JP2000193666A - Enzyme-immunoassay and reagent using absorbing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To use alkali phosphatase(ALP) for a labeled substance unaffected by various ALP-related nonspecific reaction through one kind of absorbent. SOLUTION: In an enzyme-immunoassay and reagent using alkali phosphatase for a labeled enzyme, a deactivated alkali phosphatase is used as an absorbing agent. More specifically, 0.05-50 μg of deactivated alkali phosphatase is used as an absorbing agent per 50 μl of specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a method and a reagent for enzyme immunoassay using alkaline phosphatase as a labeling enzyme. The present invention relates to a method and a reagent for enzyme immunoassay using inactivated alkaline phosphatase as an absorbent. The present invention relates to a method and a reagent for enzyme immunoassay using 0.05 to 50 μg of inactivated alkaline phosphatase as an absorbent.

2. Description of the Related Art In the field of clinical examination, a safe and simple enzyme immunoassay (hereinafter referred to as EIA) is frequently used. EIA which initially used absorbance as a measurement index
However, due to the recent development of technology, the detection index has shifted to fluorescence and luminescence, and the measurement system has also become highly sensitive accordingly. However, as a result of the widespread use of highly sensitive EIAs, the effect of trace amounts of non-specific reactions, which has not been found hitherto, has become a problem. Alkaline phosphatase (hereinafter, referred to as ALP) is one of the labeling enzymes used for EIA. When a body fluid such as serum is measured using ALP as a labeling enzyme, ALP-related non-specific reaction in serum is measured. May affect the measured value, and a correct measured value may not be obtained. A in serum
LP-related non-specific reactions include ALP-binding immunoglobulin (Clinical test, Vol. 31, No. 8, 1987, Amn. Intern. M.
ed., Vol. 90, No. 1, p30-35, 1979, Clin. Chim. Act
a., Vol.30, No.135 (1), p41-48, 1983) and polymer AL
P (Ann. Clin. Biochem., Vol. 26, p151-157, 1989)
Thyroid hormone (Bone Miner, Vol. 4, p355-363, 1988,
Endocrinology, Vol. 120, p1873-1881, 1987) and glucose (Clin. Chim. Acta, Vol. 133, p15-24, 1983) are known. ALP-binding immunoglobulin is a type of enzyme-binding immunoglobulin and has the same action as an anti-ALP antibody. This ALP binding immunoglobulin is
When bound to ALP as a label, which is one of the components of IA, the labeled ALP loses its enzyme activity, resulting in an abnormally low measured value in the case of the sandwich assay and an abnormal measured value in the case of the competition assay. Causes high prices. On the other hand, high-molecular ALP is present in the serum of patients with liver disease, and in patients with hyperthyroidism, thyroid hormone increases ALP activity,
It is known that in diabetic patients, glucose inhibits ALP activity. The biological factors affecting these ALP activities include, in EIA using ALP as a labeling enzyme,
It may interfere with the measurement of ALP activity as a labeling enzyme. In the measurement system using the solid phase method, ALP in serum
Is adsorbed to the solid phase during the immune reaction and produces the same activity as ALP on the substrate, causing an abnormally high measured value in the sandwich assay and an abnormally low measured value in the competition assay . Furthermore, although not a non-specific reaction derived from a body fluid, if the ALP-labeled antibody is non-specifically adsorbed on a solid phase, measurement noise derived from the solid phase may increase, resulting in a decrease in measurement sensitivity. Such an ALP-related non-specific reaction, particularly the presence of an ALP-binding immunoglobulin or a substance which affects ALP activity in a body fluid, may cause ALP in a conventional EIA having a low detection sensitivity, such as detection of ALP activity with a chromogenic substrate.
The influence on the ALP activity by the related non-specific reaction was relatively small, and was hardly a problem. However, in recent EIAs for detecting ALP activity with high sensitivity such as chemiluminescent substrates, ALIA by ALP-related non-specific reaction has been proposed.
Changes in P activity can now be detected, and the effect of ALP-related non-specific reactions on measured values has increased. As described above, in the sample, A which may affect a highly sensitive measurement system may be used.
Since there is an LP-related non-specific reaction, in a highly sensitive EIA using ALP, an EIA that removes all the ALP-related non-specific reactions having different causes as described above and provides an accurate measurement value is desired. I was

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an EIA and a reagent using an ALP-labeled substance which is free from the effects of ALP-related non-specific reactions.

