OIE International Standard Reagents: IZSAM experience Giovanni Savini Abu Dhabi 14-16 February

Introduction OIE-approved International Standard Sera The OIE Biological Standards Commission (Laboratories Commission) coordinates a programme for the preparation, validation and distribution of OIE-approved International Reference Standards for antibody assays for infectious diseases of animals.

Introduction OIE-approved International Standard Sera The standards are prepared by an OIE Reference Laboratory in accordance with Guidelines drawn-up by the Laboratories Commission and in collaboration with other laboratories. Such standard preparations are designated by the OIE as primary reference standards for use in conjunction with tests described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Aniamls (mammals, birds and bees).

Introduction OIE-approved International Standard Sera AIM The aim of the programme is to harmonise diagnostic testing and encourage the mutual recognition of test results for international trade.

Introduction Guidelines for the preparation, validation and distribution of antibodies as International Reference Standards for antibody assays for infectious diseases of animals http://www.oie.int/fileadmin/Home/eng/Our_scientific_expertise/docs/pdf/GUIDELINE_3_REF_STANDARDS_ANG.pdf

International Reference Standards (IRS) Definition International Reference Standards (IRS) Reagents to which all assays are standardised

International Reference Standards They are prepared by OIE Reference Laboratories (or other international reference labs) which use an internationally accepted Standard test Protocol

International Reference Standards They are necessary to ensure that an antibody assay is capable of measuring antibody activity to a specified level of diagnostic sensitivity.

Reference Standards Secondary Standards Tertiary standards are reagent calibrated by comparison with IRS normally prepared by National Reference Laboratory Commonly represent the National Standards Tertiary standards are reagents calibrated against the Secondary standards usually prepared by normal diagnostic laboratories

Reference Standards Secondary or Tertiary standards are used on a daily basis to standardize testing

International Reference Standards How to prepare them?

Matrix Usually blood serum which should be free: from haemolysis from excessive lipaemia from infectious agents (safe) it facilitates shipment between countries from TSE

Matrix Treatments with binary ethyleneimine (BEI) irradiation at 25-30 kilogray and keeping at -78°C

International reference standards Positive serum It should origin from animals with a typical humoral immune-response to the selected pathogen (experimental infection, vaccination and exceptionally from naturally infected animals). It should be free from cross-reactive antibodies It could origin from a single animal or it could derive from a pool of animals

Our experience The 10th of June 1993 IZSAM was appointed as OIE Reference Laboratory for CBPP The mandate includes : To function as a centre of expertise and standardization of techniques To store and distribute biological reference products To develop new procedures for diagnosis and control To collect, process, analyze and disseminate epizootiological data To place expert consultants at the disposal of the OIE

ISS for CBPP Constitution: it would be better if matrix shows the desired level of reactivity (without further dilution) Between October 1990-September 1993, 94 outbreaks of CBPP occurred in Italy In May 1993: Outbreak of CBPP in Piedmont Region included 600 cattle Donor animal: fattening Charolais 18-months old beef serologically positive to CBPP: CFT 1:320 (Campbell & Turner CFT- OIE Manual 2.4.9, par 2.1)

ISS for CBPP CBPP was diagnosed in the donor animal by: by serological testing post-mortem examination performed at slaughtering time Confirmatory tests (IZSAM, NRC for CBPP): isolation of MmmSC (OIE Manual Ch. 2.4.9 par 1.2) immunohistochemistry (OIE Manual Ch. 2.4.9 par 1.3) PCR (OIE Manual Ch. 2.4.9 par 1.4) No clinical signs or pathological changes other than CBPP were observed

ISS for CBPP Stability and storage: treatment to inactivate adventitious agents, freeze-dried Blood was centrifuged, the serum separated and filtered (Sartorius capsule filters 0.8, 0.6, 0.45, 0.2µm) No bacterial contaminants were detected in the serum It was freeze-dried (1ml in 5 ml rubber capped bottles) and stored at -20°C

ISS CBPP Presence of heterologous Abs and adventitious viruses on freeze-dried sera Type of test Reference Result S-VN BVD IZSTE B2.1.4SOP012 Negative S-VN Respiratory Syncytial Virus IZSTE B2.1.4SOP026 1/8 S-VN BVH4 IZSTE B2.1.4SOP027 1/4 S-VN IBR OIE Manual, Ch. 2.3.5 S-VN Adenovirus Internal standardised protocol S-VN Parinfluenza 3 1/16 ELISA FMD IZS Brescia Adventitious Virus Isolation Negative (after 3 pass. in BHK cell cultures)

ISS for CBPP Results of controls There was no evidence that heterologous Abs detected in the serum interfere with the modified CFT Vaccination against FMD had officially ceased in January 1992: no Abs to serotypes A, O, C, A22 Iraq, O Manisa and Asia were detected by ELISA Prions were not present in the blood stream

ISS for CBPP Stability and storage: treatment to inactivate adventitious agents, freeze-dried After lyophilization, serum was divided in two lots of 400 bottles each: Non-irradiated Irradiated (25 kilogray) e-beam or ɣ-irradiations could have detrimental effect on CBPP CFT titres: Mediscan GmbH, Austria Gammarad Italia

Irradiated positive reference standard ISS for CBPP Controls after treatment CFT titres have been evaluated to confirm their activity: Non irradiated positive reference standard 1:160 1:240 1:320 1:480 1:640 1:960 1:1280 Bottle 1 ++++ +++ ++ + Neg Bottle 2 Irradiated positive reference standard 1:10 1:20 1:40 1:80 1:160 1:240 1:480 Bottle 1 ++++ +++ ++ + Neg Bottle 2

ISS for CBPP Certification required Data sheets: description of donor animal, nature of Abs response, details of organism used to elicit the immune response, details of experimental infection or immunization protocol, reference tests used, presence of heterologous Abs, safety testing, sterilization methods, batch number, date of production The full technical and statistical data on the evaluation of the candidate reference standard, together with the full data sheet information, was submitted to OIE Standards Commission on 14 January 2003

ISS for CBPP The OIE Biological Standards Commission has approved the OIE International Standard Serum for CBPP on 29 September 2003 The sera are available as two lots: non-irradiated lot for CFT irradiated lot for ELISA

ISS for CBPP The OIE International Standard Serum for CBPP was added to the list of OIE-approved International Standard Sera

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