JPH0552845A - Reagent for immunoassay - Google Patents

Abstract

PURPOSE:To accurately measure an antigen to be measured without receiving the effect of the cross reaction due to a cross-reactive substance even when a polyclonal antibody is used. CONSTITUTION:A reagent for immunoassay contains an insoluble carrier having an antibody supported thereon and a part of the antibody is preliminarily reacted with the cross-reactive substance crossing an antigen to be measured with respect to the antibody and estimated to be present in a specimen. By this method, the antigen bonded region bonding the cross-reactive substance of the antibody is blocked.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent used in an immunoassay for detecting an increase in aggregation caused by an immunoreaction between an antibody carried on an insoluble carrier and an antigen to be measured, and particularly to avoid cross-reaction. The present invention relates to an immunoassay reagent that enables highly accurate detection or quantification of an antigen to be measured.

[0002]

2. Description of the Related Art Conventionally, when measuring an antigen in a sample, an antibody corresponding to the antigen is carried on an insoluble carrier, and an increase in agglutination caused by an immune reaction with the antigen is detected to detect the increase in the sample. Immunoassays for detecting or quantifying contained antigens are widely used. The reagent used in this type of assay is to immunize an animal with an antigen, purify the obtained serum by a method such as ammonium sulfate salting out, ion exchange or chromatography to obtain an antibody, which is an insoluble carrier. It was configured to be carried by.

However, when a substance having a cross-reactivity with the above-mentioned antibody against the antigen to be measured is present in the sample, the antigen to be measured cannot be measured accurately. To give an example,
For example, when luteinizing hormone (LH) is present when measuring human chorionic gonadotropin (HCG). Also, when performing routine inspections in an inspection room, etc.,
In many cases, multiple items are measured simultaneously for each sample.
In such a case, for example, if urinary human albumin is a measurement item and bovine serum albumin is included in a diluent or the like that constitutes a measurement reagent for another simultaneous measurement item, it will be contained in the sample due to carryover of the machine. May be mixed with bovine serum albumin. As a result, human albumin cannot be accurately detected due to the presence of bovine serum albumin.

On the other hand, in order to obtain a specific antibody which does not cause the above-mentioned cross-reaction, a method using a monoclonal antibody has been carried out (for example, JP-A-2-66458). However, the production of a monoclonal antibody requires a great deal of labor and time, and there is a problem that the price is significantly high. A method has also been proposed in which the antibody that cross-reacts is removed by passing the antibody through an affinity chromatography column packed with a gel in which a cross-reactive substance is immobilized on cellulose or the like. However, the complicated operation as described above is required, and the yield is not sufficient.

On the other hand, Japanese Patent Laid-Open No. 63-16266 discloses a casein hydrolyzate in a reaction system between an antibody-sensitized latex or red blood cells and an antigen as a reagent for measuring autoantibodies for diagnosing autoimmune diseases. Is disclosed, where it is shown that the addition of casein hydrolyzate prevents non-specific aggregation conditions.

Further, Japanese Patent Laid-Open No. 63-266356 discloses that the effect of cross-reaction can be suppressed by adding a cross-reactive substance or a cross-reactive analogue to the immune reaction system. In other words, paying attention to the fact that the cross-reactivity rate is larger as the concentration of the cross-reactive substance is lower, and is relatively small when the concentration of the cross-reactive substance is higher, and the cross-reactive substance is added to the above reaction system. By adding
It suppresses the effects of cross-reactivity.

However, although the immunoassay reagents described in JP-A-63-16266 can suppress the non-specific reaction, the cross-reaction cannot be suppressed among the non-specific reactions. In addition, JP-A-63-266
In the method described in No. 356, when the substance to be measured is a low molecular weight substance, a tentative effect is exhibited, but when measuring a high molecular weight protein, the substance to be measured has a low concentration in the method of this patent. In the case of, it was difficult to actually use it because it was difficult to obtain a large change in absorbance. Further, since the cross reaction is suppressed by the competitive reaction, there is a possibility that the cross reaction cannot be completely suppressed depending on the conditions such as the measurement time.

