Prima Biomedical Co

148 8th Ave., Suite E La Puente, CA 91746 Tel: (626) 333 4626 Fax: (626) 333 4696 FOR THE DETECTION OF HEPATITIS B SURFACE ANTIGEN SUMMARY AND EXPLANATION OF THE PROCEDURES The Prima HBsAg one step test is a chromatographic based immunoassay in vitro rapid, sensitive, special and simple test designed for a qualitative determination of Hepatitis B Surface Antigen (HBsAg) in human serum or plasma. The discovery of Australian antigen by Blumberg, et. al., and its subsequent identification as the surface antigen of hepatitis B virus (HBsAg) represents a significant break through in the understanding of the disease, serum hepatitis. It is known that the screening of blood donor for the presence of this antigen in serum has significantly reduced the incidence of hepatitis B in blood transfusion recipients. HBsAg has an antigenic heterogeneity. The principal determinant is called a (a1, a2, a3) and is commontoallthe different sero-types of HBsAg. Two couples of subspecific determinants have also been identified, that is d/y (1y, 2y, 3y) and w/r which seems to be mutually exclusive. Therefore the following combinations are possible: adw, adr, ayw, ayr. BIOLOGICAL PRINCIPLE OF PROCEDURE The Prima HBsAg test is a chromatographic immunoassay (CIA) for the rapid qualitative determination of human hepatitis B surface antigen (HBsAg) in serum. The membrane is pre-coated with anti-HBs capture antibody on the test band region and Rabbit anti-gaot IgG on the control band region. During testing, the serum specimen is allowed to react with the colloidal gold particles which have been coated with gaot-anti-HBs. The mixture then moves latterally on the membrane by capillary action. For a positive result, a pink colored band with a specific antibody-HBsAg-antibodycolloidal gold particle complex will form on the test band region. Absence of colored band in the test band region indicates a negative result. To serve as a procedural control, a pink colored band at the control region will always appear regardless the presence of HBsAg. STORAGE AND STABILITY The Prima HBsAg test should be stored at room temperature (up to 30 C) in the sealed pouch or desiccated container. PRECAUTION

ONE STEP HBsAg DEVICE TEST

Do not use test kit beyond expiration date. There should be no smoking or eating where antigen containing materials are being handled. Wear disposable gloves and lab caot while handling specimens. Wash hands thoroughly afterwards. Decontaminate and dispose of specimens and all potentially contaminated materials as if they contain infectious agent. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated.

SPECIMEN COLLECTION:

The Prima HBsAg test is performed on human serum or plasma. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible to avoid hemolysis. Lipemic, icteric, or hemolyzed specimens may give inconsistent test result. Specimens, if containing precipitate, should be clarified prior to testing. HBsAg is thermo-labile. If specimens are not to be tested they should be refrigerated immediately at 2 to 8 C , if storage periods greater than 3 days are anticipated , the specimen should be frozen. If specimens are to be shipped, they should be packed in compliance with federal regulations covering the transportation of etiologic agents. Do not use heat-inactive specimens. ASSAY PROCEDURE Material Provided

Test Device Specimen Pipette

Material required but not provided Specimen collection container Stop watch

For In Vitro diagnostic use only.

QUALITY CONTROL A procedural control is included in the test. A colored band appearing on the control region of the membrane indicates proper performance and reactive reagents.

Good laboratory practices include the use of external control specimens to ensure proper kit performence. Each day of testing, two level of commercial controls should be tested on the Prima HBsAg test strips The two level of control should consist of a negative control and a positive control containing low level of HBsAg. The use of the low level positive control will assure that the test strips have not been adversely affected and are detecting HBsAg at the stated sensitivity of the test system. TEST PROCEDURE Review specimen collection instructions. Test Devices together with patients samples or controls should be brought to room temperature (20 - 30C) prior to testing. Remove the test devices from its protective pouch (bring the sealed pouch to room temperature before opening to avoid condensation of moisture on the membrane). Label the strip with patient or control identifications. Use the dropper or pipette to withdraw specimen or control from the specimen collection container and dispense 5 drops (approximately 0.2 ml) into the sample well. For each sample or control, use a separate container, dropper or pipette and device. Wait for colored bands to appear. Depending on the concentration of HBsAg, positive results may be observed in as short as 60 seconds. For 5 ng/ml, read within 10 minutes, for 2ng/ml, read within 20 minutes. However, to confirm negative results, the complete reaction time, 30 minutes is required. Do not interprete result after 30 minutes. INTERPRETATION OF RESULTS

In addition to a pink colored control band, a pink colored band will appear in the patient test region. Colored bands in the patient test region of a positive sample may be any shade of pink. Invalid A total absence of color in two regions is an indication of procedure error and/or that test reagent deterioration has occurred. Add additional drops of samples to the sample well. If there is still no color band appears, the test should be voided. It should be noted that the control band may be faint when the color of the test band is intense. The result should be considered as valid. PERFORMANCE CHARACTERISTICS Sensitivity The Prima HBsAg test detects hepatitis B surface antigen (HBsAg) concentration in serum or plasma specimens higher than 2 ng/ml as indicated by the development of a colored band in the test region of the test strip. Specificity Antibodies used for Prima HBsAg test were raised against whole hepatitis B surface antigen. All subtype Hepatitis B antigens (adr, adw, ayr, ayw) show positive results with Prima HBsAg test. On the other hand samples from Hepatitis A, Hepatitis C and other Hepatologic diseases patients do not produce positive results with Prima HBsAg test. Nonspecific Reactive All highly sensitive immunoassay system have a potential for nonspecific reactives, but specificity of repeatably reactive specimens can be confirmed by neutrilization test.
BIBLIOGRAPHY: 1. Rubin, E. Acute and Chronic Viral Hepatitis. Federation Proceedings. 38 (13): 2665-2673 (1979). 2. R.D.Aach, J.W.Grisham, C.W.Parker. Detection of Australia antigen by radioimmunoassay. Proc. Nat. Ac. Sci. U.S.A. 68:1056 (1971). 3. Kim, C.Y. and Tilles, J.G. Purification and Biophysical characterization of hepatitis antigen. J. Clin. Invast. 52:1176-1186 (1973). 4. Radioimmunoassay for Hepatitis B virus markers, Manual of Clinical Immunology. Rose, N. And Friedman, H., Editors. 5. C.W.Caldwell, J.T.Barret. Enzyme immunoassay for hepatitis B and its comparison to other methods. Clin. Chem. Acta. 81: 305 (1977). 6. G.Wolters et al. Enzyme-linked immunosorbent assay for hepatitis B surface antigen. J.Infect.Dis. 136:311 (1977). 7. Magnius, L.O., A new antigen-antibody system. Clinical significance in long-term carriers of hepatitis Bsurface antigen. J. Am. Med. Assoc. 231:256-359 (1975). 9. Blumberg BS, Sutnick Al, London WT, Millman J. Australia antigen and Heptitis. New Eng. J. Med. 283:349-354 (1970). 10. Darrell L. Peterson, Structure of Hepatitis B Surface Antigen. The J. Of Biol. Chemistry. 257(17): 10414-10420 (1982).

negative positive

invalid

Negative No apparent band on the patient test region A pink colored band appears on the control region Positive