THE PRESENT DOCUMENT IS A FAC-SIMILE VERSION When performing the assay, please use the kit insert
Enzyme immunoassay for the detection of tetracyclines
(code AB754/AB752)
B ZERO® Tetra/Epimers is an ELISA kit prepared for an immunoenzymatic assay for the quantitative analysis of tetracyclines. 1 TEST PRINCIPLE The kit contains the procedure and the materials sufficient for The assay is performed in plastic microwells that have been 96 determinations (code AB754) or 48 determinations (code coated with anti-tetracycline antibody. Assay buffer, AB752). tetracycline zero standard solution or samples and the enzyme A microtiter plate photometer or a strip photometer is conjugate are added to the microplate. During the first required. incubation, free tetracycline molecules eventually contained in the sample and the enzyme-labelled tetracycline compete for Analysable samples the anti-tetracycline antibody binding sites on the solid phase. Muscle, honey, milk, shrimp, water. Any unbound molecule is then removed from the solid phase in a washing step. The binding between the enzyme conjugate Sample preparation and the antibody is detected by a chromogenic substrate. The (Homogenization), mixing, (centrifugation), dilution. enzyme converts the colourless chromogen into a blue product. The addition of the stop reagent leads to a colour Assay time: 90 minutes (sample preparation not included). change from blue to yellow. The absorbance is measured by a microplate reader at 450 nm. The colour development is Detection limit (in tetracycline equivalents) inversely proportional to the tetracycline concentration in the Honey, milk, shrimp: 4 ppb sample. Muscle: 8 ppb Water: 1.11 ppb 2 PROVIDED REAGENTS Specificity Microtiter plate: coated with anti-tetracycline antibody. Analyte Cross-reactivity (%) As the strips are breakable, the wells can be used individually. Lotto: 02090P Rev.1 del 12-10-2010 tetracycline 100 For this purpose, it is sufficient to get out the wells from the frame and to break the joint. oxytetracycline 95 Assay buffer: 1 plastic vial. 4-epi-tertacycline 95 Std 0: 1 amber plastic bottle containing the standard 0 ng/ml of tetracycline. ATTENTION: only the standard zero is demeclocycline 90 provided. B/B0 values of calibration curve (0,1-0,8ng/ml) are rolitetracycline 80 reported in the kit certificate of conformity. Enzyme conjugate: 1 amber glass bottle containing chlortetracycline 80 lyophilized enzyme conjugate. Enzyme conjugate diluent: 1 amber plastic bottle. 4-epi-oxytetracycline 70 Sample diluent 10X: 1 plastic bottle. metacycline 60 Washing-buffer 5X: 1 plastic bottle. Developing solution: 1 amber plastic bottle. doxytetracycline 50 Stop solution: 1 plastic vial. 4-epi-chlortetracycline 30
______1 Tecna S.r.l. – Società a socio unico - Area Science Park - Padriciano 99, Trieste, Italy V.3 - 01/19 tel: +39 040 3755341; fax +39 040 3755343 e-mail: [email protected] web: www.tecnalab.com
Enzyme conjugate: After the vial has reached the room temperature, resuspend the lyophilized enzyme conjugate with 1 ml of enzyme diluent. Calculate and prepare the necessary Component Code AB754 – 96 det. Code AB752 – 48 det. quantity for the experiment. Dilute the conjugate 1/25 in the Microtiter plate 96 wells 48 wells enzyme diluent (for example: take 30 l of concentrated (12 strips x 8 wells) (6 strips x 8 wells) Assay buffer 6ml 6ml enzyme conjugate + 720 l of enzyme diluent). Std 0 4 ml 3 ml ATTENTION: do not take less than 20 l of concentrate Enzyme conjugate 2 vials 2 vials enzyme conjugate from the vial. Enzyme conjugate diluent 2x12 ml 2x12 ml Sample diluent 10x 2x25 ml 25 ml Enzyme conjugate