Journal of Clinical Laboratory Analysis 5:439-442 (1991)

An for the

Enzyme lmmunoassay Screening Test Detection of Total Antinuclear Antibodies

Napoleon M. Monce, Jr.,’ Ronald T. Bogusky,2 and N. Niels Cappel’ ‘Helix Diagnostics, Inc., West Sacramento, California; ’Department of Medicine, University of California,

Davis, Medical Center, Sacramento, California

The enzyme immunoassay antinuclear anti- body (EIA ANA) screening test method is a new assay format for the qualitative determi- nation of antinuclear antibodies (ANAs) in human serum or plasma. This assay method collectively detects ANAs against double stranded DNA (dsDNA), SS-AIRo, SS-BILa, Scl-70, Sm, and SmlRNP antigens, along with serum positive for peripheral, homoge- neous, speckled, nucleolar, and centromere

Key words:

patterns. This assay correlated well with data obtained with hemagglutination testsfor spe- cific ANA antigens, with the indirect immu- nofluorescence (IFA) ANA HEp-2 test, and with the Critftidia fucifiae IFA test for anti- dsDNA. This new test procedure is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples.

enzyme-linked immunosorbent assays, autoantibodies, immunofluorescence, hem- agglutination, autoimmune disease

INTRODUCTION

The serological characterization of autoantibodies react- ing with cellular and nuclear components is of major impor- tance in the diagnosis of systemic rheumatic diseases. The most useful autoantibodies in the initial screening for rheu- matoid arthritis (RA), systemic lupus erytheinatosus (SLE), and related rheumatic diseases are rheumatoid factors and anti- nuclear antibodies (1). These antibodies are generally detected using hemagglutination techniques, immunodiffusion, and indirect immunofluorescence tests (2-4). These tests, although simple to perform, have several drawbacks. First, positive sera must be titered to obtain a semiquantitative result because low titers of these antibodies are known to occur in several non-rheumatic diseases (5). Second, they are laborious when testing large numbers of pateint samples, and third, they are subject to errors from human interpretation as well as variability in fluorescence microscopes (4).

During the past several years, enzyme-linked immuno- sorbent assays (ELISAs), performed with purified antigens isolated from natural tissue sources, increasingly have been used to detect specific antinuclear antibodies (6-8). The sen- sitivity and specificity of these newer methods have been excel- lent as demonstrated by correlation with existing methods. Similar principles were recently used to devise an enzyme immunoassay antinuclear antibody (EIA ANA) test that detects total antinuclear antibodies.

In this evaluation, we compared the performance of an EIA ANA test method with that of conventional diagnostic methods currently in use for ANA detection and charac- terization.

0 1991 Wiley-Liss, Inc.

MATERIALS AND METHODS

Serum Samples

Human serum samples were obtained from several medi- cal centers and reference laboratories. Twenty-one were pur- chased characterized serum samples, 126 were indirect immunofluorescence (IFA) ANA positive samples collected from a local reference laboratory and confirmed positive by Helix Diagnostics’ IFA ANA, 30 were known normal sam- ples, and 225 were IFA ANA negative samples collected from a local reference laboratory and confirmed negative on Helix Diagnostics’ IFA ANA. Samples were aliquoted and stored at - 29°C until tested.

lmmunofluorescence Procedure

Detection of ANA pattern and titer via indirect immuno- fluorescence was accomplished using the HEp-2 cell line (Helix Diagnostics, West Sacramento, California). The anti- body measurements were run according to the package insert instructions provided with each kit.

In general, serial dilutions of patient samples starting at 1:40 were done using phosphate buffered saline (PBS). Reac- tion wells were layered with 0.02 ml of each dilution and allowed to incubate at room temperature in a moist chamber for 30 minutes. Each slide contained a positive and negative control (included in the kit). After this initial incubation period,

Received May 13, 1991; accepted September 5 , 1991.

Address reprint requests to Napoleon M. Monck, Jr., Helix Diagnostics, Inc., 3148 Industrial Blvd., West Sacramento, CA 95691.

440 Monc6 et at.

slides were washed with a strong stream of PBS and layered with fluorescent conjugated goat antihuman IgG (heavy and light chains) and incubated again for 30 minutes at room tem- perature in a moist chamber. After this incubation period, slides were again washed with a strong stream of PBS and mounted with glycerol, coverslipped, and read. Slides were read with an Olympus BH-2 fluorescent microscope on the same day that they were prepared. A serum is considered pos- itive when the fluorescent intensity of the cells’ nuclei is greater than the negative control and there is a clearly discernible pattern in the nuclei.

dsDNA Procedure

Detection of antibodies to double stranded DNA (dsDNA) was accomplished using the Crithidiu luciliue test (Helix Diag- nostics, West Sacramento, California). The test was run according to the package insert instructions provided with each kit. The procedure is similar to that of the immunofluores- cent ANA assay, except that serial dilutions of patient sam- ples start at 1 : 10.

