LDH.sub.1 levels in serum samples can be rapidly and accurately assayed by a novel immunochemical technique. In such procedure the serum sample is treated with soluble antibodies against the M subunit and the resulting antigen-antibody reaction product is insolubilized with a second antibody supported on an insoluble material. The resulting supernatant containing only LDH, isoenzyme is assayed for enzymatic activity by conventional procedures. The assay of LDH.sub.1 levels in sera is useful in determining whether the subject has undergone a myocardial infarction.

BACKGROUND OF THE INVENTION 
U.S. Pat. No. 4,046,634 discloses an assay for isoenzymes, including LDH 
isoenzymes, which employs ion exchange column chromatography to isolate a 
mixture of LDH.sub.1 and LDH.sub.2 isoenzymes which are then detected by 
conventional techniques such as the Wacker LDH method. 
German Auslegeschrift No. 21 28 670 discloses a method for assaying for 
isoenzymes, including LDH isoenzymes by treating the test sample with a 
large excess of a specific antibody to either LDH.sub.1 or LDH.sub.5 
isoenzymes so as to effect quantitative precipitation of the 
antigen-antibody complex. After incubation and centrifugation of the 
precipitated complex, the enzyme activity of the supernatant was 
determined by the method reported in Z. Klin. Chemie und Klin. Biochemie 
8, 658 (1970). 
Sussman et al., Journal of Biological Chemistry 243, 160 (1968) have 
described an assay for individual organ specific isoenzymes of human 
alkaline phosphate using a two step procedure. In the first step the 
specific antibody to the desired isoenzyme was reacted with the test 
sample and then in a subsequent step the antibody-antigen complex is 
precipitated with a second antibody (anti-.gamma.-globulin). After 
centrifugation the supernatant is tested for residual isoenzyme activity. 
The use of a second antibody insolubilized on a solid support material in 
radioimmunoassay procedures is described in U.S. Pat. No. 4,048,298. The 
disclosure includes the use of second antibodies adsorbed to the surface 
of polymeric solid support materials. 
U.S. Pat. No. 3,843,443 relates to a method of immobilizing proteins on a 
fluorocarbon polymer support. Included within the disclosed proteins are 
antibodies and the preferred polymer support material is polyvinylidene 
fluoride (Kynar-a trademark of the Pennwalt Corp.). 
DESCRIPTION OF THE INVENTION 
The present invention relates to an improved immunochemical assay for the 
isoenzyme LDH.sub.1 which isoenzyme is a known marker for myocardial 
infarction. 
In the improved method of the present invention a test sample such as a 
serum sample, is treated with a soluble antibody specific against the M 
subunit of the LDH isoenzymes, i.e., LDH.sub.2, LDH.sub.3, LDH.sub.4 and 
LDH.sub.5. After mixing and incubating for a short time, a second antibody 
insolubilized on a solid phase support material is added and the mixture 
is mixed and then incubated for another short period. The insoluble 
antigen-antibody(1)-antibody(2)-solid support complex is centrifuged down 
and the supernatant tested for LDH enzyme activity. The activity observed 
will be essentially that of the LDH.sub.1 isoenzyme component of the 
original sample. 
The method of the present invention has substantial advantages over 
procedures utilized in the prior art for isoenzyme assay. It is very 
rapid, highly accurate and reproducible. The preparation of the LDH.sub.1 
containing supernatant can be accomplished in a matter of minutes instead 
of the substantial number of hours previously required for immunological 
techniques. Moreover, the present method provides a clean separation of 
LDH.sub.1 from the other LDH isoenzymes which is not possible by 
ionexchange column procedures. 
The specific antibody against the M subunit of LDH used in the present 
invention is known in the art. See for example the previously indicated 
German Auslegeschrift No. 21 28 670. Further disclosures relating to such 
antibody are to be found in J. S. Burd et al., Clinica Chimica Acta 46, 
205-216 (1973) and J. S. Burd et al., Biochimica, Biophysica Acta, 310, 
238-247 (1973). 
The second antibody is prepared by immunization of a different animal than 
the one in which the specific first antibody is prepared with a gamma 
globulin from the blood of the host species used for the first antibody 
preparation. Thus the second antibody will be immunoreactive for the first 
antibody and will complex with it. 
The second antibody is insolubilized by attaching said second antibody to 
an insoluble support material. Suitable support materials include water 
insoluble organic polymeric substances such as cellulose or other 
polysaccharide, a vinyl addition polymer or condensation polymer or a 
water insoluble inorganic substance of polymeric nature, such as glass or 
silicone resins or the second antibody may be adsorbed to the surface of a 
solid support such as polystyrene, polypropylene, polyfluoroethylene or 
polyvinylidene fluoride. The method of attachment of the second antibody 
to the solid support is not narrowly critical and may include (1) 
covalently coupling the soluble second antibody to any insoluble polymeric 
substance; (2) converting the soluble second antibody to an insoluble 
polymerized form, such as by reaction with an insolubilizing agent; (3) 
physical entrapment of particles of the second antibody in the pores of a 
gel polymer such as a cross-linked polyacrylamide; or (4) by physical 
adsorption on an insoluble polymeric substance. 
In a preferred embodiment of the present invention the second antibody is 
supported by adsorption on activated Kynar utilizing the general 
procedures disclosed in U.S. Pat. No. 3,843,443.

The method of the present invention is further illustrated by reference to 
the following Example. 
Example 
1. Reagents 
a. Antiserum to LDH.sub.5 : goat anti LDH.sub.5 serum is diluted to a 
concentration that binds 300-400 IU/l of purified LDH.sub.5 isoenzyme 
employing Fisher Diagnostic or equivalent reagents for the determination 
of LDH enzymatic activity. The dilution of the antiserum is made in 0.02M 
Tris pH 7.5 with 0.1% NaN.sub.3. 
b. Insoluble antiserum to goat gamma globulin. 
