Quantification of lactate dehydrogenase-1 in serum with use of an M-subunit-specific monoclonal antibody.
We have developed a rapid, one-step assay for measuring lactate dehydrogenase-1 (LD-1) activity in serum after extraction of LD-2, LD-3, LD-4, and LD-5 isoenzymes by an immobilized M-subunit-specific monoclonal antibody (D.8.1). In the assay, 100 microL of serum is mixed with 50 microL of a suspension of 0.8-micron-diameter latex particles coated with 30 micrograms of the monoclonal antibody D.8.1, then incubated at room temperature for 5 min. The latex particles, to which LD-2 through LD-5 are bound, are pelleted by centrifugation for 2 min at 12,000 X g, and the LD-1 activity is measured kinetically in the supernatant fluid. We optimized the assay for antibody immobilization, antibody concentration, and time and temperature of incubation. Serum bilirubin concentrations up to 0.33 g/L (0.56 mmol/L) did not interfere in the assay. Hemolysis interfered solely through LD-1 released from erythrocytes. The within-assay CV for low-concentration quality-control material (LD-1 33 U/L) was 3.5% (n = 9) and for high-concentration material (LD-1 185 U/L) was 1.9% (n = 8); the between-assay CVs for the two materials were 6.1% (n = 9) and 2.5% (n = 10), respectively. The LD-1 activity measured in 98 samples by our assay compared well with a two-step polyclonal antibody-based assay (Isomune-LD, Roche Diagnostic Systems; r = 0.998) and with an electrophoretic method (Paragon, Beckman Instruments; r = 0.956).
Vaidya
H C
HC
Washington University School of Medicine, Department of Pathology, St. Louis, MO 63110.
Porter
S E
SE
Landt
Y
Y
Silva
D P
DP
Dietzler
D N
DN
Ladenson
J H
JH
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000097:blood*; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011863:Radioimmunoassay; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase