JPH0429059A - Simple enzyme immunoassay of tissue plasminogen activator - Google Patents

Abstract

PURPOSE:To measure simple enzyme immunity of tissue plasmonogen activator (t-PA) without subjecting a plasma sample to an acid treatment by using a polyclonal PC antibody for an insolubilized antibody and antibody labeled with an enzyme. CONSTITUTION:The t-PA which is an antigen is conjugated with the PC antibody insolubilized on a carrier and the PC antibody labeled with the enzyme is also conjugated with the antigen to form a complex. The labeling quantity thereof is measured. The PC antibody is prepd. by dissolving, for example, the t-Pa in a physilogical salt soln., then mixing the soln. with Freund's complete adjuvant, immunizing an animl several times with this soln. to obtain anti-t-Pa anti-serum and salting out this serum to obtain the anti-t-Pa antibody globulin. An active carbon is properly used in addition to magnetic material particles, glass, etc., as the carrier to immobilize the antibody. All the enzymes normally used for EIA are included in the enzyme. The antibody labeled with the enzyme is prepd. by using various protein crosslinking agents. The quantity of the t-Pa which is the antigen is exactly measured in accordance with the common method by measuring this label.

Description

[Detailed description of the invention] 1) Industrial application field The present invention relates to an enzyme immunoassay.

INDUSTRIAL APPLICATION This invention is utilized when measuring tissue plasminogen activator (sometimes called t-PA) in basic research of medicine and life sciences, or clinical examination and diagnosis.

2) Problems to be Solved by the Prior Art and the Invention t-PA is a factor greatly involved in the fibrinolytic mechanism of thrombus, which converts plasminogen adsorbed to fibrin clots into plasmin and dissolves fibrin.

In recent years, t-PA has attracted attention not only as a fibrinolytic activator, but also as a marker for liver disease, ischemic heart disease, malignant tumors, etc., and it is being used not only in basic research but also in clinical tests. Measuring t-PA has become more common in diagnosis. However, conventionally, when measuring blood t-PA, blood anticoagulants were added immediately after blood collection to remove cellular components. The obtained plasma is used as a sample, and in this case, as typified by the report by Sakurama et al., after obtaining the plasma, it is common to perform acid treatment for the following reasons and then use it for measurement. . That is,
Since t-PA is immediately bound to t-PA inhibitor (abbreviated as FAI) in plasma, acid treatment must be performed to dissociate t-PA from this complex.

However, this acid treatment is not only complicated in terms of measurement operations, but also causes subtle changes in delicate biological components within the body, making it difficult to accurately measure t-PA.
There are many cases where it is not possible to measure the Moreover, when we focused on highly specific monoclonal antibodies and used a highly specific mouse anti-t-PA monoclonal antibody as an insolubilized antibody and a labeled antibody, FAI did not dissociate even after the conventional acid treatment. However, it was also confirmed that sufficient measurements were not possible.

That is, in view of the above problems, the present invention provides a simple enzyme immunoassay method that does not require acid treatment of plasma even in the presence of FAI.

3) Means for solving the problem The inventors of the present invention conducted intensive research from various angles in order to solve the above problem, but they were not successful, so they made a fundamental change in their thinking and solved the specificity. We focused on polyclonal antibodies rather than monoclonal antibodies, focusing on low specificity, which is contraindicated in assay systems, rather than high specificity.

When they used a polyclonal antibody as the antibody, they unexpectedly obtained an extremely useful new finding that allows for accurate measurements without acid treatment of plasma samples. The present invention is based on this new knowledge and has been completed as a result of further research.

The present invention enables a measurement method that does not require acid treatment to dissociate FAI by selecting polyclonal antibodies that have many antibodies that recognize different epitopes.

The present invention involves binding an antigen, t-PA, to a polyclonal antibody insolubilized in a carrier, and using an enzyme to perform I! l! This enzyme immunoassay method is characterized in that a labeled polyclonal antibody is also bound to an antigen to form a complex, and the amount of this label is measured.

According to the method of the present invention, the complicated acid treatment step of plasma unlike the conventional method is completely unnecessary, and the method exhibits remarkable effects.

The animal species used to produce the polyclonal antibodies used in the present invention include rabbits, guinea pigs, goats, sheep, pigs, mice, etc.; is preferred. Polyclonal antibodies can be produced by conventional methods; for example, t-PA is dissolved in physiological saline, mixed with complete Freund's adjuvant, and animals are immunized several times to obtain anti-t-PA antiserum. ,
This antiserum may be salted out with sodium sulfate to obtain anti-t-PA antibody globulin.

The carriers for insolubilizing the antibodies used in the present invention include magnetic particles, polystyrene balls, microplates, silicone rubber pieces, glass, etc., as well as activated carbon, acid clay, kaolinite, bentonite, starch, gluten, cellulose, sepharose, enzymes, etc. A carrier for immobilizing the active substance is appropriately used, and the antibody is made insoluble in the carrier by known immobilization methods.

Enzymes used in the present invention include all enzymes commonly used in EIA, for example.

Horseradish - peroxidase, β-galactosidase, bovine mucosal alkaline phosphatase, glucose oxidase, malate dehydrogenase, G-6-P-dehydrogenase, glucoamylase, acetylcholinesterase, lysozyme, as well as NAD (NADH) and N
Coenzymes such as ADP (NADPH) are also used as appropriate. Among these, for example, horseradish peroxidase is preferable when a coloring substrate or luminescent substrate is used, and β-galactosidase is preferable when a fluorescent substrate is used.

