Method of differential estimation of glutamic acid-oxalacetic acid transaminase isozyme

A method is disclosed for differential estimation of glutamic acid-oxalacetic acid transaminase isozyme which comprises estimating m-GOT by: preparing an immuno conjugate by adding an anti-s-GOT obtained by immunizing a mammal with purified s-GOT as antigen extracted from the blood or the tissue of the mammal into the human serum; adding sensitized particles which are allowed to couple said anti-s-GOT antibody to insoluble particles; and completely absorbing said s-GOT in said human serum.

This invention relates to a method of differential estimation of glutamic 
acid-oxalacetic acid transaminase isoyzme. 
BACKGROUND OF THE INVENTION 
The level of glutamic acid-oxalacetic acid transaminase (hereafter 
designated as GOT), in diseases of the liver and heart varies in 
accordance with the degree of disorder of these organs. Therefore, the 
measurement of GOT level has a very high clinical and diagnostic 
significance. 
It is known that GOT is composed of two kinds of isozymes which are located 
in different parts of the cells. One is in the supernatant fraction of 
cells (hereafter designated as s-GOT) and the other is in the mitochondria 
(hereafter designated as m-GOT). 
The differential estimation of GOT isozyme therefore also has a very high 
clinical significance just as does the measurement of total GOT. 
Conventionally, several methods including electrophoresis, ion-exchange 
chromatography, etc. have been reported for the differential estimation of 
GOT isozyme. These methods, however, necessitate complicated pre-treatment 
and are limited to use in research laboratories especially equipped to 
carry out these determinations. 
Previously, the present inventors developed a simple and rapid method of 
differential estimation of GOT isozyme (see Japanese Patent Application 
No. Sho 48-73157). The object of the present invention is to provide an 
improvement in the previous technique of determining isozyme accurately. 
According to applicants' previous invention, differential estimation of 
m-GOT in samples were determined by using the purified s-GOT extracted 
from the serum or the tissue of mammals as an antigen. The substance to be 
examined was mixed with sensitized particles coupling an antiserum 
obtained by immunizing the mammal against the antigen with insoluble 
particles in an adequate buffer solution (i.e. about neutral to not 
destroy erythrocyte), and then removing immunoconjugate of s-GOT in the 
substance and anti-serum by separating said mixed solution by 
centrifuging. 
According to this method, however, while the removal of s-GOT in human 
serum could be performed completely when immunized with purified human 
s-GOT as antigen, a small amount of unabsorbed s-GOT remained when using 
s-GOT as antigen which orginated from other mammals. Accordingly, accurate 
measurement of m-GOT was not obtained, which is particularly unfortunate 
since the normal value of m-GOT in human serum is only zero to 5 Karmen 
units. Therefore, it has become especially important to find a method by 
which an accurate determination of m-GOT is possible even at low levels. 
The present invention is, therefore, directed to a process for determining 
m-GOT accurately by absorbing the s-GOT in human serum completely with an 
anti-s-GOT antibody obtained by immunizing other mammals with purified 
s-GOT as an antigen extracted from the serum or the tissue of the mammal. 
In other words, according to the present invention, m-GOT is differentially 
estimated by preparing an immunoconjugate by adding the anti-s-GOT 
antibody obtained by immunizing the mammal with s-GOT originating from 
mammals other than human into the sample (human serum) and then adding 
sensitized insoluble particles which are allowed to couple with the 
anti-s-GOT antibody. 
By "sensitized particles" is meant carriers or coated particles such as red 
blood cells or latex particles coupled with either antigens or antibodies 
for the purpose of being used in immunological reaction. 
The "insoluble particles" employed in the present invention are 
erythrocytes, cells of microorganism, polystyrene particles, copolymer 
dextran gel insoluble in water, cellulose powders, copolymer of divinyl 
benzene, aminopropyl glass, gelatine or the mixture thereof. 
Any erythrocyte may be used if it is separated from the mammal and is 
stored in physiological saline. Suitable polystyrene particles are 
available under the name of "polystyrene latex" by Difco Company in U.S.A. 
The copolymer dextran gel, copolymer agarose and the like are also 
commercially available, called SEPHAROSE or SEPHADEX. 
Representive coupling agents used in the present invention are 
bis-diazobenzidine, bis-diazobenzidine sulfonic acid, 
tetraazo-para-phenylene-diamine, difluoro-dinitro benzene, 
difluorodinitro-phenyl sulfonate carbodiimide, toluene isocyanate, 
cyanuric chloride, and dichloro-S-triazine. 
In addition, cyano compounds such as cyanobromide, and tannic acid, the 
physical adsorbent, may be used as the coupling agents. Some of these 
coupling agents, especially cyanurization agent, dialdehyde and saturated 
or unsaturated .alpha.-.beta.-aldehyde, couple to the group on the surface 
of the cells, and at the same time, protect the cells of the erythrocyte 
and the microorganism. Therefore, these cells are stable to dissolution. 
When such coupling agents are used, there is no need to perform another 
stabilization or residual treatment. By using antibodies, insoluble 
particles and coupling agents of the present invention, the immunizing 
adsorbent which removes s-GOT is obtained. In addition, in accordance with 
the description of this invention s-GOT can be measured in reverse using 
m-GOT with insoluble particles.