[0008]

Therefore, the object of the present invention is to cross-react with a wide range of antigens to be measured, from low molecular weight substances to high molecular weight substances such as proteins, even when polyclonal antibodies are used. Another object of the present invention is to provide an immunoassay reagent capable of performing highly accurate measurement while avoiding the influence of the above and without strictly controlling the reaction conditions and the like.

[0009]

The immunoassay reagent of the present invention detects an increase in agglutination caused by an immune reaction between an antibody carried on an insoluble carrier and an antigen to be measured, thereby detecting an analyte in a sample. It is used for a method for detecting or quantifying an antigen, and includes an insoluble carrier carrying an antibody, and a part of the antibody crosses the antigen to be measured with respect to the antibody and is mixed in the sample. It is characterized in that it has been previously reacted with a cross-reactive substance which is predicted to be.

A polyclonal antibody is preferably used as the carried antibody. In the case of a monoclonal antibody, it is possible to obtain an antibody corresponding to a specific antigenic determinant, but it takes a lot of time and labor to prepare it. In addition, when a polyclonal antibody is obtained by an ordinary immunization method, an antibody corresponding to an arbitrary antigenic determinant of an immunizing substance can be produced due to its production method. Therefore, when the immunological substance has a substance similar to itself (for example, bovine albumin for human albumin or luteinizing hormone (LH) for human chorionic gonadotropin (HCG)), other than the antibody specific to the antigen to be measured. Also, an antibody against an antigenic determinant common to the antigen to be measured and these cross-reactive substances can be prepared. When measuring a sample in which such a cross-reactive substance is present, for example, when a polyclonal antibody is used as it is in the quantification of trace albumin in human urine, bovine serum albumin (BSA) is generally used as a sample diluent. If it comes into contact with a solution or the like containing, cross-reaction will occur and accurate measurement cannot be performed.

Further, also in the measurement of a trace substance in blood or urine, when a substance that crosses the measurement target with respect to the antibody is contained, it may be difficult to use the polyclonal antibody as it is. However, in the reagent according to the present invention,
Even if a polyclonal antibody is used, by supporting the polyclonal antibody on an insoluble carrier in the process of producing the reagent and then adding a cross-reactive substance to react, the antibody against the cross-reactive substance is inactivated in the carried polyclonal antibody. It Therefore, the reagent of the present invention can specifically detect or quantify a trace substance while avoiding cross-reaction. The amount of the cross-reactive substance added at the time of preparation of the reagent is not limited as long as the effect can be exerted, but it is preferably equal to or more than the total amount of the polyclonal antibody supported on the insoluble carrier.

The polyclonal antibody used in the present invention is produced by immunizing a substance with an antigen according to a conventional method. The animal species to be immunized with the antigen to obtain the polyclonal antibody is not particularly limited. Also,
The antigen is also not particularly limited, but substances that are expected to undergo many cross-reactions, such as drugs, hormones, blood proteins, and the like, more specifically, hemoglobin, albumin, HCG, and the like are suitable for the present invention. Used.

As the cross-reactive substance, a substance which causes a cross-reactivity with the antibody corresponding to the antigen used and which is expected to be present in the measurement sample is appropriately used. In this case, the cross-reactive substance that reacts with the polyclonal antibody is not limited to one kind, and may be plural kinds as necessary. Examples of the insoluble carrier used in the immunoassay reagent of the present invention include inorganic substance powder, organic substance powder including synthetic polymer powder, microorganisms, blood cells, cell membrane pieces and the like. As the inorganic substance powder, silica, alumina, or a metal piece such as gold, titanium, iron, or nickel is used. As the organic substance powder, insoluble agarose, cellulose and insoluble dextran are considered. Further, latex is used as the synthetic polymer powder, and it is usually used in the state of a latex suspension. The polymer constituting the latex, polystyrene, styrene-styrene sulfonate copolymer, (meth) acrylic acid polymer, acrylonitrile-butadiene copolymer,
Examples thereof include vinyl chloride- (meth) acrylic acid ester copolymers and vinyl acetate- (meth) acrylic acid ester copolymers. The average particle size of the latex to be used is appropriately selected depending on the detection concentration range of the substance to be measured or the measuring device, but is usually 0.05 to 1.0 μm.

