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06-02341-07

IDEXX CAV

CAV

IDEXX CAV 1

English version

Chicken Anemia Virus Antibody Test Kit

For veterinary use only.

Name and Intended Use

IDEXX CAV is IDEXX's enzyme immunoassay for the detection of antibody to Chic ken Anemia

Virus (CAV) in chicken serum.

General Information

Chicken Anemia Virus (CAV) is an important pathogen of poultry and has been found in broilers, breeders and SPF ocks. Virus isolation is difcult and time consuming. Screening for the presence of antibody will indicate exposure to the virus. The enzyme-linked immunosorbent assay has been utilized to detect antibody against CAV. This method is quite useful in large-scale testing of ocks for exposure to CAV. The IDEXX CAV assay can be used as a screen for the presence of Chicken Anemia Virus antibodies or to assess response to vaccination.

Description and Principles

The IDEXX CAV assay is performed in a microtiter well coated with Chicken Anemia Virus antigen. During the rst incubation, CAV antibodies present in the sample react with immobilized antigens. After a wash step, an Anti-CAV monoclonal antibody enzyme conjugate is added to the microwell. If no Anti-CAV antibodies are present in the test sample, the Anti-CAV conjugate is free to react with the CAV antigen. Conversely, if Anti-CAV antibodies are present in the sample, the enzyme- conjugated monoclonal antibodies are blocked from reacting with the anti gen. Following this incubation period, the unreacted conjugate is removed by washing and a s ubstrate/chromogen solution is added. In the presence of enzyme, the substrate is converted to a product which reacts with the chromophore to generate a blue color. The absorbance at 650nm, A(650), is measured using a spectrophotometer. Results are calculated by dividing the A(650) of the sample by the mean A(650) of the negative control, resulting in an S/N value.

The quantity of antibodies

to CAV is inversely proportional to the A(650) and thus, to the S/N value. The presence of CAV antibodies indicates previous exposure to Chicken Anemia Virus.

Reagent Volume

1CAV Antigen Coated Plates; preserved with Kathon5

2 Positive Control - diluted chicken anti-CAV serum; preserved with sodium azide 2 mL 3 Negative Control - diluted chicken serum non-reactive to

CAV; preserved with sodium azide

2 mL 4 Conjugate - anti-CAV: HRPO conjugate; preserved with gentamicin and Kathon 50 mL

5Sample Diluent - preserved with sodium azide235 mL

ATMB Substrate60 mL

BStop Solution 60 mL

CWash Concentrate (10X) - preserved with gentamicin235 mL

Other Components: Zip lock bag.1

NOTE:

Materials Required but Not Provided

Precision pipettes and multiple delivery pipetting device with disposable pipette tips, 96-well plate

reader, tubes for diluting samples, distilled or deionized water and device fo r the delivery and aspiration of wash solution. Reagent volumes listed in the Test Procedure require pipette precision of ±5%.

Precautions and Warnings for Users

Handle all CAV biological materials as though capable of transmitting CAV. The antigen coated plates may be a source of CAV. Prior to coating on the solid phase, the antigen has been inactivated by chemical treatment. Nevertheless, do not assume complete inactivation. Some kit components contain sodium azide as a preservative. Dispose of contents in accordance with local, regional,and national regulations. Do not expose TMB solutio ns to strong light or any oxidation agents. Store all reagents at 2-8°C. All wastes should b e properly decontaminated prior to disposal. Do not use kit serials past expiration date and do no t intermix components from kits with different serial numbers. Careful pipetting and washing t hroughout this procedure are necessary to maintain precision and accuracy. Optimal results will be obtained by strict adherence to this protocol. For veterinary use only.

Preparation of Samples

Note: Dilution of serum samples may be made with microdilution tubes or directly in the microtiter plate by adding the appropriate amount of serum to the previously pipetted Sample Diluent. Be sure to change tips for each sample. Samples must be thoroug hly mixed prior to dispensing into the coated plate. DO NOT DILUTE THE CONTROLS. A.

Field Exposure

For detection of antibody subsequent to field exposure to Chicken Anemi a Virus. Dilute serum samples ten-fold (1/10) with sample diluent prior to being assayed (e.g. diluting 10

µL of sample with 90 µL of sample diluent).

B.

Vaccination

For the detection of elevated antibody levels (e.g. subsequent to CAV vaccination), dilute serum samples 1/100 (e.g. diluting 10 µL of sample with 990 µL of sa mple diluent). This dilution will allow adequate differentiation of responses from samples w ithin a vaccinated flock. Responses of vaccination will depend upon dose, route of administration, age of flock, etc.

Preparation of Wash Solution

The Wash Concentrate (10X) should be brought to 18-26°C and mixed to ensure dissolution of any precipitated salts. The Wash Concentrate (10X) must be diluted 1/10 with distilled/ deionized water before use (e.g. 30 mL of concentrate plus 270 mL of wa ter per plate to be assayed).

