ÐÏà¡±á>þÿ !þÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿÿì¥Á)` øR¿d1bjbj€€JTâ{â{ZA²ÿÿÿÿÿÿ¤ttttÄÄÄØÀ^À^À^À^l,a”Ø´˜ Ìb^*c*c*c*c×drIh]iŒ¯—±—±—±—±—±—±—$¾šh&@Õ—™Ämµd"×dmmÕ—tt*c*c‹n˜ q q qmdt8*cÄ*c¯— qm¯— q qŠÏ¬Ä/’*cÀb €Õp•vÑÀ^kmØë‘"{’4„˜0´˜ ’"fCoºfD/’fÄ/’LéiôÝj® q‹kŒlðéiéiéiÕ—Õ—ýpéiéiéi´˜mmmmØØØ$.ü5Ä(ØØØü5ØØØttttttÿÿÿÿREAGEN"! Zilpaterol ELISA Test Kit RND99040 Product Description REAGEN"! Zilpaterol ELISA Test Kit is a competitive enzyme immunoassay for the quantitative and qualitative analysis of Zilpaterol in feed, meat/liver/kidney, milk and urine.. The unique features of the kit are: Rapid (10 - 30 minutes), and organic reagent-free extraction method for various samples with high recovery (75 - 95%) High sensitivity (0.03 ng/g or ppb) and low detection limit (0.1 ng/g or ppb for meat/tissue) A quick ELISA assay (less than 2 hours regardless of number of samples) High reproducibility Procedure Overview The method is based on a competitive colorimetric ELISA assay. The drug of interest has been coated in the plate wells. During the analysis, sample is added along with the primary antibody specific for the target drug. If the target is present in the sample, it will compete for the antibody, thereby preventing the antibody from binding to the drug attached to the well. The secondary antibody, tagged with a peroxidase enzyme, targets the primary antibody that is complexed to the drug coated on the plate wells. The resulting color intensity, after addition of substrate, has an inverse relationship with the target concentration in the sample. Kit Contents, Storage and Shelf Life REAGEN"! Zilpaterol ELISA Test Kit has the capacity for 96 determinations or testing of 42 samples in duplicate (assuming 12 wells for standards). Return any unused microwells to the foil bag and reseal them with the desiccant provided in the original package. Store the kit at 2-8°C The shelf life is 12 months when the kit is properly stored. Kit ContentsAmountStorageZilpaterol-coated Microtiter Plate 1 x 96-well plate (8 wells x 12 strips)2-8(CZilpaterol Standards: Negative control (white cap tube) 0.03 ng/mL (yellow cap tube) 0.1 ng/mL (orange cap tube) 0.3 ng/mL (pink cap tube) 0.9 ng/mL (purple cap tube) 2.7 ng/mL (blue cap tube) 50 ng/mL (spiking, optional, red cap tube) 0.8 mL 0.8 mL 0.8 mL 0.8 mL 0.8 mL 0.8 mL 0.8 mL 2-8(C 2-8(C Zilpaterol Antibody #1 12 mL2-8(C100X HRP-Conjugated Antibody #2250 (LAntibody #2 Diluent 20 mL20X Wash Solution 28 mLStop Buffer 14 mLTMB Substrate 12 mL10X PBS 25 mL20X Sample Extraction Buffer (Optional)10 mLSample Balance Buffer I (Optional) Sample Balance Buffer II (Optional)10 mL62 gLiver Extraction Buffer (Optional)10 mLBeta-Agonist Clean Up Buffer I (Optional) Beta-Agonist Clean Up Buffer II (Optional) Beta-Agonist Clean Up Buffer III (Optional)5 mL5 mL28 mL If you are not planning to use the kit for over 3 months, store Zilpaterol Antibody #1 and 100X HRP-Conjugated Antibody #2 at -20(C or in a freezer. Sensitivity (Detection Limit) Sample TypeDetection Limit (g/g or ppb)Feed2Meat/Liver/Kidney0.1Milk0.5Serum/Plasma0.3Urine0.5 Specificity (Cross-Reactivity) AnalytesCross-Reactivity (%)Zilpaterol100Mabuterol5.8Mapenterol4.2Tolubuterol2.8Terbutaline2.3Cimbuterol1.7Salbutamol1.0Cimaterol0.6Pirbuterol0.4Isoprenaline0.2Required Materials Not Provided With the Kit Microtiter plate reader (450 nm) Incubator Tissue Mixer (e.g. Omni Tissue Master Homogenizer) Rotary evaporator or nitrogen gas Vortex mixer (e.g. Gneie Vortex mixer from VWR) 10, 20, 100 and 1000 (L pipettes Multi-channel pipette: 50-300 (L (Optional) Warnings and Precautions The standards contain Zilpaterol. Handle with particular care. Do not use the kit past the expiration date. Do not intermix reagents from different kits or lots except for components with the same