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THE PRESENT DOCUMENT IS A FAC-SIMILE VERSION When performing the assay, please use the kit insert

Enzyme immunoassay for the detection of

(code AB754/AB752)

B ZERO® Tetra/Epimers is an ELISA kit prepared for an the anti- antibody binding sites on the solid phase. immunoenzymatic assay for the quantitative analysis of Any unbound molecule is then removed from the solid phase in tetracyclines. a washing step. The binding between the enzyme conjugate The kit contains the procedure and the materials sufficient for and the antibody is detected by a chromogenic substrate. The 96 determinations ( code AB754 ) or 48 determinations (code enzyme converts the colourless chromogen into a blue AB752 ). product. The addition of the stop reagent leads to a colour A microtiter plate photometer or a strip photometer is required. change from blue to yellow. The absorbance is measured by a microplate reader at 450 nm. The colour development is Analysable samples inversely proportional to the tetracycline concentration in the Muscle, honey, milk, shrimp, water. sample.

Sample preparation 2 PROVIDED REAGENTS (Homogenization), mixing, (centrifugation), dilution. Microtiter plate: coated with anti-tetracycline antibody. As the strips are breakable, the wells can be used individually. Assay time: 90 minutes (sample preparation not included). For this purpose, it is sufficient to get out the wells from the frame and to break the joint. Detection limit (in tetracycline equivalents) Assay buffer: 1 plastic vial. Honey, milk, shrimp: 4 ppb Std 0: 1 amber plastic bottle containing the standard 0 ng/ml of Muscle: 8 ppb tetracycline. ATTENTION: only the standard zero is provided. Water: 1.11 ppb B/B 0 values of calibration curve (0,1-0,8ng/ml) are reported in Specificity the kit certificate of conformity. Analyte Cross -reactivity (%) Enzyme conjugate: 1 amber glass bottle containing lyophilized tetracycline 100 enzyme conjugate. Enzyme conjugate diluent: 1 amber plastic bottle. 95 Sample diluent 10X: 1 plastic bottle. 4-epi-tertacycline 95 Washing-buffer 5X: 1 plastic bottle. Developing solution: 1 amber plastic bottle. 90 Stop solution: 1 plastic vial.

80 Component Code AB754 – 96 det. Code AB752 – 48 80 det. Microtiter plate 96 wells 48 wells 4-epi-oxytetracycline 70 (12 strips x 8 (6 strips x 8 wells ) metacycline 60 wells ) Assay buffer 6ml 6ml doxytetracycline 50 Std 0 4 ml 3 ml 4-epi-chlortetracycline 30 Enzyme conjugate 2 vials 2 vials Enzyme conjugate 2x12 ml 2x12 ml 1 TEST PRINCIPLE diluent The assay is performed in plastic microwells that have been Sample diluent 10x 2x25 ml 25 ml coated with anti-tetracycline antibody. Assay buffer, Washing buffer 5x 2x100ml 100 ml tetracycline zero standard solution or samples and the enzyme Developing solution 16 ml 12 ml conjugate are added to the microplate. During the first Stop solution 12 ml 6 ml incubation, free tetracycline molecules eventually contained in the sample and the enzyme-labelled tetracycline compete for

______1 Tecna S.r.l., - Area Science Park - Padriciano 99, Trieste, Italy /19 THE PRESENT DOCUMENT IS A FAC-SIMILE VERSION When performing the assay, please use the kit insert

