Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1994-05-02
2001-06-05
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S024000
Reexamination Certificate
active
06242208
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for the determination of an LDH
1
fraction in a sample.
2. Description of the Related Art
LDH (lactate dehydrogenase) has five isozymes, that is LDH
1
through LDH
5
. Each of organs has its own composition of the isozymes. For example, LDH
1
is present in a myocardium in the largest amount. Since LDH
1
escapes from the myocardium into blood under a condition of a myocardial infarction, a rise in serum LDR
1
level can be diagnostic for such a disease. Therefore, an LDH
1
isozyme assay is clinically significant.
In a most common LDH isozyme assay, LDH
1
, LDH
2
, LDH
3
, LDH
4
and then LDH
5
are fractionated in the order of electrophoretic mobility. An immunological LDH assay is also known. In other LDH assay disclosed in Japanese Patent Publication No. 6477/1983, a coenzyme derivative (e.g. reduced type nicotinamide adenine dinucleotide) is used. In addition, LDH assays wherein a sample is treated under an alkaline condition are described in Japanese Patent Publication No. 28280/1985 and Japanese Patent Kokai Publication No. 278997/1987.
The above-mentioned electrophoretic or immunological assay is unsuitable for clinical autoanalysis because of complicated process and long operation time. In addition, the LDH isozymes may be insufficiently fractionated by the electrophoretic method.
Strictly speaking, it is impossible to assay the LDH isozyme fractions by the method wherein the coenzyme derivative is used, while the ratio of H subunit to M subunit in the enzyme can be suitably determined by the method.
In the method comprising the alkaline treatment of the sample, more than 50% of LDH
1
may be inactivated during the inhibition of the other isozymes. In addition, the process is complicated and takes a long time.
Thus, the above-mentioned conventional LDH assays are unsatisfactory for a clinical examination.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an LDH
1
assay useful for the clinical examinations including autoanalysis.
Accordingly, the present invention provides a process for the determination of an LDH
1
fraction in a sample, which comprises inhibiting LDH
2
, LDH
3
, LDH
4
and LDH
5
in the sample with a protease in the presence of a protein-denaturating agent and then determining LDH
1
remaining uninhibited.
DETAILED DESCRIPTION OF THE INVENTION
The present process is suitable for the determination of the LDH
1
fraction in a clinical sample such as serum or plasma.
The protease used in the present invention may be any protease suitable for the purpose of the invention. Specific examples of the protease are serine proteases (e.g. trypsin, &agr;-chymotrypsin, subtilisin, proteases K, etc.), cystein proteases (e.g. papain, bromelain, clostripain, poliovirus protease, etc.), carboxy proteases (e.g. pepsin, etc.), metalloproteases (e.g. thermolysin, collagenase, dispase, etc.), thrombin, elastase, endoprotease, carboxypeptidase, pronase, and cathepsin. They may be used independently or as a mixture to two or more of them. Proteases disclosed in Japanese Patent Kokai Publication No. 149399/1985 may also be used.
Although the amount of the protease is not critical in the present invention and varies with other assay conditions, &agr;-chymotrypsin, for example, can be used in a concentration in the range of from 10 to 1,000 units/ml.
In the present invention, any protein-denaturating agent having an activity suitable for the process can be used. Specific examples of the protein-denaturating agent are bile acids (e.g. cholic acid, deoxycholic acid, taurocholic acid, taurodeoxycholic acid, etc.), guanidine derivatives (e.g. guanidine hydrochloride, guanidine sulfate, guanidine nitrate, guanidine thiocyanate, etc.), urea compounds (e.g. urea, thiourea, etc.), thiocyanate, trichloroacetic acid or its salts, perchloric acid or its salts, anionic surfactants (e.g. N-lauroyl sarcosinate, sodium dodecylsulfate, etc.), cationic surfactants (e.g. dodecyltrimethylammonium, dodecylamine hydrochloride, etc.) and mixtures thereof.
Although the amount of the protein-denaturating agent is not critical and varies with other assay conditions, for example 0.05 to 5 M guanidine can be used.
The LDH
1
isozyme remaining uninhibited can be determined according to an enzyme assay selected from a lot of conventional assays widely used in clinical examinations. Such an assay used in the present process should be easily carried out within a short time with high precision, sensitivity and accuracy.
In a preferred embodiment of the present process, the LDH
1
isozyme remaining uninhibited is determined by an enzyme assay which comprises catalytically developing a chromogen or dye precursor with the isozyme and then measuring absorption in a visible light range. It is also possible to measure ultraviolet absorption of coenzyme NADH reduced by a catalytic effect of the LDH
1
isozyme.
In another preferred embodiment, the LDH
1
isozyme is determined according to the Wroblewski method which is a well-known enzyme assay performed under a neutral condition. It is found that the inhibition of the isozymes LDH
2-5
can be effected in a wide pH range according to the present invention (cf. Example 1). Therefore, when the determination of the LDH
1
isozyme remaining uninhibited is performed under a neutral condition, the present assay can be advantageously carried out under the same neutral condition throughout the assay, while complicated pH correction is necessary in the above-mentioned conventional assay comprising the alkaline treatment of the sample.
REFERENCES:
patent: 4224406 (1980-09-01), Gomez et al.
patent: 4250255 (1981-02-01), Sanford
patent: 4803159 (1989-02-01), Smith-Lewis
patent: 5158873 (1992-10-01), Abbott et al.
patent: 0292838 (1988-11-01), None
patent: 2278997 (1987-12-01), None
patent: 9001067 (1990-02-01), None
patent: 9001067 (1990-08-01), None
Research Disclosure, vol. 163, Nov. 1977, pp. 63-67, Disclosure No. 16370.*
Patent Abstracts of Japan, vol. 9, No. 309 (Dec. 1985).
Patent Abstracts of Japan, vol. 13, No. 39 (Jan. 1989).
Boyer, P.D., The Enzymes, vol. XI, Part A, pp. 257-258 (1975).
Li., S., S.-L., et al, Eur. J. Biochem, 149, 215-225 (1985).
Selmeci, L., et al, Experimentia 27/8, pp. 888-889, Aug. 15, 1971.
Forman, H.J., et al, The Journal of Biological Chemistry, vol. 252, No. 10, 3379-3381 (1977).
Hoppe-Seyler's Z. Physiol. Chem. Abstract, Jeckel et al, 354, pp. 737-738, Jul. 1973.*
Forman et al, “Effects of Chaotropic Agents versus Detergents . . .”The Journal of Biological Chemistry, vol. 252, No. 10 (1977) pp. 3379-3381.
Isshiki Kenji
Shirahase Yasushi
Watazu Yoshifumi
Gitomer Ralph
International Reagents Corporation
LandOfFree
LDH1 assay does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with LDH1 assay, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and LDH1 assay will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2457041