part No’s within their expiration dates. ANTIBODIES AND PLATES ARE KIT-AND LOT-SPECIFIC. Make sure that the HRP Conjugate and Diluent are mixed in correct volumes. Try to maintain a laboratory temperature of 20°–25°C (68°–77°F). Avoid running assays under or near air vents, as this may cause excessive cooling, heating and/or evaporation. Also, do not run assays in direct sunlight, as this may cause excessive heat and evaporation. Cold bench tops should be avoided by placing several layers of paper towel or some other insulate material under the assay plates during incubation. Make sure you are using only distilled or deionized water since water quality is very important. When pipetting samples or reagents into an empty microtiter plate, place the pipette tips in the lower corner of the well, making contact with the plastic. Incubations of assay plates should be timed as precisely as possible. Be consistent when adding standards to the assay plate. Add your standards first and then your samples. Add standards to plate only in the order from low concentration to high concentration, as this will minimize the risk of compromising the standard curve. Always refrigerate plates in sealed bags with a desiccant to maintain stability. Prevent condensation from forming on plates by allowing them equilibrate to room temperature (20 – 25°C /68 – 77°F) while in the packaging. SAMPLE PREPARATION Be sure samples are properly stored. In general, samples should be refrigerated at 2-4°C for no more than 1-2 days. Freeze samples to a minimum of -20°C if they need to be stored for a longer period. Frozen samples can be thawed at room temps (20 – 25°C / 68 – 77°F) or in a refrigerator before use. 1 Preparation of 1X Sample Extraction Mix 1 volume of 20X Sample Extraction Buffer with 19 volumes of distilled water. Preparation of 1X PBS Mix 1 volume of the 10X PBS with 9 volumes of distilled water. Preparation of 1X Sample Balance Buffer Add 10 mL of the Sample Balance Buffer I and 62 g of Sample Balance Buffer II (Powder) to a 250-mL plastic bottle. Add 190 mL of distilled water to the bottle, mix well to obtain a homogeneous solution. Feed Homogenize a reasonable amount of feed sample by a suitable mixer. Add 20 mL of 0.1 M HCl to 2 g of the homogenized sample. Vortex for 15 minutes at a multi-tube vortexer or shaker. Centrifuge for 10 minutes at 2,000 x g at room temperature (20 – 25(C / 68 – 77(F), transfer 1 mL of the supernatant to a new tube, add ~55 (L of 1 M NaOH to adjust the pH to 6-8 (the amount of 1 M NaOH could be adjusted based on different feed samples). Centrifuge the sample for 10 minutes at 2,000 x g, transfer 0.5 mL of the supernatant to a new tube, add 1.0 mL of 1X PBS, mix well. Use 50 (L of the sample for the assay. Note: Dilution factor: 30. Meat/Chicken Liver Method I Remove fat from meat, liver or kidney. Homogenize the sample with a suitable mixer. Add 8 mL of acetonitrile and 1 mL of ethyl acetate to 3 g of the homogenized sample. Vortex for 3 minutes at maximum speed. Centrifuge for 5 minutes at 4,000 x g at room temperature (20 – 25(C / 68 – 77(F), transfer 6 mL of the supernatant to a new tube. Use a rotary evaporator to dry the sample in a 60-70(C water bath under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 60-70(C water bath. Add 1 mL of hexane to dissolve the sample and then add 1 mL of 1X PBS, vortex for 1 minute at maximum speed. Leave the tube open and heat the sample at 85(C for 3 minutes (( This step can be omitted if the lower aqueous layer is enough for the ELISA assay). Centrifuge the sample for 5 minutes at 4,000 x g. Use 50 (L of the lower aqueous layer for the assay. Note: Dilution factor: 0.5. Method II Remove fat from meat or chicken liver. Homogenize the sample with a suitable mixer. Add 2 mL of 1X Sample Extraction