Test Procedure

Allow the reagents to come to 18-26°C, then mix gently by inverting and swirling. 1. Obtain antigen coated plate(s) and record the sample position. If usin g partial plates, remove only those wells sufcient for samples to be tested. Place the remain ing wells, along with the desiccant, in the extra ziplock bag provided and return to 2-8°C. 2. Dispense 100 µL of UNDILUTED Negative Control into duplicate wells. 3. Dispense 100 µL of UNDILUTED Positive Control into duplicate wells. 4. Dispense 100 µL of diluted sample into appropriate wells. Samples may be tested in duplicate but a single well is acceptable. 5. Incubate for 60 minutes (± 5 minutes) at 18-26°C. 6. Wash each well with approximately 350 µL of Wash Solution 3-5 times. 7.

Dispense 100 µL of Conjugate into each well.

8. Incubate for 30 minutes (± 2 minutes) at 18-26°C. 9.

Repeat step 6.

10. Dispense 100 µL of TMB Substrate solution into each well. 11. Incubate for 15 minutes (± 1 minute) at 18-26°C. 12. Dispense 100 µL of Stop Solution into each well to stop the reaction. 13. Measure and record absorbance values at 650 nm, A(650).

Results

For the assay to be valid, the Negative Control optical density (650 nm) must be greater than or equal to 0.600 and the Positive Control S/N must be less than or equal to 0.50. For invalid tests, technique may be suspect and the assay should be repeated. The pr esence or absence of antibody to CAV is determined by the sample to negative (S/N) ratio for each sample.

Calculations

1.Negative Control mean (NCx)

NC1 A(650) + NC2 A(650)

= NCx 2

2.Positive Control mean (PCx)

PC1 A(650) + PC2 A(650)

= PCx 2

3.S/N Ratio

Sample A(650)

= S/N NCx

Interpretation of Results

1/10 Dilution

1. Samples with S/N ratios greater than 0.60 are considered negative within the limits of the test. 2. Samples with S/N ratios less than or equal to 0.60 are considered positi ve.

1/100 Dilution

The immune status of a vaccinated ock is best assessed by monitoring and recording antibody levels in representative samples as a function of time. The resulting ock proles allow an assessment of the distribution of antibody levels and an analysis of cha nges in status over time.

U.S. Vet. License No. 313

Product Code: 50C1.00

*IDEXX and Test With Confidence are trademarks or registered trademarks of IDEXX L aboratories, Inc. or its affiliates in the

United States and/or other countries.

©2011 IDEXX Laboratories, Inc. All rights reserved.

IDEXX Technical Services:

IDEXX USA Tel: 1 800 548 9997 or 1 207 556 4890

Fax: 1 800 328 5461 or 1 207 556 4826

IDEXX Europe Tel: 00800 727 43399

Fax: 00800 433 99329

CAV

IDEXX CAV 5

Version française

Kit de détection des Anticorps anti-Anémie Infectieuse du Poulet

Réservé à l'usage vétérinaire.

Dénition et application

IDEXX CAV est un kit de dosage immunoenzymatique mis au point par IDEXX pour la détection des anticorps de la l'Anémie Infectieuse dans le sérum de poulet.

Généralités

Le virus de l'Anémie Infectieuse du Poulet (CAV) est un agent pathogène important de la volaille;

il a été détecté chez les poulets de chair, les reproducteurs et les poulets SPF. L'isolement du

virus est long et difcile. La détection de la présence d'antic orps signale l'exposition au CAV. La technique ELISA a été utilisée pour détecter la présen ce d'anticorps du CAV. Cette technique s'est révélée utile pour la détection à grande éche lle de l'exposition au CAV dans les troupeaux de volaille. Le kit IDEXX CAV est destiné à detecter la présence des Anticorps dirigés c ontre le virus

de l'Anémie Infectieuse du Poulet ou à évaluer le statut immunitaire des animaux en réponse

à la

vaccination.

Description et principes

Le test IDEXX CAV utilise des microplaques sensibilisées avec l'antigène du virus de l'Anémie

Infectieuse du Poulet (CAV). Au cours de la première incubation, les anticorps du CAV présents dans l'échantillon réagissent avec l'antigène immobilisé . Après une opération de lavage, un

conjugué immunoenzymatique monoclonal anti-CAV est ajouté à la cupule. Si l'échantillon testé

ne contient pas d'anticorps du CAV, le conjugué anti-CAV est libre de réagir avec l'antigène du

CAV. Par contre, si l'échantillon contient des anticorps du CAV, les anticorps monoclonaux avec conjugué enzymatique ne peuvent pas réagir avec l'antigène.

Après cette période d'incubation,

le conjugué n'ayant pas réagi est éliminé par lavage et u ne solution de substrat/chromogène

est ajoutée. En présence d'enzyme, le substrat se transforme en un produit qui réagit avec le

chromophore pour donner une coloration bleue. L'absorbance à 650 nm, A(650), est mesurée à l'aide d'un spectrophotomètre. Les résultats sont obten us en divisant l'absorbance A(650) de l'échantillon par l'absorbance A(650) moyenne du contrô le négatif, ce qui donne un rapport échantillon/contrôle négatif (E/N). La quantité d'antic orps du CAV est inversement proportionnelle à l'absorbance A(650) et, par conséquent, au rapport E/N. La présence d'anticorps du CAV indique l'exposition au virus de l'anémie infectieuse du poulet.

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