an enzyme-linked immunosorbant assay (elisa) for detecting antimitochondrial antibody

0270-9139/84/0404-0727$02.00/0 HEPATOLOGY Copyright 0 1984 by the American Association for the Study of Liver Diseases

Vol. 4, No. 4, pp. '727-730.1984 Printed in U. S. A.

An Enzyme-Linked Immunosorbant Assay (ELISA) for Detecting Antimitochondrial

Antibody MARSHALL M. KAPLAN, JOHN v. GANDOLFO AND ELAINE G. QUARONI

Gastroenterology Division, Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts 021 I 1

W e have developed an enzyme linked immunosorbant assay (ELISA) for antimitochondrial antibody. Polyvinyl microtiter plate wells are coated with partially purified rat kidney mitochondria, and excess protein binding sites are blocked with bovine serum albumin. Human serum, diluted 1: 1,000, is incubated for 1 hr. Then 8-galactosidase-goat-anti-human IgG (H+L) is added followed by the substrate, p-nitrophenyl-8-D-galactopyranoside. The plates are then read at 404 nM in a microelisa autoreader. A positive result was defined as optical density z 0.100, more than 5 standard deviations above the mean of 36 normal individuals. With this techique, 56 of 60 patients with primary biliary cirrhosis were positive for antimitochondrial antibody (93%), mean O.D., 0.456 C 0.031 S.E. Seventeen of 17 patients with extrahepatic bile duct obstruction and 14 or 14 patients with alcoholic cirrhosis were negative. Only 1 of 29 patients with chronic active liver disease was positive (4%). Antinuclear antibody and antimicrosomal antibody do not bind in this assay, and activity is absorbed from sera by preincubation with suspensions of rat kidney mitochondria. The ELISA is approximately 20 times more sensitive than a quantitive microtiter complement fixation technique and more convenient than radioimmunoassay. It is rapid, quantitative and uses stable reagents. In contrast to immunofluorescence techniques, it is not affected by observer interpretation.

Determination of antimitochondrial antibody (AMA) is useful in the diagnosis of primary biliary cirrhosis. AMA is present in more than 85% of patients with primary biliary cirrhosis and is rarely present in patients with mechanical bile duct obstruction (1, 2). However, the currently used methods for determining AMA are not optimal. Immunofluorescence microscopy, the most frequently used technique, requires specialized equipment and is influenced by observer interpretation (3, Whittingham, S., Gastroenterology 1974; 66:169, Corre- spondence). Quantitative complement fixation depends upon a delicate balance of unstable reagents and is tedious to perform (Kaplan, M. M., Gastroenterology 1973; 65548, Abstract). Radioimmunoassays are not widely available and require the use of radioactive substances with relatively short half-lives (4). We have recently developed an enzyme-linked immunosorbant as-

Received November 28,1983; accepted March 27,1984. This work was supported by NIH Research Grant AM 28490, NIH

Research Training Grant AM 07024 and NIH Clinical Study Unit Grant RR 300054.

Address reprint requests to: Marshall M. Kaplan, M.D., New Eng- land Medical Center, 171 Harrison Avenue, Boston, Massachusetts 02111.

say (ELISA) to detect AMA and have evaluated its suitability for routine diagnostic procedures.

MATERIALS AND METHODS PATIENTS

Sera were obtained from the following populations: (i) 36 normal individuais all of whom had normal liver function tests and no suggestion of underlying liver disease: (ii) 60 patients with well-documented primary biliary cirrhosis; all had chronic cholestatic liver disease, patent bile ducts and liver biopsies diagnostic or consistent with primary biliary cirrhosis; (iii) 17 patients with mechanical bile duct obstruction demonstrated either by radiologic technique or a t surgery; (iv) 14 patients with alcoholic liver disease; all had histories of alcohol abuse and percutaneous needle biopsies of the liver diagnostic of alcoholic liver disease; (v) 10 patients with persistent hepatitis, and 19 with chronic active hepatitis; all had elevations of the aminotransferases and had liver biopsies consistent with chronic active liver disease; (vi) 10 patients who had circuating antibodies to other cytoplasmic organelles. Five had antinuclear antibodies in titers ranging from 1:256 to 1:8,192. Four of these patients had systemic lupus erythematosus and one had

727

728 KAPLAN, GANDOLFO AND QUARONI HEPATOLOGY

chronic active hepatitis. Five patients had antimicroso- ma1 antibodies in titers ranging from 1:1,600 to 1:102,400. Three of these patients had thyroiditis, one had ne- phrotic syndrome, and one had multiple endocrinop- athies and moniliasis.

