JPH10104232A - Assay of human plasminogen and measuring kit therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly sensitive and speedy assay of human plasminogen which does not cause coagulation due to non-specific reaction and enables accurate measurement in a wide scope even if a sample is not diluted. SOLUTION: When human plasminogen in a sample is assayed and the an assay of human plasminogen in which this sample and insoluble carrier which reacts anti-human plasminogen antibody are mixed in a solution to generate coagulation reaction due to antigen-antibody reaction and an absorbance change amount based on the above-mentioned coagulation reaction is measured and referred with standard calibration curve is done by this method, the above- mentioned insoluble carrier is latex particle and the concentration of the carrier in the above-mentioned solution is 0.02 to 2weight%. The solution contains at least one kind of polyethylene glycol having average molecular weight of 3,000 to 1,000,000 and polyvinylpyrrolidone having average molecular weight of 3,000 to 1,000,000 of 0.1 to 5weight%. The above-mentioned coagulation reaction is done at constant temperature for 5 seconds to 15 minutes, and the above- mentioned absorbance change amount is an increase amount of absorbance in accordance with the coagulation reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a highly sensitive and rapid method for quantifying human plasminogen in a sample, and a measuring kit for carrying out the method for quantifying human plasminogen.

[0002]

2. Description of the Related Art Human plasminogen (hereinafter also referred to as "human PLG") present in blood has a molecular weight of about 930.
00 is a single-chain glycoprotein and a precursor of plasmin, an enzyme involved in the control of blood coagulation. Since this human PLG has a particularly high affinity for fibrin, which is a main component of thrombus and blood coagulation, it binds to this fibrin to promote degradation, and as a result, promotes dissolution of thrombus and the like.

[0003] For this reason, human PLG is consumed and reduced when there is a thrombus or the like in the blood. Therefore, quantification of human PLG is useful for diagnosis of thrombosis and disseminated intravascular coagulation (DIC), which is a disease in which thrombosis occurs frequently throughout the body.

[0004] Clinically, it is useful to measure the amount of antigen of human PLG and its activity as an enzyme. There are various immunoassay methods for measuring the amount of antigen, and the method for measuring the activity value as an enzyme is described in “Tests and Techniques” (22 (5), (1994)), p. 245. As described above, there is a method of measuring the consumption amount using a synthetic substrate of the enzyme.

[0005] Among them, the immunoassay methods include:
In recent years, immunoassay methods using latex agglutination have been developed for the purpose of miniaturizing the reaction system, improving sensitivity, shortening the reaction time, and the like. Various latex agglutination immunoreagents used in such an immunoassay by latex agglutination are commercially available.

[0006] However, the conventional latex agglutination immunoreagent often causes agglutination by non-specific reaction, and EIA
Compared to the method, there is a problem that accurate measurement is not possible especially in a low concentration range.

In general, an immunoassay method uses an antigen-antibody reaction, so the optimal measurement range is narrow, and the measurement range of a conventional latex agglutination immunoreagent is 0.05 to 1
It was about mg / dl. However, the normal values of human PLG vary greatly depending on age and gender, and have a wide distribution of normal values. For this reason, when performing tests using conventional latex agglutination immunoreagents, "medicine and pharmacy"
As described on pages 411-417 of (29 (2), (1993)), a complicated dilution operation was required, which was inconvenient.

[0008] Latex agglutination immunoreagents capable of measuring in the range of 2 to 41 mg / dl have also been developed, but also these reagents are diluted to achieve a wide measurement range, so that the operation is complicated. Yes, it causes erroneous operation and lowers the measurement accuracy.

[0009]

SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide a highly sensitive and rapid human PLG which is free from aggregation due to non-specific reactions and can be measured accurately over a wide range without dilution. An object of the present invention is to provide a quantification method and a measurement kit for performing the quantification method of human PLG.