Means for Solving the Problems The inventors of the present invention have conducted studies on ALP-related non-specific reactions in order to solve the conventional problems. Various ALPs derived only from different absorbents
The present inventors have found that related non-specific reactions can be eliminated and completed the present invention. That is, the present invention provides an EIA and a reagent using an ALP label and an inactivated alkaline phosphatase. Hereinafter, the present invention will be described in detail. In the present invention, EIA means an EIA containing an ALP label using ALP as a labeling enzyme for an antigen or an antibody, and includes known EIAs such as a sandwich measurement method and a competition method. Preferably, the antigen or antibody binding solid phase and A
This is an EIA using an LP label. AL used for EIA
As the P-labeled substance, known substances such as an ALP-labeled antibody and an ALP-labeled antigen can be used. For example, to label ALP on an antibody or antigen, the method of Yoshitake et al.
e et al., J. Biochem., Vol.92, No.5, p1413-1424, 1
982), the amino group of ALP and the thiol group of the antibody or antigen to be bound may be bound by a dicrosslinking reagent such as GMBS. The substance to be labeled and ALP
When bonding each other's amino groups with a cross-linking agent at the time of bonding, the compound can also be prepared by introducing GMBS into one and iminothiolane dihydrochloride (manufactured by Nacalai Tesque) into the other. In the present invention, the inactivated ALP means an ALP that does not substantially cause enzymatic activity on a corresponding substrate,
The antigenicity as ALP in the immune response is maintained. Such inactivated ALP can be obtained, for example, by a method such as acid treatment, heat treatment, and zinc removal by chelating reagent treatment (Enzymologia, Vol. 35, p157, 1968, JBC, Vol. 23).
3, p1121, 1958, BBA, Vol. 52, p36, 1961), which can be suitably used in the present invention. In order to use inactivated ALP in the present invention, EI
Inactivated ALP may be used during the immune reaction of A. More preferably, it is preferable that the inactivated ALP and the labeled ALP coexist during an immune reaction. For example, a solid phase-bound antibody or antigen such as polystyrene beads, an ALP label, and a sample are added to a reaction buffer to react.
In step EIA, inactivated ALP can be added to a reaction buffer, an ALP-labeled body fluid, a sample diluent, or the like. In the EIA using fine particles such as latex or magnetic particles as a solid phase, inactivated ALP can be added to a solid phase preservation solution in addition to a reaction buffer, a sample diluent, and an ALP-labeled body solution. Further, the sample, the solid phase, and A
The sample and the solid phase or A
After a reaction with the LP label for a certain period of time, a delay one-step EIA in which an ALP-labeled substance or a solid phase is continuously added, or a washing operation after a reaction between the sample and the solid phase for a certain period of time, and AL
In the case of 2-step EIA or the like adding a P-labeled substance, ALP
Inactivated AL at the time of reacting the labeled product as an immunoreactive reagent
It is preferable to add deactivated ALP to various reaction solutions so that P is added. Specifically, inactivated ALP can be added to an ALP-labeled body fluid and used for an immune reaction. The reagent using the inactivated ALP according to the present invention refers to the EI of the present invention.
It means a reagent capable of performing A, and can include, for example, an antibody or antigen-binding solid phase, an ALP-labeled antibody or antigen, a sample diluent, a washing solution, a substrate, a reaction stop solution, and the like. The inactivated ALP which is a feature of the present invention may be prepared by adding it to the above-mentioned antigen or antibody-bound solid-phase preservation solution, ALP-labeled antibody or antigen solution, sample diluent, or the like. When the label is used as a lyophilized reagent, it can be added to the reconstituted solution. Alternatively, an inactivated ALP solution may be prepared as a reaction additive, and the inactivated ALP may be added as a reaction additive when performing an immune reaction. The amount of inactivated ALP used in the present invention can be appropriately adjusted depending on the amounts of the sample and the ALP label used. Preferably, inactivated ALP is added at a concentration of 0.1 per 50 μl of the sample.
05 to 50 μg, more preferably 50 μl of the specimen
It is advisable to add 0.5 to 5 μg of inactivated ALP per hit. In addition, the inactivated ALP was added to the ALP
It is preferable to use 000 times by weight. Samples to be measured with the EIA and the reagent of the present invention include blood, plasma, serum,
There is no particular limitation as long as the target substance such as urine, tissue fluid, culture solution, etc. is to be measured, but it is more effective when used for measurement of a biological fluid in which an ALP-related nonspecific reaction is likely to occur. In the present invention, the ALP-related nonspecific reaction refers to ELP
It means a reaction that increases or decreases ALP activity, which is a measurement index of IA, without a valid immune response, and decreases ALP activity by ALP-binding globulin, increases ALP activity by ALP-like active substance, This includes an increase in ALP activity due to specific adsorption. For example, a sample that causes such an ALP-related non-specific reaction is shown in Example 1 described later. In a sample that performs a normal immune reaction, when the sample is diluted 2 times, 5 times, and 10 times and measured, the measured value of the sample becomes 1/2, 1/5, 1/10,
If the obtained measured value is multiplied by the sample dilution ratio, the converted measured value of the undiluted solution is identical. However, Example 1
As shown in the converted values in Table 1, the non-specific samples shown in Table 1 showed results in which the measured values of the undiluted solution differed depending on the dilution ratio of the sample. The most suspicious non-specific reaction in EIA is the presence of a globulin that binds to the antibody specific to the antigen to be measured used in EIA. For example, when a mouse monoclonal antibody is used as the specific antibody, an anti-mouse IgG antibody in the sample is used. The non-specific reactions caused by are well known. Therefore, since the specific antibody used in the measurement system that showed a non-specific reaction was a goat antibody, it was assumed that the labeled body was indirectly bound to the solid phase by some anti-goat antibody globulin. As an agent, a healthy goat antibody was added to a solid phase solution to try to absorb non-specific reactions. However, the measurement value abnormality of the non-specific sample could not be eliminated. On the other hand, the inactivated AL was added to the ALP-labeled antibody solution.
The EIA of the present invention to which P was added eliminated the measurement value abnormality of the non-specific sample, and gave a consistent sample measurement value regardless of the dilution ratio of the sample. Conventionally, it has been known that an enzyme-labeled antibody directly or indirectly binds to a solid phase, but it is thought that the cause is that an enzyme-labeled antibody nonspecifically binds to a solid phase via an antibody portion therein. Was. On the other hand, the non-specific sample shown in Example 1 could not eliminate the non-specific reaction with the healthy goat antibody, indicating that it was not a non-specific reaction via the antibody portion in the enzyme-labeled product. Inactivated ALP of the invention
Has shown an effect on such non-specific reactions.
This suggests that the labeled ALP is indirectly bound to the solid phase via some substance in the sample. The sample showing the ALP-related non-specific reaction shown in Example 2 is a sample showing a high abnormal measurement value in the competition law. Since the competition method gives a downward-sloping standard curve, an increase in the measured sample value means a decrease in the ALP activity, which is a detection index, which is different from the nonspecific sample shown in Example 1. The reaction is shown. This non-specific reaction is considered to be affected by anti-ALP globulin because the ALP activity of the ALP-labeled product is suppressed. However, as shown in Example 2, a sample showing such a non-specific reaction is also present in this sample. When measured by EIA using the inactivated ALP of the present invention, it was confirmed that a normal measured value was obtained. As described above, the EIA characterized by using the inactivated ALP of the present invention can remove all the effects of ALP-related non-specific reactions of different origins and can give accurate measured values. Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

