to determine the amount of each of the two items. 56-7
As shown in No. 8598, antibodies of different items are immobilized on the reaction tube and beads used as solid phase, a sample is placed in the tube containing the beads, an antigen-antibody reaction is performed, and then the beads and tube are separated. This method attempts to determine the amount of each item from the amount of enzyme-labeled antibody that is separately immobilized.

All of these are attempts to perform common operations for each item at the same time, but then separate the reaction reagents or separate the signal detection methods to obtain the measured values for each item separately.

<Problems to be Solved by the Invention> Although it is true that measuring trace amounts of markers helps in diagnosis, it takes a lot of effort to measure each item separately. Particularly when detecting abnormalities among a large number of normal healthy people for mass medical examinations, the effort and cost required are extraordinary. , signal detection, etc. had to be performed separately, which was not very effective in terms of simplification.Therefore, as a means to reduce this labor, all operations were performed only once, and one item out of many items was However, we attempted to develop a screening reagent and measurement method that can detect any abnormal values.In other words, by using an adjusted mixture of measurement reagents for multiple target test items, if there is an abnormality in any of the - items, the signal will change. The goal is to develop reagents and methods that can detect the presence of abnormal items. Rather than measuring all items for all people (first, measure with this screening reagent or measurement method to detect abnormalities). For those who did not have any abnormal values, all of these items are normal, so no further measurements are taken, and only those with abnormal values need to be measured for each item to determine which item is abnormal. This makes it possible to significantly reduce the number of specimens to be processed without sacrificing the purpose of detecting abnormalities.

<Means for solving the problem> The present invention is comprised of the following claims (1) to (11).

(1) Two or more of one or both of the following (A) and (B) that specifically binds to each of the measurement targets,
An immunoassay reagent having the ability to simultaneously determine multiple items, comprising one or more of the following (C) and (D), as required.

(A) Antibody labeled with a non-radioactive substance (B) Antigen labeled with a non-radioactive substance (C) Unlabeled antibody of the same type as (A) (D) Unlabeled antigen of the same type as (B) (2) Labeled with a non-radioactive substance An immunoassay reagent capable of simultaneous determination of multiple items according to claim (1), wherein the antigen or non-radioactive substance-labeled antibody is an enzyme-labeled antigen or an enzyme-labeled antibody.

(3) An immune system capable of simultaneous determination of multiple items according to claim (1), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is a fluorescent substance-labeled antigen or a fluorescent substance-labeled antibody. Measurement reagent.

(4) Claims (1) to (3) that use non-radioactive substance-labeled antigen microparticles or non-radioactive substance-labeled antibody microparticles as the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody An immunoassay reagent with the ability to simultaneously determine multiple items.

(5) An immunoassay reagent capable of simultaneous determination of multiple items according to claim (4), which uses red blood cells, gelatin particles, or synthetic polymers such as latex as the fine particles.

(6) Two or more of the following (A), (B) or both that specifically bind to each of the measurement targets,
If necessary, apply homogeneous immunoassay to the object to be measured with one or more of the following (C) and (D), and measure from the obtained signal value. All of the target values are normal, or there are only a few (
A multi-item simultaneous determination immunoassay method characterized by detecting whether one or more of the values are abnormal values.

(A) Antibody labeled with a non-radioactive substance (B) Antigen labeled with a non-radioactive substance (C) Unlabeled antibody of the same type as (A) (D) Unlabeled antigen of the same type as (B) (7) Labeled with a non-radioactive substance The multi-item simultaneous determination immunoassay method according to claim (6), wherein the antigen or non-radioactive substance-labeled antibody is an enzyme-labeled antigen or an enzyme-labeled antibody.

(8) The multi-item simultaneous determination immunoassay method according to claim (6), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is a fluorescent substance-labeled antigen or a fluorescent substance-labeled antibody.

(9) Claims (6) to (8) that use non-radioactive substance-labeled antigen microparticles or non-radioactive substance-labeled antibody microparticles as the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody Multi-item simultaneous determination immunoassay method.

The multi-item simultaneous determination immunoassay method according to claim (9), in which the (lO) microparticles are red blood cells, gelatin particles, or synthetic polymers typified by latex.

(11) The multi-item simultaneous determination immunoassay method according to claim (6), which uses immunoturbidimetry as the homogeneous immunoassay method.