Buffer to 1 g of the homogenized sample, vortex for 3 minutes at maximum speed or rotorack for 10 minutes (e.g. VWR Tube Rotator). Add 3 mL of acetonitrile and 2.5 mL of 1X Sample Balance Buffer to the sample. Vortex for 1 minute at maximum speed. Centrifuge for 5 minutes at 4,000 x g at room temperature (20 25(C / 68 77(F), transfer 900 ¼L of the supernatant to a new tube. Use a rotary evaporator to dry the sample in a 60(C water bath under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 60(C water bath. Add 1 mL of hexane to dissolve the sample and then add 0.6 mL of 1X PBS, vortex for 1 minute at maximum speed. Centrifuge the sample for 5 minutes at 4,000 x g, discard the upper hexane layer. Use 50 (L of the lower aqueous layer for the assay. Note: Dilution factor: 2. Swine/Bovine Liver and Kidney Remove fat from beef or pig liver/kidney. Homogenize the sample with a suitable mixer. Add 2 mL of 1X Sample Extraction Buffer to 1 g of the homogenized sample, vortex for 3 minutes at maximum speed or rotorack for 10 minutes (e.g. VWR Tube Rotator). Add 3 mL of acetonitrile and 2.5 mL of 1X Sample Balance Buffer, 100 ¼L of Liver Extraction Buffer to the sample. Vortex for 1 minute at maximum speed. Centrifuge for 5 minutes at 4,000 x g at room temperature (20 25(C / 68 77(F), transfer 900 ¼L of the supernatant to a new tube. Use a rotary evaporator to dry the sample in a 60(C water bath under reduced pressure. Alternatively, the sample can be dried by blowing nitrogen gas in a 60(C water bath. Add 1 mL of hexane to dissolve the sample and then add 0.6 mL of 1X PBS, vortex for 1 minute at maximum speed. Centrifuge the sample for 5 minutes at 4,000 x g, discard the upper hexane layer. Use 50 (L of the lower aqueous layer for the assay. Note: Dilution factor: 2. Milk Take out 1 mL of milk sample, add 0.1 mL of 0.5 M HCl and 50 (L of Beta-Agonist Clean Up Buffer I to all of the samples, vortex for 5 seconds, then add 50 (L of Beta-Agonist Clean Up Buffer II to each sample, vortex for 30 seconds. Centrifuge at 2,000 x g at room temperature (20 – 25(C / 68 – 77(F ) for 5 minutes. Take 100 (L of the supernatant, add 320 (L of Beta-Agonist Clean Up Buffer III, mix well. Use 50 (L per well for the assay. Note: Dilution factor: 5. Serum/Plasma Dilute 0.1 mL of serum or plasma with 0.9 mL of 1X PBS, mix well. Use 50(L of the diluted sample per well for the assay. Note: Dilution factor: 10. Urine Method I Centrifuge 0.5 mL of the urine sample at 4,000 x g for 5 minutes. Use 50(L of the supernatant per well for the assay. Note: Dilution factor: 1. To avoid high background, we recommend diluting the sample with 1X PBS 5 fold (1:4). Method II (Suitable for urine sample with high background) Take out 1 mL of urine sample, add 100(L of 0.5 M HCl and 50(L of Beta-Agonist Clean Up Buffer I to all of the samples, vortex for 5 seconds, then add 50 (L of Beta-Agonist Clean Up Buffer II to each sample, vortex for 30 seconds. Centrifuge at 2,000 x g at room temperature (20 – 25(C / 68 – 77(F) for 5 minutes. Take 100(L of the supernatant, add 320(L of Beta-Agonist Clean Up Buffer III, mix well. Use 50(L per well for the assay. Note: Dilution factor: 5. ZILPATEROL ELISA TEST KIT PROTOCOL Reagent Preparation IMPORTANT: All reagents should be brought up to room temperature before use (1 – 2 hours at20 – 25°C / 68 – 77°F); Make sure you read “Warnings and Precautions” section on page 3. Solutions should be prepared just prior to ELISA test. All reagents should be mixed by gently inverting or swirling prior to use. Prepare only the volumes that are needed for the number of wells being run. Do not return the reagents to the original stock tubes/bottles. It is recommended that disposable reservoirs be used when handling reagents to minimize the risk of contamination and