[0010]

According to the present invention, when quantifying human plasminogen in a sample, the sample is mixed with an insoluble carrier sensitized with an anti-human plasminogen antibody in a solution to obtain an antigen. An agglutination reaction due to an antibody reaction is caused, and a method for quantifying human plasminogen, comprising measuring the change in absorbance based on the agglutination reaction and comparing it with a calibration curve, wherein the insoluble carrier is latex particles, The concentration of the insoluble carrier in the solution is from 0.02 to
2% by weight, and an average molecular weight of 3,000 to 3,000 in the solution.
One million polyethylene glycol and at least one of polyvinylpyrrolidone having an average molecular weight of 3,000 to 1,000,000 are contained in an amount of 0.1 to 5% by weight, and the aggregation reaction is performed at a constant temperature for 5 seconds to 15 minutes. The absorbance change is a method for quantifying human plasminogen, which is an increase in absorbance associated with an agglutination reaction. Hereinafter, the present invention will be described in detail.

In the present invention, when quantifying human PLG in a sample, first, the sample and an insoluble carrier sensitized with an anti-human PLG antibody are mixed in a solution to cause an agglutination reaction by an antigen-antibody reaction. .

The sample is not particularly limited, and includes, for example, serum, plasma and the like. In addition, the human P of the present invention
The LG quantification method can be applied to various other biological samples and non-biological samples.

In the present invention, latex particles having a relatively constant particle size and capable of industrially producing products of constant quality and performance in a large amount are used as the insoluble carrier. The latex particles are not particularly limited, and include, for example, styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates, methacrylates, and other vinyl-based monomers such as homopolymers and copolymers; styrene-butadiene copolymers, Fine particles such as a butadiene-based copolymer such as a methyl methacrylate-butadiene copolymer are included. Among these, polystyrene-based latex particles are preferably used because they have excellent antibody-adsorbing properties and can stably maintain biological activity for a long period of time.

The latex particles have a particle size of 0.02 to 0.02.
0.5 μm is preferred. If it is too small, dispersion becomes difficult when freeze-drying, and if it is too large, self-aggregation proceeds and dispersibility decreases. More preferably, 0.05 to
0.2 μm.

The above insoluble carrier is one to which an anti-human PLG antibody has been sensitized. The anti-human PLG antibody is not particularly limited, and any of a polyclonal antibody and a monoclonal antibody can be used. Examples of the polyclonal antibody include animal species derived from humans, rabbits and goats, globulin fractions, purity of affinity-purified fractions and the like,
The processing method of Fab ', F (ab) 2, etc. is not limited. The sensitization of the anti-human PLG antibody to the insoluble carrier can be performed by a method of physically or chemically adsorbing the antibody.

In the present invention, the sample and the insoluble carrier are mixed in a solution. The above solution is not particularly limited, and examples thereof include a phosphate buffer, a glycine buffer, a Tris-HCl buffer, and a good buffer.

In the present invention, the concentration of the insoluble carrier in the solution is 0.02 to 2% by weight. If it is too low, the formation of aggregates is insufficient, and the required sensitivity cannot be obtained.If it is too high, the absorbance as the background is too high, and accurate quantification cannot be performed. You. Preferably, it is 0.03-0.3% by weight.

In the present invention, the solution contains at least one of polyethylene glycol and polyvinylpyrrolidone. When these are contained in the above solution, the sensitivity can be improved and the reaction can be promoted.

The average molecular weight of the polyethylene glycol and the polyvinyl pyrrolidone is 3000
~ 1,000,000. If it is too small, the effect of improving sensitivity or accelerating the reaction is insufficient, while if it is too large, the solubility in the solution becomes extremely low, and a problem arises in solubility, so that it is limited to the above range. Preferably, it is 4000 to 30,000 for polyethylene glycol and 10,000 to 100,000 for polyvinylpyrrolidone.

The concentration of at least one of the polyethylene glycol and polyvinylpyrrolidone in the solution is 0.1 to 5% by weight. If it is too low, the sensitivity is low, and accurate measurement in a low concentration range cannot be performed. If it is too high, aggregation due to nonspecific reaction occurs, and accurate measurement cannot be performed. Therefore, the range is limited to the above range. Preferably, it is 1 to 4% by weight for polyethylene glycol and 0.2 to 3% by weight for polyvinylpyrrolidone.

The polyethylene glycol and the polyvinyl pyrrolidone are preferably used by dispersing and dissolving in an appropriate medium. In this case, the insoluble carrier and at least one of the polyethylene glycol and polyvinylpyrrolidone may be used as a one-pack type latex reagent by dispersing and dissolving in the same medium. May be used as a two-part reagent of a latex reagent and a solution reagent by dispersing and dissolving in a medium.