The examples according to this invention are as follows: 
EXAMPLE I 
One ml of a suspension of anti-s-GOT serum sensitized blood cells was 
placed into a small test tube, washed with 5 ml physiological saline, the 
supernatant of which was discarded after centrifugation. 
A solution of 0.6 ml containing known quantities of purified human s-GOT 
and m-GOT was added to a lyophilized anti-s-GOT antibody, and after 
standing for 5 minutes at room temperature, it was thoroughly shaken and 
stirred adding 0.5 ml of said solution to said anti-s-GOT serum sensitized 
blood cells and allowed to stand for 5 minutes additional at room 
temperature. Then, the level of m-GOT in the supernatant was measured with 
a method of UV absorption after separating with a centrifuge. 
The results are shown in Table I 
TABLE I 
__________________________________________________________________________ 
Solution including 
s-GOT 
200 
200 
200 
200 
200 
200 
200 
200 
0 
s-GOT and m-GOT 
m-GOT 
0 2 5 10 20 40 80 160 
200 
m-GOT level of 
supernatant 0 2 5 10 21 40 81 162 
200 
__________________________________________________________________________ 
Remark: The unit is shown in Karmen units 
EXAMPLE II 
One ml suspension of anti-m-GOT serum sensitized blood cells was placed 
into a small test tube, washed with 5 ml physiological saline, the 
supernatant of which was discarded after centrifugation. 
A solution of 0.6 ml containing known quantities of purified s-GOT and 
m-GOT was added to a lyophilized anti-m-GOT antibody, and after standing 
for 5 minutes at room temperature, it was thoroughly shaken and stirred 
adding 0.5 ml of said solution to said anti m-GOT serum sensitized blood 
cells and allowed to stand for 5 additional minutes at room temperature. 
Then, the level of s-GOT in the supernatant was measured with a method of 
UV absorption after separating with a centrifuge. 
The results are shown in Table II. 
TABLE II 
__________________________________________________________________________ 
Solution including 
s-GOT 
200 
200 
200 
200 
200 
200 
200 
200 
0 
s-GOT and m-GOT 
m-GOT 
0 2 5 10 20 40 80 160 
200 
s-GOT level of 
supernatant 200 
200 
202 
200 
198 
201 
200 
202 
0 
__________________________________________________________________________ 
Remark: The unit is shown in Karmen units 
EXAMPLE III 
The level of m-GOT and s-GOT in human serum was measured respectively with 
UV techniques using the anti-s-GOT lyophilized antibody and the anti-s-GOT 
serum sensitized blood cells, anti m-GOT lyophilized antibody and anti 
m-GOT serum sensitized blood cells respectively with the same methods as 
in EXAMPLES I and II. In addition, the total GOT level (m-GOT and s-GOT) 
was measured with UV method. 
The results are shown in Table III. 
TABLE III 
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Sample Total GOT level 
s-GOT level 
m-GOT level 
______________________________________ 
blood serum 1 
63 57 8 
blood serum 2 
67 62 6 
blood serum 3 
71 62 8 
blood serum 4 
88 75 12 
blood serum 5 
183 156 28 
blood serum 6 
303 280 25 
______________________________________ 
Remark: The unit is shown in Karmen units