3 MATERIALS REQUIRED BUT NOT PROVIDED 3. Mix 164.7 ml of 0.2M Na 2HPO 4 solution with 35.3 ml of - Distilled water 0.1M citric acid solution. - Methanol (muscle) 4. Adjust pH to 7.0 with NaOH or HCl if necessary. - Methanol 80% (shrimp) - McIlvain Buffer pH 7.0 (muscle) 7 SAMPLES PREPARATION Equipment 7.1 Honey - Balance - Dilute 0,5g of honey with 19,5 ml of sample diluent - For grinding: grinder or blender “Osterizer” like (muscle, shrimp) 1x. - For extraction: vortex (muscle, honey, shrimp), shaker - Mix on vortex until complete dissolution (muscle, shrimp) (centrifugation at 3000 g for 5 minutes will help with - Centrifuge for 15ml vials (muscle, shrimp, optional for samples exhibiting precipitates) honey) The dilution factor is 40. - 0,2µm polysulfone filter (water) - 20-200 ml micropipette with tips. 7.2. Muscle - 200-1000 ml micropipette with tips. - Finely grind the sample. - 500-300 ml multichannel micropipette with tips. - Prepare the sample extraction solution mixing 1 part - Microtiter plate or strip reader equipped with a 450 nm McIlvain Buffer and 1 part of methanol. filter. - Weigh 1 g of sample and add 3 ml of sample extraction solution. 4 WARNING AND PRECAUTIONS FOR THE USERS - Vortex thoroughly for 1 minute. - For in vitro diagnostic use only. - Mix on shaker for 40 minutes. - Some reagents contain solutions that may be identified as - Centrifuge at 2000 x g for 10 minutes dangerous substance by the Regulation (EC) No - Dilute supernatant 20x with the sample diluent 1x 1272/2008. Please refer to Safety Data Sheet available on (for example: 50ml of milk + 950ml of buffer). Tecna web site. The dilution factor is 80. - Handle the reagents with caution, avoiding contact with skin, eyes and mucous membranes. 7.3. Milk - Dilute the sample 40x with the sample diluent 1x (for 5 HANDLING AND STORAGE INSTRUCTIONS example: 50 ml of milk + 1950 ml of buffer) - Store the kit at +2/+8 °C. The dilution factor is 40 . - Enzyme conjugate provided is lyophilized. Once reconstituted, the concentrated enzyme conjugate 7.4. Shrimp solution will remain viable for 4 weeks (store at -20°C) . - Finely grind the sample. - Reseal the unused strips of the anti-tetracycline microtiter - Weigh 1 g of sample and add 3 ml of methanol 80%. plate in the bag together with the desiccant bag provided. - Vortex thoroughly for 1 minute. - Do not use components after the expiration date. - Do not intermix components between different kit lots. - Mix on shaker for 20 minutes. - Do not use photocopies of the instruction booklet. Follow - Centrifuge at 2000 x g for 10 minutes. always the instruction booklet that is included inside the kit - Transfer 2 ml of supernatant in a clean vial. box. - Centrifuge at 2000 x g for 10 minutes. - Dilute supernatant 10x with the sample diluent 1x 6 WORKING SOLUTIONS PREPARATION (for example: 100 ml + 900 ml of buffer). Assay buffer: ready to use. The dilution factor is 40. Std 0: ready to use. Enzyme conjugate: After the vial has reached the room 7.5. Water temperature, resuspend the lyophilized enzyme conjugate with - Filter the sample using a 0.2µm polysulfone filter. 1 ml of enzyme diluent. Calculate and prepare the necessary - Prepare sample diluent 1x using the sample to dilute quantity for the experiment. Dilute the conjugate 1/25 in the 10x buffer (for example: 100 ml of sample diluent 10x enzyme diluent (for example: take 30 ml of concentrated + 900 ml of sample). enzyme conjugate + 720 ml of enzyme diluent). The dilution factor is 1.11 . ATTENTION: do not take less than 20 ml of concentrate enzyme conjugate from the vial. Enzyme conjugate diluent: ready to use; 8 ASSAY PROCEDURE Sample diluent 10x: dilute the dilution buffer 1:10 (for example: 8.1 Preliminary comments 1+9) with distilled water. - Bring all reagents to room temperature before use; keep Washing buffer 5x: dilute the concentrated 1:5 with distilled them at room temperature at least for one hour. water (for example 25 ml + 100 ml of distilled water). - Store reagents at +2/+8°C immediately after use. The diluted washing buffer is stable at room temperature for 24 Concentrated enzyme conjugate should be stored at - hours and at +2/+8°C for two weeks. 20°C. Developing solution: ready to use. - Do not change the assay procedure, in particular: Stop solution: ready to use. - do not prolong the incubation time; McIlvaine buffer (only for muscle extraction): - do not incubate the plate at temperatures higher than 1. Prepare a 0.2M sodium phosphate dibasic solution: 14.2g 25°C or lower than 18°C; - do not shake the plate during the incubations; of Na 2HPO 4 to 500 ml of distilled water. 2. Prepare a 0.1M citric acid solution: 2.94 g of citric acid - use for dispensing accurate and precise micropipettes trisodium salt to 100 ml of distilled water. with suitable tips;

