GUIDELINES TO BLOCKING ENZYME IMMUNOSORBENT ASSAY FOR THE DETECTION OF AFRICAN HORSE SICKNESS VIRUS ANTIBODIES

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1 GUIDELINES TO BLOCKING ENZYME IMMUNOSORBENT ASSAY FOR THE DETECTION OF AFRICAN HORSE SICKNESS VIRUS ANTIBODIES Content 1. PURPOSE SCOPE REFERENCES TEST PRINCIPLE SAFETY PRECAUTIONS GENERAL REQUIREMENTS FOR TEST S PERFORMANCE Protocol Samples Traceability Precautions Quality control Proficiency test ASSAY VALIDATION Plate validation Interpretation of sample results... 7 Page 1/ 7

2 1. PURPOSE The purpose is to provide general guidance to the best implementation and quality control of the Blocking- Enzyme Immunosorbent Assay (Blocking-ELISA) for the detection of antibodies against African horse sickness virus. These guidelines take into account applying principles and rules under the accreditation scope of ISO/IEC 17025, General Requirements for the competence of testing and calibration laboratories of the International Organization for Standardization. 2. SCOPE To detect the presence of antibodies against African horse sickness virus in sera samples of animal species of Equidae. 3. REFERENCES 3.1. Council Directive 2009/156/EC on animal health conditions governing the movement and importation from third countries of Equidae as last amended Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products as last amended INgezim AHSV Compac Plus: Blocking immunoenzymatic assay for detection of specific antibodies to African horse sickness virus in equine sera. INGENASA. Manufacturer s instruction manual ISO/IEC 17025:2017 General Requirements for the competence of testing and calibration laboratories. International Organization for Standardization OIE Chapter Biosafety and biosecurity: standard for managing biological risk in the veterinary laboratory and animal facilities. Manual for Diagnostic Tests and Vaccines for Terrestrial Animals OIE Chapter Quality Management in Veterinary testing Laboratories. In: Manual for Diagnostic Tests and Vaccines for Terrestrial Animals. Page 2/ 7

3 3.7. OIE Chapter African horse sickness (Infection with African horse sickness virus). In: Manual for Diagnostic Tests and Vaccines for Terrestrial Animals WHO Laboratory Biosafety Manual, Third Edition. WHO, Geneva, Switzerland WHO International Health Regulations, Second Edition. WHO, Geneva, Switzerland. 4. TEST PRINCIPLE The competitive- blocking ELISA is designed to detect specific AHSV antibodies in sera from animals of any equine species, i.e. horses, donkeys, zebra and their crosses, preventing the problem of specificity experienced occasionally using indirect ELISA. The principle of the test is the blocking of the reaction between the recombinant VP7 protein absorbed to the ELISA plate and a conjugated AHS-VP7 specific monoclonal antibody (Mab). Antibody in tested serum will block the reaction between the antigen and the Mab resulting in a reduction in colour. Because the Mab is directed against the VP7, the assay will give a high level of sensitivity and specificity. Reagents are supplied by the manufacture in kit format that includes specific reagents and test s control for the correct performance (INgezim AHSV Compac Plus ). The assay is one of the serological tests prescribed for the control of movement and importations to European Union, according to requirements of Directive 2009/156/EC, as last amended. It is also recommended by OIE Manual among other ELISA methods for the specific AHS antibodies detection as well as for the implementation of checks to prior international movements. Page 3/ 7

4 5. SAFETY PRECAUTIONS The assay shall be carried out following Biosafety, Biocontainment and Bioprotection guidance, and rules established according to international and national regulations (see references). According to OIE Manual, there is no evidence that humans become infected with any field strain of AHSV, either through contact with naturally or experimentally infected animals or by virus manipulation in laboratories. 6. GENERAL REQUIREMENTS FOR TEST S PERFORMANCE 6.1. Protocol The manufacturer s kit instructions shall be followed as the basis for test implementation. The laboratory will keep records of the current instruction edition in force and, with this purpose, will revise systematically the booklet inserted in each purchased kit to ensure that always the information of latest edition is used. Obsolete instruction editions shall be archived for the period of time determined by the corresponding Accreditation Body. The laboratory shall made available a Standard Operating Procedure (SOP) that, taking as the basis manufacturer s kit instructions, adapts operations to the laboratory quality system established according to ISO 17025, as well as to every aspect that might be relevant for a correct assay performance (e.g. quality control of assay, interpretation of results, actions to take in case of doubtful results, etc). According to ISO requirements, the use of kits after the expiring date is strongly not recommended Samples Sample s biological properties shall be guaranteed by applying correct sampling, preservation and transport procedures until arrival at the laboratory. The laboratory should implement a protocol to reject samples in poor conditions to be analyzed (e.g. strongly hemolization, putrefaction). Page 4/ 7

5 Once sera are obtained from un-clotted blood samples, they shall be kept at 5ºC (3-8) until their assay. For prolonged periods, samples shall be frozen in capped tubes/vials (or similar) at -20 º C or below Traceability The traceability system in the laboratory shall preserve the correct identity of samples during laboratory operations Precautions Please, refer to the manufacturer s kit instructions. In particular: Reagents and samples must be kept at room temperature for 30 minutes before testing. Frozen samples might need extra time to reach the required temperature. Plates and other kit components must be stored at 5 ºC (3-8). Use clean and sterile material (e.g. pipettes, tips and vessels) in contact to kit reagents. Such practices avoid contamination and preserve kits reagents until expiring date. Do not mix reagents from different kit batches. Use spectrophotometer correctly maintained, and periodically verified and calibrated Quality control The kit includes positive and negative controls that shall be assayed in duplicates for the best reliability. A variation less than 10% between duplicates will be considered as the best performance, although sporadic greater variation ( 20%) might be accepted. The average of Optical Density (ODs) obtained for duplicates will be used for the calculation of samples blocking value, If available, a weak positive serum assayed in duplicate should be included in each test run as internal control. A variation of % blocking less than 10% between duplicates will be considered as the best performance, although sporadic greater variation ( 20%) might be accepted. Page 5/ 7

6 In addition, to test repeatability it is advisable to test a number of samples in duplicate (e.g. 2 samples per assay). The results of replicates must be solid by showing the same qualitative result, e.g. either both replicates positive or both negative. In case of samples closed to grey zone (inconclusive), a variation of blocking percentage less than 10% between duplicates will be considered as the best performance, although sporadic greater variation ( 20%) might be accepted Proficiency test The laboratory shall participate regularly in external proficiency testing schemes. Participation in such scheme is a requirement for accredited laboratories. According to EU rules, European Union National Reference Laboratories have to participate in the Annual Proficiency Test organized by the European Union Reference Laboratory (EU-RL). Proficiency testing material has been well characterized and any spare material, once the proficiency testing has been completed, can be useful to demonstrate staff competence, or to regularly check test performance. 7. ASSAY VALIDATION It is advisable to develop an excel sheet for calculations that avoid mistakes. A template is available at the EURL under request Plate validation Each assayed plate must be validated according to manufacturer s kit instructions. When a weak positive serum is used as internal control, this serum shall give expected result. In case of not acceptance, the assay must be repeated for all samples contained in the plate. Page 6/ 7

7 7.2. Interpretation of sample results Once the plate has been validated, sample blocking value shall be calculated according to manufacturer s kit instructions. Doubtful results should be further investigated by retested the sample, or by sampling again the animal. Page 7/ 7