MATERIALS Bovine serum albumin (BSA), radioimmunoassay

Grade, p-nitrophenyl-0-D-galactopyranoside and poly- oxyethylene sorbitan (Tween 20) were obtained from Sigma (St. Louis, Mo.). 0-galactosidase-goat-anti-human IgG (H+L) was obtained from Zymed (Burlingame, Calif.). Antisheep hemolysin and guinea pig complement- C’ were obtained from the Colorado Serum Company (Denver, Colo.). Sheep red blood cells were obtained from Donald Gaulitz, of the Massachuesetts General Hospital (Boston, Mass.). Fetal calf serum was obtained from Gibco Laboratories (Grand Island, NY). Mixed goat and burro-fluoresceinated antihuman y -globulin was obtained from Meloy Laboratories, Inc. (Springfield, Va.). MicroTest I11 flexible assay plates (Falcon 3911) were obtained from Becton Dickinson Labware (Oxnard, Calif.). Optimal densities were determined either on a Gilford Spectrophotometer Model 240 or on a microelisa Autoreader manufactured by Dynatech (Alexandria, Va.).

METHODS Rat kidney mitochondria were prepared according to

the method of Hogeboom (5). For use in the ELISA, mitochondria were diluted to a concentration of 46 pg protein per ml in phosphate-buffered saline (PBS), 0.015 M (pH 7.2), 0.1% sodium azide. This protein concentration was found to be optimal after testing varying mitochondrial protein concentrations. Diluted mitochondria (0.1 ml) were incubated at room temperature for 1 hr in wells of microtiter plates. The mitochondria suspension was removed with suction and the plate washed twice with PBS-l% BSA, 0.1% NaN3 (pH 7.2). The second wash was left on the plate for approximately 30 min. This step blocks the remaining protein binding sites on the well. At this point, the plate may be stored at 4°C for at least 1 month if kept dry and covered with Saran wrap. All subsequent dilutions and washes were made with PBS-0.05% Tween, 0.05% BSA, 0.1% NaN3 (pH 7.2).

In additional experiments, microtiter plate wells were coated with equal concentrations of sonicated rat kidney mitochondria and with antigens other than rat kidney mitochondria. For this latter purpose, radioimmunoassay grade bovine serum albumin and fetal bovine serum were diluted to concentrations of 46 pg protein per ml in PBS, and wells were coated and prepared as described above.

For assay, human sera were diluted 1:1,000 in 0.1 ml of the PBS buffer and incubated for 1 hr at room temperature in the appropriate well. Each sample was run in duplicate together with control wells which contained no mitochondria. After the incubation, the sera were removed by suction and the plate washed three times with the above PBS. Then, 0.1 ml P-galactosidase-goat- anti-human IgG (H+L), diluted 1:500 in PBS was added

to each well, incubated for 1 hr a t room temperature, and removed by suction. The plates were then washed three times with PBS. Next, 0.1 ml of substrate, 5 mM p - nitrophenyl-0-D-galactopyranoside in 10 mM MgC13- PBS, 0.1% NaN3 (pH 7.5), was incubated for 1 1/2 hr at 37°C in a water bath. Optical density at 404 nM was read in a Dynatech Microelisa Autoreader.

Sera positive and negative for AMA were run as controls on each plate. The positive control serum value was used to correct for day-to-day variations between plates. These variations were typically small, 3.9% +_ 1.0% S.E. range 0.3 to 9%, when eight positive samples were run weekly for 1 month.

Protein was determined by the method of Lowry (6). The microtiter complement fixation method for determination of AMA was performed as previously described (Kaplan, M. M., Gastroenterology, 1973; 65548, Ab- stract). Antinuclear antibodies, AMAs and antimicroso- ma1 antibodies were detected by indirect immunofluorescence for which purpose a Leitz Ortholux fluorescent microscope was used (7). Unfixed cryostat sections of mouse liver were used to detect antinudear antibodies, mouse heart to detect antimicrosomal antibodies and mouse kidney to detect AMAs.