Hemagglutination Procedure

Antibodies against SS-A/Ro, SS-B/La, Sm, and Sm/RNP were determined using hemagglutination (Hemagen Diagnos- tics, Waltham, Massachusetts). The test was run according to the package insert instructions provided with each kit.

In general, samples were diluted 150, 1:100, and 1:200 in SeraTest Serum Diluent. A 0.05 ml aliquot of each dilu- tion was added into a V-well microtiter plate. Each plate contained a positive and negative control (included in the kit). One free-falling drop of Sera Cell (red cell suspension) was then added into each well. After a 90 minute undisturbed incubation period at room temperature, each sample was read according to the criteria given.

EIA ANA Procedure

Samples were screened for the presence of ANAs using the EIA ANA screening test (Helix Diagnostics, West Sacramento, California). The antigen used in the EIA ANA preparation is a nuclear extract of HEp-2 cells with additional SS-A added. In general, after 1:40 dilutions of patient samples with serum diluent were done, 0.1 ml of each sample was added into antigen coated polystyrene microtiter wells and allowed to incubate for 30 minutes at room temperature. Wells were then washed five times with PBS-Tween followed by five washes with deionized (DI) water. Horseradish peroxidase (HRP) con- jugated antihuman IgG (0.1 ml) was then added to each well. After another 30 minute incubation period, wells were again washed as before. Color development solution (0.1 ml) was then added to each well and the reaction was stopped after 10 minutes by adding 0.1 ml of stopping solution. Wells were read using a Bio Tek microplate reader set at 450 nm with a maximum optical density (O.D.) reading capability of 2,000.

RESULTS

Comparison of the EIA Test With IFA Test

Serum samples (n = 402) were tested with the IFA test and with the EIA test to screen for the presence of antinu- clear antibodies. After determination of IFA ANA pattern and titer, the 147 IFA positive samples were then tested for the presence of dsDNA by the Crithidiu luciliue test, and for S S - A / Ro, SS-B/La, Sm, and Sm/RNP antibodies by hemagglutination.

A 2 X 2 table of positive and negative results for the EIA ANA test versus the IFA test is shown in Table 1. Of the 402 samples tested, 135 were positive and 232 were negtive on both the EIA and IFA tests. The average EIA O.D. reading, at 450 nm, of the 147 IFA positive and the 232 IFA negative samples was 0.999 and 0.101, respectively. The O.D. range for the 30 known normal samples was 0.004 to 0.089. From this data, a cutoff value of 0.235 was assigned by taking the mean negative value plus two standard deviations. Using this cutoff value, the EIA test detected 23 additional positive sam- ples which the IFA test did not, while the IFA test detected 12 additional positives which the EIA test did not. Defining sensitivity as the ability of a test to give a positive result for positive samples [true positives/true positives + false nega- tives (TP/TP + FN)], and specificity as the ability of a test to give a negative result for negative samples [true negatives/ true negatives + false positives (TN/TN + FP)], we found the EIA test to be 91 .O% specific and 91.8% sensitive when compared to the IFA test.

A linear regression diagram comparing the EIA O.D. value with the IFA titer of the 402 samples was graphed and a cor- relation coefficient (r = .792) was calculated (Fig. 1).

Comparison of the EIA Test With IFA Staining Patterns

The main IFA staining patterns observed were homoge- neous, centromere, speckled, peripheral, and nucleolar. The average EIA O.D. reading for each pattern is shown in Table 2. Peripheral staining consistently gave the highest readings with the smallest standard deviation. This pattern is known to correlate well with high titer ANA testing.

Comparison of the EIA Test With Hemagglutination and Crithidia luciliae

Of the 147 IFA positive samples, 17 were positive for dsDNA, 38 for SSA/Ro, 8 for SSB/La, 3 for Sm, and 16 for

TABLE 1. Comparison of EIA ANA and IFA ANA Test

IFA - +

EIA + 135 23 12 232 -

Agreement (TP + TN)/total: 91.3%

Enzyme lmmunoassay Antinuclear Antibody Screening 441

Optical Density

1.9 t 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1

'40 '80 '160 '320 '640 '1280 '2560 IFA Titer

(Reciprocal) 1 to

TABLE 3. Average EIA O.D. Reading for Specific Antibodies'

Antibody Numberdetected Average EIA O.D. reading f S.D.

dsDNA 17 1.848 - + 0.287 SS-A 38 1.271 ? 0.526 SS-B 8 1.433 i 0.644 Sm 3 1.837 * 0.282 S d R N P 16 1.233 2 0.585

'0.D. readings at 450 nm

Fig. 1. calculated correlation coefficient.

Linear regression diagram comparing IFA titer with EIA O.D. and

Sm/RNP. All of these samples were detected and gave a pos- itive reading on the EIA ANA test: 100% agreement. The average O.D. reading for each antibody is shown in Table 3.