Among biological components, many substances that can serve as diagnostic markers increase in amount during disease. Each marker is provided with a cutoff value that separates normal values from abnormal values. Utilizing the aforementioned homogeneous immunoassay, using a mixture of measurement reagents for two or more types of items,
- A reagent that shows an abnormal value when it exceeds the cutoff value in any item can be a screening reagent. The present invention is a reagent and method that embodies such a concept.

To give a more specific example, alphafnidoprotein (AFP) is known to be elevated in primary liver cancer, and alphafnidoprotein (AFP) is considered to be one of the main markers because it is elevated in various malignant tumors. If ferritin is selected as a measurement item and a mixed measurement reagent is used to detect an increase in any of the ferritin levels, it becomes a screening method that can detect a wide range of malignant tumors. Also, carcinoembryonic antigen (C
Similar to the combination of EA) and AFP, it is a screening method that can detect malignant tumors.
V), by selecting hepatitis B virus (HBV) antigens and antibodies as markers and measuring them in combination, it can be used in a wide range of ways, such as as a screening method for post-transfusion infections.

However, the cut-off values of individual markers vary, and on the other hand, the sensitivity of one measurement method is almost constant. Therefore, in the present invention, in order to equalize the sensitivity of signals for various cut-off values with one measurement method, If necessary, use a mixture of labeled and unlabeled antibodies. In other words, the range of normal values is wide (for items with high cutoff values, add enough unlabeled antibody to the measurement reagent to absorb the measurement substance in the normal range, and use it only when the antigen enters the abnormal range). , adjust so that the signal obtained increases.Conversely, the range of normal values is narrow (for items with low cutoff values, do not add unlabeled antibody, and the signal increases even with a small increase in the amount of antigen). In this way, when using a mixture of labeled antibody reagents containing antibodies corresponding to each cutoff value, if there is an antigen above the cutoff value in any of the items, there will be no signal. Indicates high value.

The various methods described above are effective for homonidia immunoassay using this screening method, but
Homogeneous enzyme immunoassay (homogeneous enzyme immunoassay) disclosed in Japanese Patent No. 122459.

For example, if a reagent containing peroxidase-labeled anti-ferritin, unlabeled anti-ferritin, and peroxidase-labeled anti-alpha-fetoprotein is used, ferritin or alpha-fetoprotein will exceed the cut-off value. When this happens, abnormalities can be detected from the increase in coloration of the enzyme substrate. Furthermore, when using the latex agglutination method, abnormalities can be similarly detected from the decrease in transmitted light using a combination of latex-sensitized anti-ferritin, anti-ferritin, and latex-sensitized anti-alphafetoprotein. In addition, in the case of items with high abundance, immunoturbidimetry can also be used, and antibodies against each can be used to make screening reagents by combining many items with high cut-off values (and few items with low cut-off values). can.

<Example 1> (A) Preparation of horseradish peroxidase-labeled anti-large alpha fetoprotein 5 mg of horseradish peroxidase (HRP) was added to 1 mj
0.1 M sodium metaperiodate (NaIO4), which was prepared on the spot by dissolving it in distilled water from Step 2, was added to 0.2rr+j.
Add 2. After shaking for 20 minutes at room temperature, a Sephadex G-25 column (1,5x12cm column, 1mM
Desalt through acetate buffer, pH 4.2 equilibration). The obtained N a I O 4 treated HRP 2 mJ2 (4
mg) prepared in 0.3M Na HCOs.
M Add to 2 mρ of tetramethylenediamine. After shaking for 2 hours at room temperature, 4 m
g/mβ of sodium borohydride 0.5rr+
12 was added and left to stand at 4°C for 1 hour, then 20mM phosphate buffer pH 7.0 (PB
Dialyze against S). The obtained amino group-introduced HRP was applied to a Cefycryl S-200 column (2.5 x 90 cm, P
BS equilibration) to pool and concentrate the fractions that have not self-polymerized.

Next, 0.1 mJ2 of 22mM N-hydroxysuccinimide ester of N(4-carboxycyclohexylmethyl)maleimide prepared with dioxane was added to the above amino group-introduced HRP 1mJ2 (3 mg) at 30°C.
React for 1 hour at . Sephadex G-2 in which the reaction solution was equilibrated with 50mM phosphate buffer pH 7.0
Maleimidized HR through 5 columns (1.5 x 12 cm)
Get P.