EXAMPLES Reference Example 1 Preparation of Ferritin Measurement Reagent A reagent used for ferritin measurement was prepared as follows. That is, the antibody-bound solid phase was sensitized with an anti-ferritin goat antibody using magnetic particles (manufactured by Nippon Paint Co., Ltd.) for the solid phase alone. The prepared antibody-bound magnetic particles were prepared using a suspension (hereinafter referred to as “particle solution”) using 50 mM Tris buffer pH 7.2 containing bovine serum albumin (manufactured by Oriental Yeast Co., Ltd.) (hereinafter, referred to as BSA) as a particle diluent. 1)). An ALP-labeled anti-ferritin antibody is prepared by preparing an anti-ferritin antibody goat antiserum (manufactured in-house) according to a conventional method, and analyzing the globulin fraction of the antiserum by the method of Yoshitakest al., Yoshitakest al., J. biochem., Vol. .Five,
p1413-1424, 1982) and bound to ALP derived from bovine small intestine (manufactured by Oriental Yeast). The enzyme-labeled antibody was prepared by using a 50 mM female buffer (pH 6.8) containing BSA as a buffer for diluting the enzyme-labeled antibody, and having a concentration of 0.1 μg / ml (hereinafter, referred to as “buffer”).
ALP-labeled antibody solution 2).
As a standard antigen, human ferritin was added to Tris buffer pH 7.2 containing 50 mM BSA and used as a standard solution. Example 1 Effect of Absorbent by Sandwich System In serum ferritin measurement by a two-step sandwich method using an anti-ferritin goat antibody-bound solid phase and an anti-ferritin goat antibody-ALP label, a sample with poor dilution linearity appeared. This sample was a sample whose measured value decreased as the dilution factor of the sample increased and the concentration of serum-derived components decreased. For this sample, the effect of the absorbent using the reagent prepared in Reference Example 1 was examined. As an absorbent to be examined, 30 μg / ml of healthy goat serum (manufactured in-house) was added to the particle solution 1 prepared in Reference Example 1, and 5 μg / ml of inactivated ALP (manufactured by Oriental Yeast Co., Ltd.) was added to the ALP-labeled antibody solution 1. Was added and the dilution ratio of the sample was changed, and the measurement of ferritin in the sample was measured using a fully automatic chemiluminescence immunoassay system Lumipulse (manufactured by Fujirebio). That is, after adding 250 μl of the particle solution 1 and 50 μl of the standard solution or the sample and reacting at 37 ° C. for 10 minutes, magnetic separation is performed to wash the antibody-bound magnetic particles, and 250 μl of the ALP-labeled antibody solution 1 is added, and the mixture is added at 37 ° C. for 10 minutes. Reacted. After washing the antibody-bound magnetic particles again, the luminescent substrate AMPP
D was added, and luminescence was measured 5 minutes at 37 ° C. Table 1 shows the results. The converted values in the table were obtained by multiplying the measured value of ferritin by the sample dilution ratio and expressed as the ferritin concentration in the sample stock solution. 30 μg / m final concentration of healthy goat antibody in Particle Solution 1
When it was added so as to be 1, no effect of suppressing the dissociation of the measured value was observed, and no tendency to alleviate the effect was observed.
On the other hand, inactivated ALP was added to labeled antibody solution 1 at a final concentration of 5 μg / ml.
When it was added so as to become, a remarkable decrease in the measured value and an improvement in the dilution linearity were observed. Further, 80 samples with a known ferritin concentration were measured, and the presence or absence of the influence of the addition of inactivated ALP was confirmed. The results are shown in FIG. ALP inactivates X axis
The measured values were plotted under the condition of no addition and the condition of adding the inactivated ALP at 5 μg / ml on the Y axis. As shown in FIG. 1, the addition of the inactivated ALP did not affect the measured value in the general sample, and was shown to be effective only in the non-specific sample as shown in Table 1.

[Table 1] Reference Example 2 Preparation of HBc Measurement Reagent A reagent used for HBc antibody measurement was prepared as follows. That is, the HBc antigen was bound to magnetic particles (manufactured by Nippon Paint Co., Ltd.) in the same manner as described in Reference Example 1 to obtain a solid phase carrier. The prepared HBc-bound magnetic particles were diluted with the same particle buffer as in Reference Example 1 and used for measurement (hereinafter, referred to as Particle Liquid 2). The ALP-labeled anti-HBc antibody is an anti-HBc antibody.
c Monoclonal antibody (made in-house) was bound to ALP in the same manner as described in Reference Example 1 (hereinafter referred to as ALP-labeled antibody solution 2). ALP-labeled antibody solution 2 used the same diluent as described in Reference Example 1 and was used at 0.05 μg / ml for measurement. There are two types of standards, a negative control and a positive control. As a negative control, human serum containing no antibody was used, and as a positive control, a human serum prepared such that the signal of the positive control was always 1% or less of the signal of the negative control. The signal obtained when the sample was measured was quantified by a percentage (inhibition value) using the negative control signal as the denominator. Inhibition is 0
% Means a response equivalent to the negative control, ie, negative, and 10%
0% indicates a response equivalent to the positive control, that is, a positive response. The cut-off value for distinguishing negative from positive was set so that the inhibition value was 50%. Example 2 Effect of Absorbent by Competitive Reaction System In an HBc antibody detection system using a competitive reaction, as in the case of a general sample and a negative control, the signal is maximized when no antibody is present, and the measured value of each sample is , The relative value (inhibition value (%)) when the signal of the negative control is taken as 100%. In the case of a negative sample, the inhibition value (%) is high, and as the amount of antibody or antibody activity in the sample increases, the signal decreases and the inhibition value (%) decreases. However, when the rheumatoid patient sample was measured by this measurement system, there were cases where the signal was higher than that of the negative control (Table 2). This was suspected to be caused by non-specific light emission via the solid phase, and it was confirmed whether the addition of the absorbent suppressed the signal. Mouse Ig as an absorbent
G, polymerized mouse IgG, anti-IgM antibody, and inactivated ALP were used. Then, a labeled body fluid to which each was added was prepared, and the measurement was performed using a fully automatic chemiluminescence immunoassay system Lumipulse (Fujirebio). The measurement was performed using 50 ALP-labeled antibody solutions 2
250 μl of the standard solution or 50 μl of the sample and the particle solution 2
After mixing at 37 ° C. for 20 minutes, magnetic separation was performed to wash the antibody-bound magnetic particles, and the luminescence substrate AMP was added.
The measurement was performed by adding PD and measuring the amount of luminescence after 5 minutes at 37 ° C. As shown in Table 2, a decrease in the analyte signal was observed only under the condition where the inactivated ALP was added to the ALP-labeled antibody solution, and no effect was obtained with other absorbents. The decrease in signal due to the addition of inactivated ALP
Although depending on the concentration of P added, 50 to 100 μg /
Around 100 ml, the negative control signal was 100% in all cases.
It showed a signal within the ± 10% range and did not vary. Further, 150 cases of the negative sample and the positive sample were measured in total, and the presence or absence of the influence by the addition of the inactivated ALP was confirmed. FIG.
The results are shown in a correlation diagram. The X axis is the condition without addition of inactivated ALP, the Y axis is the condition with addition of 50 μg / ml of inactivated ALP,
The inhibition value (%) of each sample was plotted. Samples that were 100% or more under the inactive ALP non-addition condition were all within 100% under the addition condition, and none exceeded the negative control signal.

[Table 2] Reference Example 3 Preparation of Free T3 Measurement Reagent A reagent used for free T3 measurement was prepared as follows. That is, the T3 analog-bound solid phase was bound to magnetic particles (Nippon Paint Co., Ltd.) via the support. The prepared T3 analog-bound magnetic particles were diluted with the same particle diluent as in Reference Example 1 and used for measurement (hereinafter, referred to as Particle Liquid 3). The ALP-labeled anti-T3 antibody is an anti-T3 goat antibody (manufactured by Roche Diagnostics Co., Ltd.) Reference Example 1
In the same manner as described in (1), diluted and used for measurement (hereinafter referred to as ALP-labeled antibody solution 3).
The standard antigen was 50 mM Tris buffer pH 7.2 containing T3 / T4 depleted serum (manufactured by AVTI) containing human T3.
To a standard solution. Example 3 Effect of Absorbent by Competition Method Using a sample having an abnormally high measured value, free T3 was measured by a competition method. As an absorbent to be examined, a solution obtained by adding 10 and 50 μg / ml of inactivated ALP (manufactured by Oriental Yeast Co., Ltd.) to the ALP-labeled antibody solution 3 prepared in Reference Example 3 was prepared. Automatic chemiluminescence immunoassay system Lumipulse (Fujirebio)
It measured using. That is, ALP-labeled antibody solution 3
Mix 0 μl with 70 μl of the standard solution or sample and mix.
After the reaction at 10 ° C. for 10 minutes, 250 μl of the particle liquid 3 was mixed with 100 μl of the reaction solution, and further reacted at 37 ° C. for 10 minutes. The antibody-bound magnetic particles were washed by magnetic separation, and the luminescence substrate AMPPD was added, and the luminescence after 5 minutes at 37 ° C. was measured. Table 3 shows the results. When inactivated ALP is added to the labeled antibody at a final concentration of 0, 10, 50 μg / ml,
Under the addition condition of 50 μg / ml, the measured value of free T3 decreased. As a control, a free T3 measuring reagent Amarex-Mab free T by the RIA method using no ALP as a measuring system was used.
Using 3 kits (manufactured by Ortho Clinical Diagnostics Co., Ltd.), the measured value of the same sample was determined, and it was in agreement with the measured value when 50 μg / ml of inactivated ALP was added.