ABSORPTION OF POSITIVE SERA WITH MITOCHONDRIA Sera from four patients with primary biliary cirrhosis

which were strongly positive for AMA by ELISA were diluted 1:105 in PBS-0.5% BSA and incubated with serial dilutions of partially purified rat kidney mitochondria at 37°C for 60 min. At the end of the incubations, the mitochondria were sedimented at 24,000 x g for 10 min. The supernatants were then assayed for AMA by the ELISA method.

RESULTS The mean optical density of sera from 36 normal

individuals was 0.022 f 0.015 S.D. (Figure 1). The upper limit of normal was arbitrarily defined as an optical density less than 0.100. This is more than 5 standard deviations greater than the mean of normals and in- cluded all 36 normal individuals. Fifty-six of the 60 patients with primary biliary cirrhosis had positive AMA tests by this criteria. The positive values ranged from optical density 0.126 to 1.0, mean 0.456 f 0.031 S.E. The four patients with primary biliary cirthosis with negative results had values less than 0.067, that is less than 3 standard deviations above the mean of the normals, Thus, lowering the upper limit of normal to a value 3 standard deviations above the mean would not have influenced our results.

The results were identical if sonicated rat kidney mitochondria were used instead of the intact mitochondria. Hence, a sonciation step was not used in subsequent experiments. Activity could be absorbed out of the serum of primary biliary cirrhosis patients by preincubation of serum with the mitochondria1 antigen (Table 1). Ten sera, selected because they had circulating antibodies to other cytoplasmic organelles (five sera positive for antinuclear antibody and five positive for antimicrosomal antibody) were all negative for AMA by ELISA. There

Vol. 4, No. 4, 1984 AN ELISA METHOD FOR DETECTING ANTIMITOCHONDRIAL ANTIBODY 729

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Normal PBC BDO ALD CALD N.36 N.60 N.17 N.14 N.28

FIG. 1. AMA by ELISA technique. Results below the horizontal line at O.D. = 0.100 are normal. PBC, primary biliary cirrhosis; BDO, bile duct obstruction; ALD, alcoholic liver disease; CALD, chronic active liver disease. Sera were diluted 1:1,000 for the ELISA.

TABLE 1. ABSORPTION OF AMA BY MITOCHONDRIA 96 AMA activity absorbed/mitochondria dilution 1:lO 1:loo 1:1,000 1:10,000

Patient Initial O.D.

1 0.327 90 35 9 12 2 0.373 85 29 2 13 3 0.108 91 65 48 31 4 0.225 97 83 2 7

positive value, 0.130. The mean of this group was 0.028 f 0.010 S.D., very similar to that of the controI population.

Although the ELISA method was 12 to 28 times more sensitive than the microtiter complement fixation technique (Figure 2), the overall percentage of positives with the two tests were similar. However, two patients who were positive by the ELISA technique (O.D. = 0.217 and 0.174) were negative with the microtiter complement fixation test and, two patients who were negative by ELISA (O.D. = 0.001 and 0.048) were positive with the microtiter complement fixation technique. The reason for these differences was unclear. Of these four patients, the two who were positive by ELISA were also positive by immunofluorescence while 1 of the 2 positive by complement fixation was positive by immunofluorescence.

DISCUSSION The ELISA technique for the detection of AMA is

simple to perform, highly reproducible and quantitative. It is as sensitive and specific as a recently described ELISA method (9) but requires fewer steps. Nagai et al. used a sandwich test system in which microtiter plates were first coated with IgG prepared from a “reference” serum from a patient positive for AMA by immunofluo-

was no reactivity of ELISA positive sera if the microtiter plates were coated with antigens other than partially purified rat kidney mitochondria, i.e., radioimmunoassay grade BSA and fetal calf serum.

All 17 patients with bile duct obstruction had normal values. The mean of this group, 0.013 k 0.007 S.D., was slightly below that of the 36 normal individuals. All 14 patients with alcoholic liver disease had normal values. The mean of this group was 0.022 f. 0.016 S.D., identical to that of the normal controls. The results of the 10 patients with persistent hepatitis, and 19 patients with chronic active hepatitis were combined into a group referred to as chronic active liver disease (8). Only 1 of these 29 Datients. a 58-vear-old man with asvmtA,omatic

FIG. 2. Comparison between the ELISA and quantitative microtiter complement fixation (CF) techniques for the detection of AMA. Normal ” *

chronic active hepatitis and a seizure disorder, had a values are indicated by the stippled areas.