Comparison of the Intra- and Inter-Assay Variability

Three samples were run 24 times on the €CIA ANA assay to test for any intra-assay variability. Their respective O.D. range and coefficient of variation (C.V.) are given in Table 4. The same three samples were run on five different produc- tion lots to test for any inter-assay variabiity. The C.V. range for each sample is also given in Table 4. These results sug- gest a close correlation within and between production lots.

DISCUSSION

Detection of ANAs in human serum has proven useful in the confirmation of autoimmune disease diagnoses. Conse- quently, sensitive methods for the detection and character- ization of these antibodies are required. The IFA is commonly used for the detection of ANAs. However, several disadvan- tages exist; standardization is difficult both intra- and inter- laboratory; results are subjective on the part of tihe technologist; and it is extremely time consuming to screen large numbers of patient samples. The recently developed EI,4 ANA screen- ing test offers distinct advantages over the current technol- ogy. The EIA ANA test provides objective results as judged by an automatic plate reader, thus eliminating observer bias. It can also be automated, making it more convenient to screen large numbers of patient samples with minimal intra- and inter-assay variability which is appropriatc: for hospitals and large clinics.

TABLE 2. Average EIA O.D. Reading for Each IFA Staining Pattern'

IFA staining Number Average O.D. pattern detected reading 2 S.D.

Nucleolar i l 0.1584 ? 0.474 Speckled 55 0.126 * 0.427 Centromere 8 0.071 ? 0.241

Peripheral 4 1 .I986 * 0.029

'0.D. readings at 450 nm.

Homogeneous 49 1.470 ? 0.690

In this study, we have applied the EIA ANA test to screen for the presence of ANAs. We tested 402 human serum sam- ples on both the IFA and EIA test. The IFA test detected 147 positive and 255 negative samples, while the EIA test detected 158 positive and 244 negative samples (Table 1). Using the established O.D. value of 0.235 as a cutoff, the EIA test also detected 23 samples which were negative on the IFA test (23 false positives), but did not detect 12 samples which were positive on the IFA test (12 false negatives). The mean O.D. value for the 23 false positive samples was 0.367 and had the following disease associations: discoid lupus, hypertension, cerebral vascular accident, adult diabetes, leucopenia, cere- bral palsy, hypothyroid, streptococcus infection, and rheu- matoid arthritis. Their detection was probably due to the greater sensitivity of the EIA format (9). The 12 false negative sam- ples detected were low IFA ANA titers (1:40, 1:80, and 1:160), with nucleolar, speckled, and homogeneous patterns. These false negatives were not specific for SS-A/Ro, SS-B/La, Srn, Sm/RNP, or dsDNA. From these results, the EIA ANA test method was found to have a 91.8% sensitivity and a 91 .O% specificity. It was also found to have a high correlation coef- ficient of .792 and a 91.3% agreement [(TP + TN)/total] when compared to the IFA test (Fig. 1).

The main IFA staining patterns observed were homoge- neous, centromere, speckled, peripheral, and nucleolar. Detec- tion of these staining patterns is important for patient diagnosis because different staining patterns have been associated with specific diseases (10). The EIA test was able to detect these patterns along with two mixed patterns (speckled-homoge- neous and speckled-nucleolar) with little or no difficulty. Only a few of the very low IFA titered homogeneous, speckled, and nucleolar patterns fell below the cutoff value (false nega- tives). The lowest pattern average O.D. value was 0.684, with peripheral staining having the highest O.D.

Crithidia luciliae and hemagglutination tests detected antibodies for dsDNA, SS-NRo, SS-B/La, Sm, and SmlRNP

TABLE 4. Intra- and Inter-Assay O.D. and Coefficient of Variation'

Intra-assay Inter-assay

Samule number O.D. ranee C.V. (%) C.V. ranee (%)

1 1.253-1.583 6.0 2.4-7.0 2 0.275-0.366 7.4 5.5-8.5 3 0.21 7-0.268 6.1 4.9-7.3

'0.D. readings at 450nm.

442 Monce et al.

from our positive samples. All specific antigen positives were detected on the EIA test (100% agreement); the lowest average O.D. reading was 1.233 (Table 3).

In summary, the EIA ANA screening test procedure demon- strated excellent performance in comparison with the immuno- fluorescence, hemagglutination, and Crithidia luciliae IFA test. Furthermore, we found this antinuclear antibody detection method to be very specific, very sensitive, and very easy to perform when screening large numbers of patient samples.

REFERENCES

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3. Friou GJ: Antinuclear antibodies: Diagnostic significance and meth- ods. Arthritis Rheum 10:151-159, 1967.

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9. Reeves WH, Fisher DE, Lahita RG, Kunkel HG: Autoimmune sera reac- tive with Sm antigen contain high levels of RNP-like antibodies. J Clin Invest 75580-587, 1985.

10. ANA Atlas; An Interpretive Guide to Immunojluoresceni HEp-2 Anti- nuclear Antibodies. Helix Diagnostics, Inc., West Sacramento, Cali- fornia, 1990.