is recommended. Preparation of 1X HRP-Conjugated Antibody #2 Mix 1 volume of 100X HRP-Conjugated Antibody #2 with 99 volumes of Antibody #2 Diluent. Preparation of 1X Wash Solution Mix 1 volume of the 20X Wash Solution with 19 volumes of distilled water. ELISA Testing Protocol Label the individual strips that will be used and aliquot reagents as the following example: ComponentVolume per Reaction24 ReactionsZilpaterol Antibody #1100 (L2.4 mL1 x HRP-Conjugated Antibody #2150 (L3.6 mL1 x Wash Solution2.0 mL48 mLStop Buffer(optional)100 (L2.4 mLTMB Substrate100 (L2.4 mL Qualitative: Add 50 (L of Negative control and 0.3 ng/mL Standards in duplicate into different wells Add 50 (L of each sample in duplicate into different sample wells. Add 100 (L of Antibody #1 and mix well by gently rocking the plate manually for 1 minute. Incubate the plate for 30 minutes at room temperature (20 – 25(C / 68 – 77(F). Wash the plate 3 times with 250 (L of 1X Wash Solution. After the last wash, invert the plate and gently tap the plate dry on paper towels (Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). Add 150 (L of 1X HRP-Conjugated Antibody #2. Incubate the plate for 30 minutes at room temperature (20 – 25(C / 68 – 77(F) ( Avoid direct sunlight and cold bench tops during the incubation. Covering the microtiter plate while incubating is recommended). Wash the plate 3 times with 250 (L of 1 x Wash Solution. After the last wash, invert the plate and gently tap the plate dry on paper towels (Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). Add 100 (L of TMB substrate. Time the reaction immediately after adding the substrate. Mix the solution by gently rocking the plate manually for 1 minute while incubating (Do not put any substrate back to the original container to avoid any potential contamination. Any substrate solution exhibiting coloration is indicative of deterioration and should be discarded. Covering the microtiter plate while incubating is recommended). Incubate the plate for 15 minutes at room temperature , visually examine the intensity of the color of the standards and sample. Interpretation of results: = 1 \* GB3 `$Color of sample which is more intense or equal to Negative control indicates the sample is negative for presence of zilpaterol. = 2 \* GB3 a$Color of sample which is less intense than Negative control and more intense than 0.3 ng/mL Standards indicates the presence of zilpaterol in the sample is less than 9ppb(MRL). = 3 \* GB3 b$ Color of sample which is less intense than 0.3 ng/mL Standards indicates the presence of zilpaterol is more than 9ppb(MRL). Quantitative: Add 50 (L of each Zilpaterol Standards in duplicate into different wells ( Add standards to plate only in the order from low concentration to high concentration). Add 50 (L of each sample in duplicate into different sample wells. Add 100 (L of Antibody #1 and mix well by gently rocking the plate manually for 1 minute. Incubate the plate for 30 minutes at room temperature (20 – 25(C / 68 – 77(F). Wash the plate 3 times with 250 (L of 1X Wash Solution. After the last wash, invert the plate and gently tap the plate dry on paper towels (Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). Add 150 (L of 1X HRP-Conjugated Antibody #2. Incubate the plate for 30 minutes at room temperature (20 – 25(C / 68 – 77(F) ( Avoid direct sunlight and cold bench tops during the incubation. Covering the microtiter plate while incubating is recommended). Wash the plate 3 times with 250 (L of 1 x Wash Solution. After the last wash, invert the plate and gently tap the plate dry on paper towels (Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps). Add 100 (L of TMB substrate. Time the reaction immediately after adding the substrate. 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