HBsAg CONFIRMATORY TEST

Transcription

1 HBsAg CONFIRMATORY TEST For the confirmation of positive samples with UMELISA HBsAg PLUS CLINICAL INTEREST After the discovery of Hepatitis B surface Antigen (HBsAg)(1), increasingly sensitive and specific methods have been developed in order to allow its identification. Despite the great specificity of screening tests, false positive results may occur because of technical error or non-specific interference (2-5). The HBsAg CONFIRMATORY TEST has been designed to ensure that a positive reaction is caused by the presence of HBsAg in the sample. PRINCIPLE OF THE TEST The HBsAg CONFIRMATORY TEST uses the principle of specific antibody neutralization to confirm the presence of HBsAg. The Confirmatory Reagent (rabbit antibodies to HBsAg) is incubated with all repeatedly reactive samples. After pre-treatment, the samples are tested with the UMELISA HBsAg PLUS. The neutralized HBsAg cannot bin to the antibody-coated ultramicrowells. This results in a reduction of fluorescence signal when compared to the non-neutralized sample (control) in which control reagent (normal rabbit serum) is used instead of confirmatory reagent.

2 2 KIT CONTENTS Code UM 2003-C (20 Tests) Contents: Reagent 1 (R1): Control Reagent (1 x 1 ml) Reagent 2 (R2): Neutralizing Reagent (1 x 1 ml) Reagent 3 (R3): Positive Control (1 x 4,5 ml) All reagents are ready to use and contain sodium azide (0,2 g/l) as preservative. (*) R3 contains 10 ± 1 IU/mL HBsAg subtype ad inactivated with heat-treatment, calibrated against the reference preparation of the Paul Ehrlich Institute (Germany). It has been tested for Anti-HIV 1+2 and Anti-HCV by reliable methods and found to be negative. Storage and stability All reagents must be stored at 2-8 ºC. Under these conditions they are stable until the expiry date. After using some of the contents of vials, the remaining contents are stable until the expiry date, if kept at 2-8 ºC in the closed original container and microbial contamination is avoided. MATERIALS REQUIRED All reagents and equipment for UMELISA HBsAg PLUS are needed. Additional equipment: - Plastic tubes, disposable strips or plates. - Isotonic saline solution. Cautions - Before start work, make sure all reagents and samples are homogeneous and at room temperature.

3 3 - Use separate tips for control and neutralizing reagents to avoid cross contamination, which lead to incorrect results and to make useless the kit. - Pipette tips should be changed for each well when applying samples and conjugate. - Do not interchange reagents from different lots. - All the warnings for the UMELISA HBsAg PLUS should be taken into account. TECHNICAL PROCEDURE 1- Preparation of the Positive Control and samples The Positive control is provided ready to use. Use undiluted and 1:101 diluted samples (10 µl of serum µl of isotonic saline solution). Mix each undiluted and diluted sample 5:1 with Control Reagent (R1) and Neutralizing Reagent (R2). Recommended Volume: 100 µl of sample + 20 µl of R1 100 µl of sample + 20 µl of R2 Analyze in each test the Positive Control like and undiluted sample. 2- Pre-incubation with Control and Neutralizing Reagents Incubate at ºC for hours. It must be carried out in a humidity chamber if plastic tubes, disposable strips or plates are not hermetically sealed. 3- Immunoenzymatic test After pre-treatment in the above manner, test the samples in the UMELISA HBsAg PLUS including the Positive Control.

4 4 a) Laboratories with SUMA readers requiring wells for setting 0 and 100 fluorescent values must use the following protocol: A B C D E F G H M2n M3c M6n M7c M10n M11c M14n M15 M18n M19c M22n FRS M2c M3n M6c M7n M10c M11n M14c M15n M18c M19n M22c Pc M2n M3c M6n M7c M10n M11c M14n M15c M18n M19c M22n Pc M2c M3n M6c M7n M10c M11n M14c M15n M18c M19n M22c Pc M1n M4c M5n M8c M9n M12c M13n M16c M17n M20c M21n Pn M1c M4n M5c M8n M9c M12n M13c M16n M17c M20n M21c Pn M1n M4c M5n M8c M9n M12c M13n M16c M17n M20c M21n Pn M1c M4n M5c M8n M9c M12n M13c M16n M17c M20n M21c b) Laboratories with SUMA readers not requiring wells for setting 0 and 100 fluorescent values must use the following protocol: A B C D E F G H Pc M1c M3c M5c M7c M9c M11c M13c M15c M17c M19c M21c Pc M1n M3n M5n M7n M9n M11n M13n M15n M17n M19n M21n Pc M1c M3c M5c M7c M9c M11c M13c M15c M17c M19c M21c Pc M1n M3n M5n M7n M9n M11n M13n M15n M17n M19n M21n Pn M2c M4c M6c M8c M10c M12c M14c M16c M18c M20c M22c Pn M2n M4n M6n M8n M10n M12n M14n M16n M18n M20n M22n Pn M2c M4c M6c M8c M10c M12c M14c M16c M18c M20c M22c Pn M2n M4n M6n M8n M10n M12n M14n M16n M18n M20n M22n Where: Pc: Positive Control with R1 Pn: Positive Control with R2 Mc: Sample with R1 Mn: Sample with R2 The marked positions correspond to the diluted samples (1:101) and those not marked to the undiluted samples. Diluted and undiluted samples should be analyzed in duplicate.

5 5 QUALITY CONTROL Optimum quality of an assay is obtained when the following criteria are met: a) Pn/Pc < 0,5 Where: Pn: Median of the Positive Control with R2 Pc: Median of the Positive Control with R1 b) Pc 75 fluorescence units INTERPRETATION OF RESULTS A sample is considered as HBsAg POSITIVE if diluted or undiluted accomplish the following condition: Mn 0,7 Mc and Mc 4 fluorescence units A sample is HBsAg NEGATIVE in all other cases, except when the diluted sample with R1 shows fluorescence values greater than 150 units, in such condition the result is not valid and the sample should be diluted. TEST CHARACTERISTICS Sensitivity and Specificity HBsAg CONFIRMATORY TEST ORGANON POSITIVE ORGANON NEGATIVE TOTAL POSITIVE 244 (TP) 0 (FP) 244 NEGATIVE 0 (TN) 29 (TP) 29 TOTAL Sensitivity = TP x 100 = 100% TP + FN Specificity = TN x 100 = 100% TN + FP Where: TP: True positive TN: True negative FP: False positive FN: False negative

6 6 BIBLIOGRAPHY 1-Sherlock, S.: The natural history of Hepatitis B. Postgrad Med J. 63:7, Sapin, R.: Interférence des anticorps dans les immunodosages avec marqueur. Ann Biol Clin. 48:361, Kenna, J.G. et al.: Methods for reducing non-specific antibody binding in Enzyme-Linked Immunosorbent Assays. J Imm Methods. 85:409, Boscato,L.M.,Stuart, M.: Heterophilic antibodies: a problem for all immunoassays. Clin Chem. 34:27, Maxei, M.K. et al.: Interference in Enzyme Immunoassays. J Clin Imm. 15:2, October, HBsAg CONFIRMATORY TEST. Código UM 2003-C. Centro de Inmuno Ensayo, Calle 134 y Ave. 25, Apdo Postal 6653, La Habana, CUBA. Phone: Telex: Fax: (537)285614