The medium is not particularly restricted but includes, for example, phosphate buffer, glycine buffer, Tris-HCl buffer, Good buffer and the like. The pH of the medium is
5.5 to 8.5 are preferred. More preferably, 6.5 to 6.5
8.0.

In the one-pack type latex reagent, bovine serum albumin, sucrose, sodium chloride and the like may be appropriately dissolved for adjusting the salt concentration. Also, when used as a two-part reagent consisting of the latex reagent and the solution reagent, bovine serum albumin, sucrose, sodium chloride or the like may be appropriately dissolved in each to adjust the salt concentration.

In the present invention, the agglutination reaction by the antigen-antibody reaction is performed at a constant temperature. Preferably, 2
5-37 ° C. The agglutination reaction is performed for 5 seconds to 15 seconds.
Do for a minute. If the length is too short, accurate measurement cannot be performed. If the length is too long, aggregation due to nonspecific reaction occurs, and accurate measurement cannot be performed.

In the present invention, the amount of change in absorbance based on the agglutination reaction is measured and compared with a calibration curve to quantify human PLG. The amount of change in absorbance is an increase in absorbance due to the agglutination reaction.

The measurement wavelength at the time of measuring the change in the absorbance is usually 500 to 1000 nm, preferably 500 to 1000 nm.
An appropriate wavelength is selected from the range of ８００800 nm, more preferably 550-650 nm. The measuring device used for measuring the change in absorbance is not particularly limited as long as it can measure the absorbance of the solution over time, and examples thereof include a general-purpose biochemical automatic analyzer. The measurement wavelength, sample amount, reagent amount, and the like can be appropriately selected according to the measurement device.

As a specific method of comparing with the above-mentioned calibration curve, for example, a change in absorbance is measured for a sample of a known amount of human PLG such as a human PLG standard serum and its dilution series, and the measured value is compared with the human PLG. This can be carried out by preparing a calibration curve from the amounts and determining the corresponding amount of human PLG in the above-mentioned calibration curve from the measured value of the change in absorbance measured under the same conditions for an unknown sample of human PLG.

The present invention 2 provides a latex particle sensitized with an anti-human PLG antibody, polyethylene glycol having an average molecular weight of 3000 to 1,000,000, and an average molecular weight of 3000 to 1
The human PLG of the present invention 1 comprising a sample diluent containing at least one of a million pyrrolidones
It is a measurement kit for performing the quantification method.

Human PLG using the assay kit of the present invention 2
As a specific method of quantifying, for example, if a general-purpose biochemical automatic analyzer is used to measure the change in absorbance, the measurement kit of the present invention 2 is set at a predetermined position,
The measurement can be performed by setting a sample to be measured and a sample of a known amount of human PLG, and appropriately setting conditions for measuring absorbance. If an absorbance meter is used to measure the change in absorbance, the measurement kit of the present invention 2, the sample to be measured, and a sample of a known amount of human PLG are mixed and stirred, and after a certain period of time, the absorbance is measured. It can be performed by measuring. Preferably, a method using a general-purpose biochemical automatic analyzer is used. Such a biochemical automatic analyzer is not particularly limited.
150 type automatic analyzer (manufactured by Hitachi, Ltd.), MIRA (C
OBAS).

According to the method for quantifying human PLG of the present invention,
The insoluble carrier sensitized with the anti-human PLG antibody and the sample were mixed in a solution to cause a latex agglutination reaction triggered by the antigen-antibody reaction, and the amount of change in absorbance of the solution was measured and prepared in advance. Using a calibration curve, the amount of human PLG in a sample of unknown concentration can be accurately measured. Further, by using the measurement kit for performing the method for quantifying human PLG of the present invention, the amount of human PLG in a sample can be accurately measured.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Examples 1 to 40, Comparative Examples 1 to 40 1) Reagents and Materials The reagents and materials used in Examples 1 to 40 and Comparative Examples 1 to 40 are as follows.

A polystyrene latex having an average latex particle size of 0.304 μm (solid content: 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) was used. Latex dilution buffer 50 mM Na ₂ HPO ₄ and 50 mM NaH ₂ PO
₄ was mixed so as to have a pH of 7.50. From antiserum anti-human PLG antibody goat to the immunoglobulin fraction with purified goat anti-human PLG antibody (1mg / ml).