diluent: ready to use; Washing buffer 5x 2x100ml 100 ml Sample diluent 10x: dilute the dilution buffer 1:10 (for Developing solution 16 ml 12 ml example: 1+9) with distilled water. Stop solution 12 ml 6 ml Washing buffer 5x: dilute the concentrated 1:5 with distilled water (for example 25 ml + 100 ml of distilled water). The diluted washing buffer is stable at room temperature for 3 MATERIALS REQUIRED BUT NOT PROVIDED 24 hours and at +2/+8°C for two weeks. - Distilled water Developing solution: ready to use. - Methanol (muscle) Stop solution: ready to use. - Methanol 80% (shrimp) McIlvaine buffer (only for muscle extraction): - McIlvain Buffer pH 7.0 (muscle) 1. Prepare a 0.2M sodium phosphate dibasic solution: 14.2g Equipment of Na2HPO4 to 500 ml of distilled water. - Balance 2. Prepare a 0.1M citric acid solution: 2.94 g of citric acid - For grinding: grinder or blender “Osterizer” like (muscle, trisodium salt to 100 ml of distilled water. shrimp) 3. Mix 164.7 ml of 0.2M Na2HPO4 solution with 35.3 ml of - For extraction: vortex (muscle, honey, shrimp), shaker 0.1M citric acid solution. (muscle, shrimp) 4. Adjust pH to 7.0 with NaOH or HCl if necessary. - Centrifuge for 15ml vials (muscle, shrimp, optional for honey) 7 SAMPLES PREPARATION - 0,2µm polysulfone filter (water) 7.1 Honey - 20-200 l micropipette with tips. - Dilute 0,5g of honey with 19,5 ml of sample diluent 1x. - 200-1000 l micropipette with tips. - Mix on vortex until complete dissolution (centrifugation - 500-300 l multichannel micropipette with tips. at 3000 g for 5 minutes will help with samples exhibiting - Microtiter plate or strip reader equipped with a 450 nm precipitates) filter. The dilution factor is 40. 7.2. Muscle 4 WARNING AND PRECAUTIONS FOR THE USERS - Finely grind the sample. - For in vitro diagnostic use only. - Prepare the sample extraction solution mixing 1 part - Some reagents contain solutions that may be identified as McIlvain Buffer and 1 part of methanol. dangerous substance by the Regulation (EC) No - Weigh 1 g of sample and add 3 ml of sample extraction 1272/2008. Please refer to Safety Data Sheet available on solution. Tecna web site. - Vortex thoroughly for 1 minute. - Handle the reagents with caution, avoiding contact with - Mix on shaker for 40 minutes. skin, eyes and mucous membranes. - Centrifuge at 2000 x g for 10 minutes - Dilute supernatant 20x with the sample diluent 1x (for 5 HANDLING AND STORAGE INSTRUCTIONS example: 50l of milk + 950l of buffer). - Store the kit at +2/+8 °C. The dilution factor is 80. - Enzyme conjugate provided is lyophilized. Once reconstituted, the concentrated enzyme conjugate 7.3. Milk solution will remain viable for 4 weeks (store at - - Dilute the sample 40x with the sample diluent 1x (for 20°C). example: 50l of milk + 1950l of buffer) - Reseal the unused strips of the anti-tetracycline microtiter The dilution factor is 40. plate in the bag together with the desiccant bag provided. - Do not use components after the expiration date. 7.4. Shrimp - Do not intermix components between different kit lots. - Finely grind the sample. - Weigh 1 g of sample and add 3 ml of methanol 80%. - Do not use photocopies of the instruction booklet. Follow always the instruction booklet that is included inside the - Vortex thoroughly for 1 minute. kit box. - Mix on shaker for 20 minutes. - Centrifuge at 2000 x g for 10 minutes. - Transfer 2 ml of supernatant in a clean vial. 6 WORKING SOLUTIONS PREPARATION - Centrifuge at 2000 x g for 10 minutes. Assay buffer: ready to use. - Dilute supernatant 10x with the sample diluent 1x (for Std 0: ready to use. example: 100l + 900l of buffer).