730 KAPLAN, GANDOLFO AND QUARONI HEPATOLOGY

rescence (9). Sonicated mitochondrial antigen was then bound to the IgG layer and was detected by peroxidase conjugated-“reference” IgG that bound to the mitochondrial antigen. Identification of AMA in patients’ sera was by a blocking method in which test serum was applied before the addition of the conjugated “reference” IgG second antibody. In our assay we examined the binding of patients’ antibody directly to mitochondria which thereby eliminated the need to isolate and conju- gate IgG from a reference patient source, and allowed the use of a commercially available p-galactosidase-conjugated goat antihuman IgG second antibody.

The ELISA is more sensitive and easier to perform than the microtiter complement fixation trechnique which we have been using, and unlike immunofluorescence techniques, is not affected by observer interpretation ( 3 , Whittingham, S., Gastroenterology 1974; 66:169, Correspondence).

It is more convenient than a recently described radioimmunoassay for AMA because it uses stable reagents, and does not require radioactive substances and expensive counting equipment (4).

The ELISA detects AMAs of all major isotypes because the second antibody is both heavy and light chains- specific and recognizes IgG, IgM, and IgA. Although we cannot explain the few observed discrepancies between the ELISA, complement fixation and immunofluorescence results, similar discrepancies among assays have been noted by others (4, 9). It is unlikely that these differences influence the clinical value of the method. Although our data suggest that the M2 antigen described previously is being detected in our assay, we made no further attempt to verify this (10, 11).

In summary, we have decribed a convenient, reproducible, sensitive and specific assay for AMA that can be used by any laboratory with a spectrophotometer. Be-

cause the assay is quantitative and not subject to observer variability as is immunofluorescence, it will lend itself to longitudinal studies of AMA titers in patients with primary biliary cirrhosis.

Acknowledgments: We thank Mary Calderazzo and Florence Hagins for their assistance in typing the man- uscript and Dr. Andrew Plaut for his helpful suggestions.

REFERENCES 1. Walker JG, Doniach D, Roitt IM, et al. Serological tests in diag-

nosis of primary biliary cirrhosis. Lancet 1965: 77:533-541. 2. Klatskin G, Kantor FS. Mitochondrial antibody in primary biliary

cirrhosis and other diseases. Ann Intern Med 1972; 77:533-541. 3. Lam KC, Mistilis SP, Perrott N. Positive tissue antibody tests in

patients with prolonged biliary obstruction. N Engl J Med 1972;

4. Manns M, Meyer zum Buschenfelde K-H. A mitochondrial antigen-antibody system in cholestatic liver disease detected by radioimmunoassay. Hepatology 1982; 2:l-7.

5. Hogeboom GH. Fractionation of cell components of animal tissues. In: Kaplan NO, Colowick SP, eds. Methods in enzymology. New York: Academic Press, Inc., 1955: 1:16-19.

6. Lowry OH, Rosebrough MJ, Farr AL, et al. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-275.

7. Klatskin G, Kantor FS. Mitochondrial antibody in primary biliary cirrhosis and other diseases. Ann Intern Med 1972; 77:533-54.

8. Soloway RD, Summerskill WHJ, Baggenstoss AH, et al. Clinical, biochemical, and histological remission of severe chronic active liver disease: a controlled study of treatments and early prognosis. Gastroenterology 1972; 63:820-833.

9. Nagai S, Manns M, Meyer zum Bushenfelde K-H, et al. Detection of mitochondrial antibodies directed against the primary biliary cirrhosis (M2) antigen by an enzyme-linked immunosorbant assay (ELISA). J Immunol Methods 1983; 6077-88.

10. Miyachi K, Gupta RC, Dickson ER, et al. Precipitating antibodies to mitochondrial antigens inpatients with primary biliary cirrhosis. Clin Exp Immunol 1980; 39599-606.

11. Meek F, Khoury EL, Doniach D, et al. Mitochondrial antibodies in chronic liver diseases and connective tissue disorders: further characterization of the autoantigens. Clin Exp Immunol 1980;

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an enzyme-linked immunosorbant assay (elisa) for detecting antimitochondrial antibody

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