On the other hand, anti-human alpha-fetoprotein (AF) at 2m℃
P) specific antibody F (ab') 2 fractions (5 mg/
m 12 , 50 m M acetate buffer, pH 5,
A 0.1 mM 0.25M 2-mercaptoethylamine solution is added to 0), and a photoreaction is performed at 37°C for 90 minutes. The reaction solution was diluted with 50mM phosphate buffer (pH 7.0).
Sephadex G-25 column (1,5X
12 cm) to obtain the anti-human AFPF Fab' fraction.

2 mg of maleimidated HRP (2 m℃) and the anti-human AF
8 mg (3 m[) of the PFab' fraction were mixed and reacted at 30° C. for 1 hour, and then left to react overnight. Sephacryl S-200 column (2
, 5x90cm), and H with a molecular weight of 120,000 or more.
Pool the RP-labeled human anti-AFP fractions.

(B) Preparation of HRP-labeled anti-human ferritin In the same manner as in (A), tetramethylenediamine was bonded to HRP to prepare amino group-introduced HRP, and further N-(4-carboxycyclohexylmethyl)maleimide was added to N- Hydroxysuccinimide ester is bonded to prepare maleimide group-introduced HRP.

Furthermore, the anti-human ferritin-specific antibody F (ab') 2 fraction is also reduced in the same manner as in (A) to obtain a Fab' fraction.

After reacting 2 mg of maleimide group-introduced HRP with 8 mg of anti-human ferritin-specific antibody Fab' fraction, HRP-labeled anti-large ferritin fractions with a molecular weight of 120,000 or more are pooled by gel filtration.

(C) Sensitivity adjustment of ferritin measurement The HRP-labeled anti-human ferritin prepared in (B) above was
% polyethylene glycol 6000 (PEG) in PBS and adjusted to a concentration of 2 μg/m°C. A dilution series of the large ferritin standard is then prepared in PBS. Furthermore, PE of anti-human ferritin rabbit IgG (ammonium sulfate purified product)
Prepare a dilution series with PBS containing G. Equivalent amounts of diluted HRP-labeled anti-human ferritin and anti-large ferritin rabbit IgG are mixed to prepare labeled antibodies containing unlabeled antibodies at each concentration. Add 100 μβ of the labeled antibody reagent described above to a dilution series of human ferritin standards at 50 μ°C, and react at 37°C for 20 minutes. Enzyme substrate solution (25mM phenol, 0.75mM 4-aminoantipyrine, 35mM
Add 0.5 mJ2 of PBS (containing hydrogen peroxide solution), react for another 10 minutes, and finally add 2 mg of PBS containing 1.8% formaldehyde to stop the reaction.
Measure the absorbance at 00 m.

Absorbance obtained at each concentration of ferritin and On g/
Differences in absorbance obtained when using mβ are shown in Table 1 (at the end of the book) according to the content of unlabeled antibody.

The results in Table 1 show that by increasing the amount of unlabeled antibody added, the absorbance difference can be kept low even when ferritin (antigen) is added.

(D) Preparation and measurement method of screening reagent for simultaneous measurement of AFP and ferritin HRP-labeled anti-human AFP prepared in (A) and HRP-labeled anti-human ferritin prepared in (B) were each 1 μg/mρ.
and anti-human ferritin rabbit IgG with a titer of 75μg/
A measurement reagent containing m42 is prepared in PBS containing 3% PEG. Add 100 μβ of the above measurement reagent to 50 μβ of the sample containing each concentration of AFPI quasi and ferritin standard,
React for 20 minutes at 7°C. Add the enzyme substrate solution used in (C) at 0.5 m°C and react for an additional 10 minutes. Finally, PBS containing 1.8% formaldehyde at 2 m°C is added to stop the reaction, and the absorbance at 500 nm is measured.

Table 2 (at the end of the book) shows the difference between the absorbance obtained by measuring a standard substance containing AFP and ferritin at various concentrations and the absorbance obtained by measuring a buffer solution containing neither of the two.

In Table 2, less than 20 ng/m 12 is defined as a normal value for AFP, less than 200 ng/mβ is defined as a normal value for ferritin, and the cut-off value according to this screening method is defined as an absorbance difference of 0.015. If the absorbance difference is less than this value, from Table 2 AFP is 20 ng/
It can be determined that it is less than mβ and ferritin is less than 200 ng/mρ. On the contrary, AFP is 20ng/m12 or more,
Furthermore, if ferritin is 200 ng/mρ or more, the absorbance difference will always exceed 0.015. Therefore, if it is below the cutoff value, both AFP and ferritin are normal, and if it is above the cutoff value, either AFP or ferritin may be abnormal. Therefore, it is sufficient to perform a single test for AFP and ferritin only on specimens having a cutoff value or higher. The above is summarized in Table 3 (at the end of the book).

In Table 3, since the majority of specimens have normal AFP and ferritin, many normal specimens can be removed by this screening method.

(E) Measurement of large serum sample A sample whose AFP concentration and ferritin concentration had been measured by radioimmunoassay was measured in the same manner as in (D). FIG. 1 shows the relationship between the absorbance difference obtained by this method and the AFP value obtained by radioimmunoassay. Furthermore, the relationship between the absorbance difference and the ferritin value according to this method is shown in FIG. From Figures 1 and 2, when AFP is 20 ng/m 12 or more and ferritin is 200 ng/m 12
In the above cases, it always exceeds 0.015, and it can be said that this screening method is effective.

In Figure 2, the three points with abnormally high absorbance differences are A
FP is 9900.10500.18600ng/m respectively
The sample had a very high beta.

<Effects of the Invention> Since this invention is configured as described above, it is possible to
It is possible to quickly identify abnormal persons (those with an abnormality in at least one of the items) from two or more trace components that are indicators of disease in urine and other biological components. It is particularly useful for saving labor in testing in mass medical examinations, etc.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between the absorbance difference according to this method and the AFP value obtained by radioimmunoassay, and FIG. 2 is a diagram showing the relationship between the absorbance difference according to this method and the ferritin value. Sniff (Z1 return OL (5 (jOII-): S

Claims (11)

[Claims] (1) Two or more of one or both of the following (A) and (B) that specifically binds to each of the measurement targets,
An immunoassay reagent having the ability to simultaneously determine multiple items, comprising one or more of the following (C) and (D), as required. (A) Antibody labeled with a non-radioactive substance (B) Antigen labeled with a non-radioactive substance (C) Unlabeled antibody of the same type as (A) (D) Unlabeled antigen of the same type as (B) (2) For immunoassay having multi-item simultaneous determination ability as set forth in claim (1), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is an enzyme-labeled antigen or an enzyme-labeled antibody. reagent. (3) An immune system capable of simultaneous determination of multiple items according to claim (1), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is a fluorescent substance-labeled antigen or a fluorescent substance-labeled antibody. Measurement reagent. (4) Claims (1) to (3) that use non-radioactive substance-labeled antigen microparticles or non-radioactive substance-labeled antibody microparticles as the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody An immunoassay reagent with the ability to simultaneously determine multiple items. (5) An immunoassay reagent capable of simultaneous determination of multiple items according to claim (4), which uses red blood cells, gelatin particles, or synthetic polymers such as latex as the fine particles. (6) Two or more of the following (A), (B) or both that specifically bind to each of the measurement targets,
If necessary, contact the object to be measured with one or more of the following (C) and (D),
A multi-item method characterized by applying a homogeneous immunoassay method and detecting from the obtained signal values whether all of the measurement targets are normal values or whether at least one or more of the measurement targets is an abnormal value. Simultaneous determination immunoassay method. (A) Antibody labeled with a non-radioactive substance (B) Antigen labeled with a non-radioactive substance (C) Unlabeled antibody of the same type as (A) (D) Unlabeled antigen of the same type as (B) (7) The multi-item simultaneous determination immunoassay method according to claim (6), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is an enzyme-labeled antigen or an enzyme-labeled antibody. (8) The multi-item simultaneous determination immunoassay method according to claim (6), wherein the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody is a fluorescent substance-labeled antigen or a fluorescent substance-labeled antibody. (9) Claims (6) to (8) that use non-radioactive substance-labeled antigen microparticles or non-radioactive substance-labeled antibody microparticles as the non-radioactive substance-labeled antigen or non-radioactive substance-labeled antibody Multi-item simultaneous determination immunoassay method. (10) The multi-item simultaneous determination immunoassay method according to claim (9), which uses red blood cells, gelatin particles, or synthetic polymers typified by latex as the fine particles. (11) The multi-item simultaneous determination immunoassay method according to claim (6), which uses immunoturbidimetry as the homogeneous immunoassay method.