______2 Tecna S.r.l. – Società a socio unico - Area Science Park - Padriciano 99, Trieste, Italy tel: +39 040 3755341; fax +39 040 3755343 e-mail: [email protected] web: www.tecnalab.com
The dilution factor is 40. - Remove the remaining droplets by tapping the 7.5. Water microplate upside down vigorously against - Filter the sample using a 0.2µm polysulfone filter. absorbent paper. - Prepare sample diluent 1x using the sample to dilute 10x Do not allow the wells to dry out. buffer (for example: 100l of sample diluent 10x + 900l 7. Using the multichannel micropipette, add 150 of sample). The dilution factor is 1.11. l of developing solution to each well and shake the plate gently with rotatory motion for 8 ASSAY PROCEDURE few seconds. 8.1 Preliminary comments 8. Incubate for 30 minutes at room temperature. - Bring all reagents to room temperature before use; keep 9. Using the multichannel micropipette, add 100 them at room temperature at least for one hour. - Store reagents at +2/+8°C immediately after use. l of stop solution to each well and mix Concentrated enzyme conjugate should be stored at - thoroughly with rotatory motion for few 20°C. seconds. - Do not change the assay procedure, in particular: 1. Measure the absorbance at 450 nm. Read - do not prolong the incubation time; within 15 minutes. - do not incubate the plate at temperatures higher than 25°C or lower than 18°C; - do not shake the plate during the incubations; 9 RESULTS CALCULATION - Divide the absorbance value of each sample by the - use for dispensing accurate and precise micropipettes absorbance of the standard 0 (B ) and multiply by 100; with suitable tips; 0 - once started, complete all the steps without the Maximum Binding (B0) is thus made equal to 100% interruption; and the absorbance values are quoted in percentage: - avoid to dry out the wells; - The reproducibility of the results depends largely upon Sample absorbance B ------X 100 = ---- (%) the efficiency and uniformity of microwells washing; always keep to the described procedure. standard 0 (B0) absorbance B0 - Use a single disposable tip for each sample to avoid cross-contamination. - Enter the (%) B/B0 values provided in the kit lot - Do not allow tips to contact the liquid already in the conformity certificate for each standard (0.1; 0.2; 0.3; 0.4; microwells or the internal microwells surface. 0.6; 0.8 ng/ml) in a semi-logarithmic system of coordinates against the standard tetracycline - Avoid direct sunlight during all incubations and cover the microtiter plate without using sealing tapes. concentration and draw the standard curve. - Interpolate the B/B0 value for each sample to the corresponding concentration in the calibration curve. 8.2 Assay procedure - The concentration of tetracycline equivalents presents in 1. Predispose the assay layout, taking into account the samples in ppb (g/kg) is obtained from the that one well is required for zero standard (B0) concentration read from the calibration curve multiplied and each sample. by the dilution factor. The dilution factor for honey, milk and shrimp is 40; for muscle is 80. 2. Using the multichannel micropipette, add 50 l Please note: For results calculation, Excel spreadsheets are of the assay buffer solution in each well. available on Tecna website www.tecnalab.com and can be 3. Add 100 l of zero standard/sample into the downloaded directly from the bottom of the product page upon registration. wells. 4. Using the multichannel micropipette, add 50 l 10 RESULT EVALUATION of enzyme conjugate in each well and shake the After results elaboration, it is necessary to verify the assay plate gently with rotatory motion for few performance. The verification is performed by comparison of seconds. obtained data with those given in kit specifications. If the values are out from the specifications given, it is advised to 5. Incubate 60 minutes at room temperature. check the expiry date of the kit, the wavelength of absorbance 6. Washing sequence filter, as well as the procedure employed. If no errors are - Pour off the liquid from the wells. found, contact our technical assistance. - Fill completely all the wells with working WARNING: substitution will be possible just in case of washing buffer using a squeeze bottle. Pour rendered kit. The kit must be conserved in its integral version and at the temperature indicated in this booklet. the liquid out from the wells. In order to avoid false non compliant results, it is necessary to - Repeat the washing sequence for 4 times. adopt a decision limit (CC). It is suggested to determine a decision limit for each matrix. In alternative, contact the technical assistance.
______3 Tecna S.r.l. – Società a socio unico - Area Science Park - Padriciano 99, Trieste, Italy tel: +39 040 3755341; fax +39 040 3755343 e-mail: [email protected] web: www.tecnalab.com
11 KIT SPECIFICATIONS 11.1. Assay specifications
Mean B0 absorbance ≥ 0.7 OD450nm
13 LIABILITY Samples evaluated as positive using the kit have to be re- tested with a confirmation method. Tecna shall not be liable for any damages to the customer caused by the improper use of the kit and for any action undertaken as a consequence of results. Tecna shall not be liable for the unsafe use of the kit out of the current European safety regulations.
______4 Tecna S.r.l. – Società a socio unico - Area Science Park - Padriciano 99, Trieste, Italy tel: +39 040 3755341; fax +39 040 3755343 e-mail: [email protected] web: www.tecnalab.com