The reagent of the present invention is generally used by suspending it in a buffer. The buffer solution has a pH of 5 to 10, preferably 6 to 8, and any buffer solution such as a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, or an ammonia buffer solution is used. It is also possible to use a buffer solution containing a sensitizer such as polyethylene glycol or dextran.

As described above, the immunoassay reagent of the present invention detects an increase in aggregation caused by an immune reaction between an antibody carried on an insoluble carrier and an antigen to be measured,
It is used for a method of detecting or quantifying an antigen to be measured in a sample. Such measurement methods include a method of visually determining the presence or absence of aggregation, and a method of irradiating the reaction solution with light to measure scattered light or transmitted light. Can also be used for
Generally, the method of determining the aggregation state by the naked eye, the antibody carried on an insoluble carrier, and the state of aggregation of the antigen to be measured in the sample is determined visually, the determination of the presence or absence of the antigen in the sample or semi-quantitative method Is used as. Further, the optical measurement method is used to quantify the antigen in the sample.

In the method for visually observing the degree of aggregation of the insoluble carrier, the sample and the solution containing the reagent of the present invention are usually mixed on a determination plate and shaken for 1 to 5 minutes, and then the presence or absence of aggregation is measured. To do. In the case of semi-quantifying the antigen contained in the sample, the sample is reacted with the sample of the present invention diluted with a solution containing an appropriate buffer solution such as a phosphate buffer solution. It is also possible to react by adding a sensitizer such as polyethylene glycol or dextran to the reaction solution.

In the method for optically detecting the increase in aggregation of the insoluble carrier, the sample is usually diluted with a solution containing an appropriate buffer such as a phosphate buffer, and then the solution containing the reagent of the present invention is mixed. To do. At this time, it is also possible to react by adding a sensitizer such as polyethylene glycol or dextran to the reaction solution. The measurement is scattered light intensity, absorbance,
Alternatively, use an optical instrument that measures the number of particles. The measurement wavelength may be 300 to 2400 nm. Regarding the photometric method, it is possible to measure the increase in scattered light intensity or absorbance by selecting the particle size or concentration of the insoluble carrier to be used and the reaction time according to a known method. Further, when it is desired to measure in a shorter time, it is possible to quantify by using the absolute values of the agglutination reactivity and the absorbance. Also, the reaction time can be appropriately selected.

The pH during the reaction is preferably 5-10, particularly 6-8. As the buffer solution during the reaction, a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution, an ammonia buffer solution or the like is used. The reaction temperature is preferably 4 to 50 ° C, particularly preferably 20 to 40 ° C.

[0019]

In the immunoassay reagent of the present invention, a part of the antibody is pre-reacted with the cross-reactive substance, and therefore the antigen-binding site that reacts with the cross-reactive substance of the antibody is blocked, so When contacted, cross-reaction effects can be avoided. Therefore, it becomes possible to accurately detect the antigen to be measured in the sample.

[0020]

Description of Examples The present invention will be clarified by giving examples of the present invention. Example 1 Human albumin (Miles Laboratorie)
Rabbits were immunized with Ss (hereinafter referred to as HSA) according to a standard method. The antiserum obtained by immunization was purified by ammonium sulfate salting out to obtain a γ-globulin fraction. The obtained γ-globulin fraction was diluted with 0.1 M glycine buffer (pH = 9.0) to a concentration of 1 mg / ml.