The following specific steps are followed in preparing an insoluble 
antiserum to goat gamma globulin (second antibody). The starting material 
is unsintered Kynar (vinylidene fluoride) resin powder, grade 301 F, 
Pennwalt Corp. The powder is dispersed in isopropanol (2-propanol) in the 
proportions of 50 grams Kynar in 1000 ml of isopropanol. The suspension is 
then homogenized by a Brinkmann Polytron for 5 minutes at a 
pulse-frequency of 4000 c.p.s. The Kynar-isopropanol mixture is then 
transferred to a cylinder containing ten liters of saline and stirred 
until dispersed. The Kynar is then allowed to settle out and most of the 
supernatant is decanted. After two water washes, the Kynar is resuspended 
in phosphate buffered saline (pH 7.0) with merthiolate (0.01%) to yield a 
2% Kynar concentration. The Kynar is now in the activated state and able 
to accept protein. While the isopropanol activated Kynar is stirring, 0.5 
ml of donkey antigoat gamma globulin serum per gram of Kynar is added. The 
mixture is then homogenized again by the Polytron for 5 minutes at the 
same pulse-frequency as before. The suspension is then continuously 
stirred at room temperature for a minimum of 6 hours followed by stirring 
at 4.degree. C. for a minimum of 12 hours. The suspension is now ready to 
be washed. This is accomplished by centrifugation at 1500.times.g for 10 
minutes followed by resuspension in 0.02M Tris Hydroxymethylaminomethane 
(Tham) pH 7.5 with 0.1% NaN.sub.3. This process is repeated once more and 
the final material resuspended in 0.02M Tris pH 7.5 with 0.1% NaN.sub.3 to 
100 grams of Kynar per 1000 ml of buffer. The mixture is again stirred and 
5 grams of bovine serum albumin (BSA) per 100 grams of Kynar added. 
Homogenization with the Polytron at 4000 c.p.s. for 5 minutes is the final 
step in this procedure. 
2. Procedure 
a. To 200 .mu.l of patient's serum add 10 .mu.l of soluble goat anti 
LDH.sub.5 serum and vortex. Wait 5 minutes. 
b. Add 200 .mu.l of insoluble second antibody* and vortex. Wait 5 minutes. 
FNT *Make sure that the insoluble antibody suspension is mixed well before use. 
Place a small stirring bar in the reagent bottle and keep mixing while 
delivering the antibody. 
c. Spin at approximately 1000 g for 5 minutes. 
d. Withdraw from the supernatant whatever amount is needed for a 
conventional LDH activity assay. Use same assay procedure. 
3. Calculations 
LDH.sub.1 activity=Activity in supernatant.times.2.05 
4. Interpretation 
To decide on a cut-off point for LDH.sub.1 an arbitrary value is 
established. This value will differ for each individual laboratory 
depending on the normal total LDH range of the enzyme assay being used. To 
establish an upper limit of normal for LDH.sub.1 (H.sub.4) take 30% of the 
upper level of normal for the total LDH enzyme activity assay. For 
example, the Fisher Diagnostic LDH Assay provided an upper limit of normal 
of 149 I.U./l. (The normal range is 52-149 I.U./l.) Therefore, the cut-off 
point adopted turned out to be 30% of 149 or 45 I.U./l. Any serum showing 
an LDH.sub.1 activity above 45 I.U./l will be considered positive for 
myocardial infarct. 
Clinical Results 
Sera from 106 patients from a Cardiac Care Unit were examined for LDH.sub.1 
elevation. In all 72 patients where a myocardial infarct was diagnosed, an 
LDH.sub.1 elevation was observed. However, LDH.sub.1 remained non-elevated 
for all 34 non-myocardial infarct patients. A cutoff of 45 IU/l was 
designated for biochemical diagnoses of infarct. The range for LDH.sub.1 
activity in MI patients was 46-470 IU/l. The range for the non-MI patients 
was 9-44 IU/l. Presently most laboratories determine the LDH.sub.1 
/LDH.sub.2 "flip" by electrophoresis. This technique is tedious and time 
consuming. By contrast the present immunochemical procedure is simple and 
fast. In addition, determining LDH.sub.1 elevation is more sensitive than 
the flip. In 59 of the 72 MI patients, the LDH flip occurred the same day 
as the LDH.sub.1 evaluation. However, in 8 cases the LDH flip occurred one 
day after the LDH.sub.1 elevation, and in 5 MI patients a flip was not 
obtained (see Tables I, II and III). 
TABLE I 
______________________________________ 
LDH Flip 
Same day Day after 
Number of 
LDH-1 as LDH-1 LDH-1 
MI patients 
Elevated elevation elevation 
Not present 
______________________________________ 
72 72 59 8 5 
______________________________________ 
TABLE II 
______________________________________ 
Number of 
Non-MI LDH-1 LDH-Flip 
patients Elevated Present 
______________________________________ 
34 None None 
______________________________________ 
TABLE III 
__________________________________________________________________________ 
Part A 
LDH-1 Elevated LDH Flip Present 
Number of 
CPK-MB 
Same Day 
One Day Same Day 
One Day 
MI patients 
Present 
as CPK 
after CPK 
Not at All 
as CPK 
after CPK 
Not at All 
__________________________________________________________________________ 
58 55 43 12 None 31 19 5 
__________________________________________________________________________ 
Part B 
LDH Flip Present 
Same Day 
One Day 
Number of 
DPK-MB 
LDH-1 as LDH-1 
after LDH-1 
MI patients 
Absent 
Elevated Elevation 
Elevation 
Not at All 
__________________________________________________________________________ 
58 3 3 2 1 None 
__________________________________________________________________________