Antibody Dilution Buffer Latex dilution buffer was used as antibody dilution buffer. Blocking buffer 100 mM Na ₂ HPO ₄ and 100 mM NaH ₂
PO ₄ and pH 7.40, and mixed with bovine serum albumin (Bovine serum album).
in, FractionV, Reagent Grad
e, MilesCorp. And NaN ₃ (special reagent grade, manufactured by Nacalai Tesque, Inc.) to a concentration of 0.1% (W / V).

Plasma Specimens Plasma from patients with a history of thrombosis and healthy subjects was used. Human PLG standard human serum Human PLG was removed from human pool serum by affinity chromatography, and a precisely weighed purified antigen was added thereto to prepare a serum containing 20 mg / dl human PLG. Furthermore, about 10, 5,
It was used after being diluted to 2.5 and 1.25 mg / dl, respectively. In addition, the physiological saline only containing no human PLG was set to 0 mg / dl.

Sample dilution solution (R1 solution) Polyethylene glycol 60 was added to the blocking buffer.
00 (average molecular weight 7500, manufactured by Wako Pure Chemical Industries) 3%
(W / V) was used. A gel base was prepared using agarose commercially available from SRID , and purified human P
A calibration curve was prepared using LG (manufactured by ADI), and human PLG in the sample was quantified. In addition, a goat polyclonal antibody was used as the antibody. Latex agglutination immunoreagent for human PLG measurement Commercially available latex agglutination immunoreagent (Elpiace APL
G, manufactured by Diatron).

2) Method (1) Preparation of Latex Reagent One volume of polystyrene latex (average particle size: 0.304 μm, solid content: 10% (W / V)) was diluted with 3 volumes of a latex diluting buffer. A 0.5% (W / V) latex solution was used. The anti-human PLG antibody has a protein concentration of 5 mg /
The antibody was diluted with a buffer solution for antibody dilution to give a sensitized antibody solution. 2.5% (W / V) latex liquid 200μ1
Was stirred in a 25 ° C. incubator with a magnetic stirrer, 800 μl of the antibody sensitizing solution was quickly added, and the mixture was stirred at 25 ° C. for 1 hour. Thereafter, 2.0 ml of a blocking buffer was added, and the mixture was continuously stirred at 25 ° C. for 2 hours. Then, at 15 ° C and 15000 rpm,
Centrifuged for minutes. 4.0 ml of blocking buffer was added to the obtained precipitate, and the precipitate was washed by centrifugation in the same manner. The washing operation was performed three times. 2.0 ml of a blocking buffer was added to the precipitate, and the mixture was stirred well, and then subjected to a dispersion treatment with an ultrasonic crusher to obtain a solid content of 0.1 ml.
A 25% (W / V) latex reagent was used. The latex reagent thus prepared was stored at 4 ° C.

(2) Measurement of Human PLG Amount Using Latex Reagent The measurement of human PLG amount using a latex reagent was performed using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). Solid content 0.25% (W / V) obtained in (1) above
R2 solution (solid content 0.25%)
(W / V)). The measurement conditions are as follows.

Sample volume 3 μl Sample diluent (R1 solution) 300 μl Reagent (R2 solution) 100 μl Measurement wavelength 570 nm Measurement temperature 37 ° C.

Difference between the absorbance approximately 80 seconds after the addition of the reagent (R2 solution) and the absorbance approximately 320 seconds (ΔOD570)
Was measured, and a value obtained by multiplying the difference in absorbance by 10,000 times was defined as an absorbance change amount. Perform the same measurement with a standard serum for blood items of known concentration in place of the sample, prepare a calibration curve in advance, extrapolate the change in absorbance of the sample to the calibration curve, and determine the amount of human PLG in the sample. Was calculated.

(3) Measurement of Human PLG Amount by SRID Method For all samples measured in (2) above,
Using the method, the amount of human PLG in the sample was measured.

(4) Measurement of the amount of human PLG using a commercially available latex agglutination immunoreagent For all the samples measured in the above (2), samples were obtained using a commercially available latex agglutination immunoreagent (Elpiace APLG, Diatron). The amount of human PLG in it was measured. The measurement was carried out using Elpia 100, a special-purpose machine, according to the operation method attached to the kit.