10 g of the obtained γ-globulin fraction dilution solution
1 ml of polystyrene latex having an average particle diameter of 0.13 μm (manufactured by Sekisui Chemical Co., Ltd., solid content: 10% by weight) was added to 3 l.
Stir at 7 ° C for 60 minutes, then centrifuge at 4 ° C (18,0).
00 rpm × 60 minutes). After centrifugation, the supernatant was discarded, and the precipitate contained 0.1% of bovine serum albumin (BSA), which is a cross-reactive substance, at a concentration of 1% by weight.
80 ml of M glycine buffer (pH = 9.0) was added,
The mixture was stirred at 37 ° C for 60 minutes. After stirring, centrifugation (18,000 rpm × 60 minutes) was performed at 4 ° C. After centrifugation, 80 ml of 0.1 M glycine buffer solution (pH = 9.0) was added to the precipitate containing the supernatant to remove latex. Was suspended to prepare a latex reagent.

Normal human urine, and normal human urine 5, 10,
Samples were prepared by adding HSA at 50, 100 and 500 μg / ml. For the specificity assay, BSA was added at a concentration of 100 μg / ml as a sample. Further, as a sample containing a substance to be measured and a similar substance as a cross-reactive substance, BSA concentration of 100 μg / ml and HSA of 50 μg were added to normal human urine.
A sample was added so that the concentration would be / ml.

Each of the above-mentioned samples was diluted 10-fold with a 50 mM phosphate buffer solution (pH = 7.0) using a glass test tube, and 10 μl of the diluted sample and the above-mentioned latex reagent 120 were diluted.
0 μl was mixed, and the change in absorbance at a wavelength of 570 nm for 5 minutes was determined using a glass cell having an optical path length of 5 mm. The results are shown in Table 1 below. Comparative Example 1 In the same manner as in Example 1, a dilution of the γ-globulin fraction (anti-human albumin antibody (derived from rabbit)) with 0.1 M glycine buffer was obtained. 10 ml of the obtained γ-globulin fraction diluted solution had an average particle size of 0.13 as in Example 1.
1 ml of polystyrene latex (manufactured by Sekisui Chemical Co., Ltd., solid content 10% by weight) of μm was added, and the mixture was stirred at 37 ° C. for 60 minutes,
Next, centrifugation (18,000 rpm × 60 minutes) was performed at 4 ° C. After centrifugation, 80 ml of 0.1 M glycine buffer solution (pH = 9.0) was added to the precipitate obtained by discarding the supernatant, and the latex was suspended to prepare a latex reagent.

On the other hand, each sample was prepared in the same manner as in Example 1. Each sample was diluted 10-fold using a glass test tube with 50 mM phosphate buffer (pH = 7.0) containing 1 wt% casein hydrolyzate. Diluted sample 10
μl and 1200 μl of the latex reagent were mixed, and the change in absorbance at a wavelength of 570 nm for 5 minutes was obtained in the same manner as in Example 1. The results are shown in Table 1.

[0025]

[Table 1]

As is clear from Table 1, in Comparative Example 1,
The sample was diluted with a diluent containing casein hydrolyzate, but the antibody-derived cross-reaction could not be removed and B
It can be seen that it is reacting with SA. On the other hand, it can be seen that the reagent of Example 1 can accurately measure the sample concentration without causing cross-reaction. Example 2 Human chorionic gonadotropin (manufactured by Cosmo Bio Inc., hereinafter referred to as H
Rabbits were immunized with CG) according to a standard method. The antiserum obtained by immunization was purified by ammonium sulfate salting out to obtain a γ-globulin fraction. This γ-globulin fraction (anti-HCG antibody)
Was diluted with 0.2 M phosphate buffer (pH = 7.2) to a concentration of 1 mg / ml. To 10 ml of the obtained γ-globulin fraction diluted solution, 1 ml of polystyrene latex having an average particle size of 0.26 μl (Sekisui Chemical Co., Ltd., solid content 10% by weight) was added, and the mixture was stirred at 37 ° C. for 60 minutes, then 4 ° C
Was centrifuged (18,000 rpm × 60 minutes).
After centrifugation, the supernatant was discarded, and the precipitate contained a concentration of 0.05% by weight.
0.2M containing luteinizing hormone (LH)
Add 10 ml of phosphate buffer (pH = 7.2), 30 ℃
And stirred for 60 minutes. Then centrifuge at 4 ° C (18,0
(00 rpm x 60 minutes), the supernatant was discarded, and the precipitate was added with 0.2 M bovine serum albumin at 0.2 M.
A latex reagent was prepared by adding 10 ml of a phosphate buffer solution (pH = 7.2) to suspend the latex.