______2 THE PRESENT DOCUMENT IS A FAC-SIMILE VERSION When performing the assay, please use the kit insert

- once started, complete all the steps without Please note: For results calculation, Excel spreadsheets are interruption; available on Tecna website and can be downloaded directly - avoid to dry out the wells; from the bottom of the product page upon registration. - The reproducibility of the results depends largely upon the efficiency and uniformity of microwells washing; always 10 RESULT EVALUATION keep to the described procedure. After results elaboration, it is necessary to verify the assay - Use a single disposable tip for each sample to avoid performance. The verification is performed by comparison of cross-contamination. obtained data with those given in kit specifications. If the - Do not allow tips to contact the liquid already in the values are out from the specifications given, it is advised to microwells or the internal microwells surface. check the expiry date of the kit, the wavelength of absorbance - Avoid direct sunlight during all incubations and cover the filter, as well as the procedure employed. If no errors are microtiter plate without using sealing tapes . found, contact our technical assistance. WARNING: substitution will be possible just in case of 8.2 Assay procedure rendered kit. The kit must be conserved in its integral version 1. Predispose the assay layout, taking into account that one and at the temperature indicated in this booklet. In order to avoid false non compliant results, it is necessary to well is required for zero standard (B 0) and each sample. 2. Using the multichannel micropipette, add 50 ml of the adopt a decision limit (CC a). It is suggested to determine a assay buffer solution in each well. decision limit for each matrix. In alternative, contact the 3. Add 100 ml of zero standard/sample into the wells. technical assistance.

4. Using the multichannel micropipette, add 50 ml of enzyme 11 KIT SPECIFICATIONS conjugate in each well and shake the plate gently with rotatory motion for few seconds. 11.1. Assay specifications 5. Incubate 60 minutes at room temperature. 6. Washing sequence Mean B 0 absorbance ≥ 0 .7 OD 450nm - Pour off the liquid from the wells. - Fill completely all the wells with working washing buffer 13 LIABILITY using a squeeze bottle. Pour the liquid out from the Samples evaluated as positive using the kit have to be re- wells. tested with a confirmation method. - Repeat the washing sequence for 4 times. Tecna shall not be liable for any damages to the customer - Remove the remaining droplets by tapping the caused by the improper use of the kit and for any action microplate upside down vigorously against absorbent undertaken as a consequence of results. paper. Tecna shall not be liable for the unsafe use of the kit out of the Do not allow the wells to dry out. current European safety regulations. 7. Using the multichannel micropipette, add 150 ml of developing solution to each well and shake the plate gently with rotatory motion for few seconds. 8. Incubate for 30 minutes at room temperature. 9. Using the multichannel micropipette, add 100 ml of stop solution to each well and mix thoroughly with rotatory motion for few seconds. 1. Measure the absorbance at 450 nm. Read within 15 minutes.

9 RESULTS CALCULATION - Divide the absorbance value of each sample by the absorbance of the standard 0 (B 0) and multiply by 100; the Maximum Binding (B 0) is thus made equal to 100% and the absorbance values are quoted in percentage:

Sample absorbance B ------X 100 = ---- (%) standard 0 (B 0) absorbance B0

- Enter the (%) B/B 0 values provided in the kit lot conformity certificate for each standard (0.1; 0.2; 0.3; 0.4; 0.6; 0.8 ng/ml) in a semi-logarithmic system of coordinates against the standard tetracycline concentration and draw the standard curve. - Interpolate the B/B 0 value for each sample to the corresponding concentration in the calibration curve. - The concentration of tetracycline equivalents presents in the samples in ppb ( mg/kg) is obtained from the concentration read from the calibration curve multiplied by the dilution factor. The dilution factor for honey, milk and shrimp is 40; for muscle is 80.

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