ANA (ANTI-NUCLEAR ANTIBODIES)
ANA (ANTI-NUCLEAR ANTIBODIES)
PREPARATION FOR ASSAY
1. Before beginning the test, bring all samples and reagents to room temperature and mix each gently.
2. Have all reagents and samples ready before the start of the assay. Once the test has begun it must be performed without any interruption to get the most reliable and consistent results
3. Use new disposable tips for each sample.
ASSAY PROCUDURE (30/30/15)
1. Secure the desired number of coated wells in holder.
2. Prepare 1:40 dilutions of test samples, Cutoff control, and Positive control by adding 10 ul of sample to 0.4 mL sample diluent in the separate tubes.
3. Dispense 100 ul of diluted Cutoff Control, diluted Positive Control , and diluted patient samples in duplicate into appropriate wells.
4. Incubate 30 minutes at room temperature.
5. Rinse the wells 5 times with washing solution.
6. Dispense 100 ul of enzyme conjugate into each well and mix for 5 seconds and incubated at room temperature for 30 minutes.
7. Remove mixture and rinse the wells 5 times with washing solution. (Be sure to wash the wells thoroughly and completely dry the wells. Improper wash may cause false positive results).
8. Dispense 100 ul of Solution A and 100 ul of Solution B into each well. Mix for 5 seconds and incubated in the dark for 15 minutes.
9. Stop reaction by adding 50 ul of 1N H2SO4 to each well and read at 450 nm with microwell reader against Blank well (only Solution A and Solution B).
Microtiter strip must be read with an ELISA reader set at 450 nm. Results should be read after stopping solution. Step 9 and reported as follows:
Negative: A negative response is indicated by an O.D.
Reading less than the O.D. of the Endpoint Cutoff control wells.
Positive: A positive response is indicated by an O.D. reading equal to or higher than the Endpoint Cutoff Control wells.
For Quantitative Report:
If quantitative report is desirable, the following is intended as a guide for the interpretation:
The ANA Index (AI) for a patient sample is defined as O.D. ratio between patient sample and that of endpoint cut Control. For example:
O.D. of patient sample: 1.00
O.D. of Endpoint Cutoff: 0.5
AI of patient sample = 1.00/0.50 =2
AI<1: ANA is Negative
AI=1, or>1 ANA is low positive
AI=2, or >2.0: can be run on ANA slide for pattern and titer or specific ANA follow-up tests to determine specific ANA involved.
The approximate incidence of positive ANA is 5% in the general normal population, 40% in normal old age and 25% in healthy relatives of SLE patients. The incidence of a positive ANA has been demonstrated in several disease states.
Disease Positive ANA # Incidence
Juvenile RA 20%
1. Reichlin, M. Current Perspectives on Serological Reactions in SLE patients Clin. Exp. Immunol. 44:1-10, 1981.
2. Venables, P.J.W., Erhardt, C.C., and Maini, R.N. Antibodies to Extractable Nuclear Antigens in Rheumatoid Arthritis: Relatioship to Vasculitis and Circulating Immune Complexed. Clin. Exp. Immunol. 39:16, 1980.
3. Harmon, C.E. Antinuclear Antibodies in Autoimmune Diseases; Significance and Pathogenecity. Medical Clinics of North America 69:547, 1985..
4. Rothschild B.M., Jones J.V., Chesney C., Pifer D. Thompson, L.D., James, K.K., and Badger, H. Relationship of Clinical findings in Systemic Lupus Erythematosus to Sero-Reactivity. Arthritis and Rheumatism 26:45-51, 1983.