Normal human urine, and normal human urine
A sample was prepared by adding HCG to 50,100 IU / ml. Also, for the test of specificity, LH
Was also added as a sample. Furthermore, as a sample containing the substance to be measured and the cross-reactive substance, 5 IU / m of HCG was added to normal human urine.
Samples were prepared by adding 1 and LH to 100 mIU / ml.

Each sample is 50 m containing 1% by weight of BSA.
Diluted 10 times with M phosphate buffer (pH = 7.0) using a glass test tube. 50 μl of the diluted sample was mixed with 50 μl of the above latex reagent, and after shaking for 3 minutes, the presence or absence of aggregation was visually determined. The results are shown in Table 2 below. Comparative Example 2 In 10 ml of the γ-globulin fraction (anti-HCG antibody) diluted solution obtained in the same manner as in Example 2, the average particle size was 0.26 μm.
l polystyrene latex (Sekisui Chemical Co., Ltd., solid content 1
0% by weight) (1 ml) was added, the mixture was stirred at 37 ° C. for 60 minutes, and then centrifuged (18,000 rpm × 60 minutes) at 4 ° C. After the centrifugation, 10 ml of 0.2 M phosphate buffer (pH = 7.2) containing no LH was added to the precipitate containing the supernatant, and the mixture was stirred at 30 ° C. for 60 minutes. Then, after centrifugation (18,000 rpm × 60 minutes) at 4 ° C.,
A latex reagent was prepared by adding 10 ml of 0.2 M phosphate buffer (pH = 7.2) containing 1.2% by weight of BSA to suspend the latex.

On the other hand, each sample was prepared in the same manner as in Example 2. The sample was diluted with 50 mM phosphate buffer containing 1% by weight of BSA of Example 2 and 1% by weight of casein hydrolyzate. 50 μl of each sample diluted in this way and 50 μl of the above latex reagent were mixed and shaken for 3 minutes, and then the presence or absence of aggregation was visually determined. The results are shown in Table 2.

[0030]

[Table 2]

In Table 2, "-" indicates that the latex does not aggregate, and "+" indicates that the latex aggregates. As is clear from Table 2, the reagent of Comparative Example 2 using the diluted solution containing the casein hydrolyzate cannot remove the cross-reaction and reacts with LH.
On the other hand, with the reagent of Example 2, it can be seen that the sample can be accurately measured without causing a cross reaction.

[0032]

As described above, according to the present invention, a part of the antibody is pre-reacted with the cross-reactive substance, and the antigen-binding site of the antibody that binds the cross-reactive substance is blocked. The cross reaction can be surely suppressed.
Therefore, by using the reagent of the present invention, the antigen to be measured can be detected with high accuracy by the immunoassay method.

Moreover, in the immunoassay reagent of the present invention, a part of the antibody is reacted with the cross-reactive substance to block the antigen-determining site which binds the cross-reactive substance of the antibody as described above. Therefore, since the reagent can be prepared using a polyclonal antibody which is inexpensive and can be easily obtained, the cost of the immunoassay reagent is not increased.

Claims (1)

[Claims] 1. A reagent used in a method for detecting or quantifying an antigen to be measured in a sample by detecting an increase in aggregation caused by an immune reaction between an antibody carried on an insoluble carrier and the antigen to be measured. And a cross-reactive substance that includes an insoluble carrier carrying an antibody, and a part of the antibody crosses the antigen to be measured with respect to the antibody and is expected to be mixed in the sample in advance. An immunoassay reagent characterized by being reacted.