Enzyme-labeled antibodies can be prepared using known techniques, such as the hinge method of Ishikawa et al.
.

J. Immunoassay, 4, 209-327.
．． 1983), the method of Kato et al.
al., FEBS Lett,, 56, 370-372.
(1975), etc., and methods using various protein crosslinking agents can be used as appropriate. For example, glutaraldehyde method, periodic acid method, maleimide method, pyridyl
Conventional methods for enzyme labeling, such as the disulfide method, penzoquinone method, and avidin method, are widely used, but among these, labeling that utilizes selective bonding between thiol groups and amino groups using divalent bonding reagents is widely used. In the present invention, the amount of t-PA, which is an antigen, is accurately measured by measuring the amount of S+. 41Wt
The amount may be measured according to a conventional method, and an electrochemical method or an optical method can be used as appropriate. As the optical method, conventional methods such as absorptiometric method, chemiluminescence method, and fluorescence method can be used as appropriate.

4) Examples and Comparative Examples The present invention will be specifically explained by the following Examples and Comparative Examples, but the present invention is not limited in any way by these Examples.

Example 1 Purified rabbit anti-t-
PA antibody solution (5 μg/n+Il 200 pQ
was dispensed into wells and left at room temperature for 3 hours. Cleaning liquid (
0.15M Sodium Chloride, 0.05% Tween 2
0 aqueous solution) three times. Blocking solution (5% rabbit serum, 1% bovine serum albumin, 0.15M sodium chloride, 0.1M sodium phosphate buffer, pH 7,
3) 250 μQ was dispensed and left overnight at room temperature. A rabbit anti-t-PA antibody immobilized plate was prepared by washing three times with a washing solution. Add reaction buffer (1% rabbit serum, 1.0% bovine serum albumin, 0.15M sodium chloride, 0.1M sodium phosphate buffer, 0.02% methylosal) to this rabbit anti-t-PA antibody immobilized plate. , pH7,
0) Plasma and standard t-PA solution containing 180 μg of t-PA and t-PA inhibitor (manufactured by FAI-1 Biopurple Co., Ltd.) were dispensed in 20 μQ each, and left overnight. After further washing three times with washing solution, 200 μQ of horseradish peroxidase-labeled Tsusagi anti-t-PA antibody solution was dispensed.
After leaving it at room temperature for 3 hours, it was washed 3 times with washing solution, and the substrate solution (0
, 04% ortho-phenylenediamine, 0.01% hydrogen peroxide, 0.1M phosphoric acid.

200 μg of sodium citrate buffer (pH 5) was dispensed, and after standing for 30 mL, 50 μQ of 1 reaction stopper (4,5 M sulfuric acid aqueous solution) was dispensed, and the absorbance at 490 μL was measured.

Comparative Example 1 Mouse anti-t-PA monoclonal antibody (lyophilized product, trade name PAM-1, manufactured by Biopool) was added to 0.15M sodium chloride and 0.1M sodium phosphate buffer (ρ) 1
7.0), the mouse anti-t-PA monoclonal antibody concentration was adjusted to 5 μg/mQ to obtain a mouse anti-t-PA monoclonal antibody solution. Using a 96-well microplate, 200 μQ of this mouse anti-t-PA monoclonal antibody solution was dispensed into wells.

Left at room temperature - overnight. It was washed three times with a washing solution (0.15M sodium chloride, 0.05% Ti1een 20 aqueous solution). Blocking solution (5% rabbit serum, 1% bovine serum albumin, 0.15M sodium chloride.

0.1 sodium aphosphate buffer, pH 7.3) 25
0 μQ was dispensed and left overnight at room temperature. The plate was washed three times with a washing solution to prepare a mouse anti-t-PA monoclonal antibody-immobilized plate. Using this mouse anti-t-PA monoclonal antibody immobilized plate, the measurement method shown in Example 1 above was applied mutatis mutandis to the samples used in Example 1 and those treated with acid.

Table 1 shows the measured values of plasma samples obtained in Examples and Comparative Examples.

As shown in Table 1, when the above sample was measured using a measurement system using a carrier with insolubilized monoclonal antibodies, sufficient measurement results were not obtained even when the sample was treated with acid. In the measurement system used, P
It was found that t-PA could be measured without the influence of AI-1.

Table 1 Measurement results of t-PA in plasma sample (triplicate measurement) Sample components t-PA measurement value (ng/mQ) t-PA
PAI-1 5) Effects of the Invention According to the present invention, by using a polyclonal antibody as an insolubilized antibody and an enzyme-labeled antibody, a simple new t-
It has the remarkable effect of providing a PA enzyme immunoassay.

Claims (2)

[Claims] (1) Bind an antigen, tissue plasminogen activator, to a polyclonal antibody insolubilized in a carrier, and then bind an enzyme-labeled polyclonal antibody to the antigen to form a complex, and measure the amount of this label. A simple enzyme immunoassay method for tissue plasminogen activator that does not require acid treatment of plasma. (2) The measuring method according to claim 1, characterized in that the amount of the label is measured optically.