Evaluation of newly developed ELISA using "MESACUP-2 test mitochondrial M2" kit for the diagnosis of primary biliary cirrhosis.
OBJECTIVE
An enzyme-linked immunosorbent assay (ELISA) using MESACUP-2 Test Mitochondria M2 kit (new-M2 ELISA) has recently become commercially available. The aim of this study was to evaluate the clinical utility of this newly developed ELISA for the diagnosis of primary biliary cirrhosis (PBC).


METHODS
We tested the immunoreactivity of sera from 82 Japanese PBC patients to the 2-oxo-acid dehydrogenase complex (2-OADC) enzymes by indirect immunofluorescence, enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit, commercial ELISAs using MESACUP Mitochondria M2 kit (old-M2 ELISA) and new-M2 ELISA, and immunoblotting on bovine heart mitochondria.


RESULTS
Each test gave the following positive results; antimitochondrial antibodies (AMA) by immunofluorescence in 71 (87%) out of the 82 sera, enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) by EMA in 61 (74%), immunoglobulin (Ig) G class anti-PDC antibody by old-M2 ELISA in 55 (67%), IgG/M/A class anti-E2 subunit of PDC (PDC-E2)/anti-E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADC-E2)/anti-E2 subunit of 2-oxoglutarate dehydrogenase complex (OGDC-E2) antibodies by new-M2 ELISA in 73 (89%), and IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes by immunoblotting in 82 (100%). Fifty-three of the 82 sera (65%) were all positive by these five assays. Of the 18 sera that were positive by new-M2 ELISA but negative by old-M2 ELISA, 12 were theoretically interpretable. Of the 11 sera that were negative for AMA by immunofluorescence but positive for at least one of anti-2-OADC enzymes by immunoblotting, four (36%) were positive by new-M2 ELISA, whereas only two and one sera were positive by EMA and old-M2 ELISA, respectively.


CONCLUSIONS
Our results indicated that the sensitivity of the newly developed new-M2 ELISA was higher than that of EMA and old-M2 ELISA, and comparable with that of immunofluorescence. However, it is still unclear whether the new-M2 ELISA could replace the conventional immunofluorescence testing for routine assay requests because six (7%) sera showed discrepant results between these two assays.
Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.
Yoshiko
Y
Kadokawa
Katsuhisa
K
Omagari
Hiroaki
H
Hazama
Kazuo
K
Ohba
Jun-ichi
J
Masuda
Hideki
H
Kinoshita
Kenji
K
Hayashida
Hajime
H
Isomoto
Yohei
Y
Mizuta
Kunihiko
K
Murase
Ikuo
I
Murata
Shigeru
S
Kohno
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000906:Antibodies / Q000032:analysis / Q000097:blood; D001323:Autoantibodies / Q000097:blood; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D007655:Ketoglutarate Dehydrogenase Complex; D008105:Liver Cirrhosis, Biliary / Q000097:blood / Q000175:diagnosis*; D008875:Middle Aged; D008928:Mitochondria / Q000276:immunology*; D011768:Pyruvate Dehydrogenase Complex; D012372:ROC Curve; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
D000906:Antibodies; D001323:Autoantibodies; D011768:Pyruvate Dehydrogenase Complex; D011933:Reagent Kits, Diagnostic; D007655:Ketoglutarate Dehydrogenase Complex; D051047:Dihydrolipoyllysine-Residue Acetyltransferase