[Table 3]

Industrial Applicability According to the present invention, it is possible to provide an enzyme immunoassay method and a reagent using ALP as a label which is not affected by various ALP-related nonspecific reactions by one kind of absorbent.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of the addition of inactivated ALP on a sample with a known ferritin concentration.

FIG. 2 is a diagram showing the effect of the addition of inactivated ALP on the HBc measurement system.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Isao Nishizono Fujirebio Co., Ltd. 2-62-5 Nihonbashihamacho, Chuo-ku, Tokyo

Claims (7)

[Claims] 1. An enzyme immunoassay using an alkaline phosphatase as a labeling enzyme, wherein an inactivated alkaline phosphatase is used during an immune reaction. 2. The immunoassay according to claim 1, wherein 0.05 to 50 μg of inactivated alkaline phosphatase is used per 50 μl of the sample. 3. The immunoassay according to claim 1, wherein the inactivated alkaline phosphatase is used in an amount of 10 to 5000 times by weight the alkaline phosphatase of the labeling enzyme. 4. The immunoassay according to claim 1, wherein the enzyme immunoassay is a sandwich assay or a competitive reaction assay. 5. An immunoassay reagent for use in the method according to claim 1. 6. The reagent according to claim 5, comprising an antigen or antibody-bound solid phase and an alkaline phosphatase-labeled antibody or antigen. 7. The reagent according to claim 5, wherein the inactivated alkaline phosphatase is contained in the solid phase buffer or the alkaline phosphatase-labeled antibody solution.