3) Tests and Results (1) Examples 1 to 40 Human PLG using latex reagent as described in (2) above, using sera of patients with a history of thrombosis and healthy subjects as specimens
According to the measurement of the amount, the amount of human PLG in the sample was measured.
Further, the amount of human PLG in the sample was measured according to the above 2) (3) Measurement of the amount of human PLG by the SRID method. Table 1 shows the results of these measurements. FIG. 1 shows the correlation between the amount of human PLG by the latex reagent shown in Table 1 and the amount of human PLG by the SRID method.

(2) Comparative Examples 1 to 40 Serum of a patient with a history of thrombosis and a healthy person was used as a sample, and the amount of human PLG in the sample was determined according to the above (2) (4) Measurement of the amount of human PLG using a commercially available latex agglutination immunoreagent. Human PLG
The amount was measured. Table 2 shows the measurement results. In addition, the amount of human PLG by a commercially available latex agglutination immunoreagent shown in Table 2 and the amount of human PLG by the SRID method shown in Table 1
The correlation with the amount of G is shown in FIG.

[0045]

[Table 1]

[0046]

[Table 2]

As is clear from Tables 1 and 2, and FIGS. 1 and 2, the amount of human PLG measured by the method for quantifying human PLG of the present invention was determined by the SRID method.
Although the LG amount agreed well with the low-concentration region and the high-concentration region, the amount of human PLG by the commercially available latex agglutination immunoreagent was not necessarily the same as the amount of human PLG by the SRID method in the low concentration region and the high concentration region. I didn't do it. From the above results, the method for quantifying human PLG of the present invention has higher sensitivity than conventional latex agglutination
It was confirmed that the method was an excellent quantitative method which was quicker and simpler than the ID method.

[0048]

Since the method for quantifying human PLG of the present invention has the above-mentioned constitution, it suppresses non-specific reactions due to coexisting substances in a sample as seen with conventional latex agglutination immunoreagents, and reduces Even in the concentration range, the same detection sensitivity as that of the SRID method is exhibited. In addition, by using the measurement kit for performing the method for quantifying human PLG, human PLG in human serum can be accurately and quickly quantified accurately. Can be used effectively for the determination of

[Brief description of the drawings]

FIG. 1 shows a sample used in Examples 1 to 40,
It is a figure which shows the correlation between the amount of human PLG by a latex reagent, and the amount of human PLG by SRID method. The vertical axis is SR
The amount of human PLG by the ID method is shown, and the horizontal axis represents the amount of human PLG by the latex reagent.

FIG. 2 shows a sample used in Comparative Examples 1 to 40,
Human PLG Amount and S by Commercially Available Latex Agglutination Immunoreagent
It is a figure which shows the correlation with the human PLG amount by the RID method.
The vertical axis represents human PL using a commercially available latex agglutination immunoreagent.
The abscissa represents the amount of human PLG according to the SRID method.

Claims (2)

[Claims] When quantifying human plasminogen in a sample, the sample is mixed with an insoluble carrier sensitized with an anti-human plasminogen antibody in a solution to cause an agglutination reaction by an antigen-antibody reaction. A method for quantifying human plasminogen, comprising measuring the change in absorbance based on the agglutination reaction and comparing it with a calibration curve, wherein the insoluble carrier is a latex particle, and the insoluble carrier in the solution is The concentration is 0.02 to 2% by weight, and in the solution, at least one of polyethylene glycol having an average molecular weight of 3000 to 1,000,000 and at least one of polyvinylpyrrolidone having an average molecular weight of 3000 to 1,000,000 is 0.1 to 5% by weight. %
And the agglutination reaction is carried out at a constant temperature for 5 seconds to 1
A method for quantifying human plasminogen, wherein the method is performed for 5 minutes, and the amount of change in absorbance is an increase in absorbance due to an agglutination reaction. 2. Latex particles sensitized with an anti-human plasminogen antibody, polyethylene glycol having an average molecular weight of 3000 to 1,000,000, and an average molecular weight of 3000 to 1
The assay kit for performing the method for quantifying human plasminogen according to claim 1, comprising a sample diluent containing at least one of a million polyvinylpyrrolidone.