1. INTENDED USE

Clenbuterol  ELISA Kit is a direct enzyme-linked  immunosorbent  assay for the quantitative detect the presence of Clenbuterol in samples of animal’s tissue, urine and feed.

Sensitivity:  0.05 ppb

Detection Limit:     Animal’s tissue            0.2 ppb / 0.05 ppb

                                Urine                            0.05 ppb

Feed                            2.5 ppb   

Recovery Rate:  90% ±　15% (pork liver, feed 80%±15%)

1. KIT COMPONENT

• 1 X Microtiter plate (8 microwells X 12 removable strips)
• 6 X Standard solutions (1 mL each): 0 ng/ mL, 0.05 ng/ mL, 0.15 ng/ mL, 0.45 ng/ mL, 0.9 ng/ mL, 1.8 ng/ mL
• 1 X Clenbuterol antibody (6 mL)
• 1 X HRP conjugated antibody (6 mL)
• 1 X Sample dilution buffer (30 mL)
• 1 X Washing buffer concentrate (10 X , 50 mL)
• 1 X Substrate A (6 mL)
• 1 X Substrate B (6 mL)
• 1 X Stopping solution (6 mL)
• 1 X Product Manual

1. MATERIALS REQUIRED BUT NOT PROVIDED

• ELISA Microtiter plate reader equipped with 450 nm filters
• 50 – 200 μL multichannel micropipette
• 50, 100 and 200 μL precision micropipette
• Microplate washer or squeeze bottle
• Homogenizer
• Centrifuge
• Centrifugal tubes
• Distilled water
• HCl, NaOH

1. PREPARATION OF WORKING SOLUTIONS

• ashinWg buffer: dilute 10X concentrate with distilled water (i.e. 1 mL + 9 mL).
• 2% NaCl: dissolve 2.0±0.01 NaCL with 100 mL distilled water into a beaker
• 0.1M HCl: dilute 8.33mL concentrated hydrochloric acid with distilled water and  make the constant volume to 1 L, OR dilute 10X 1M HCl with distilled water (i.e. 1 mL + 9 mL).
• Animal’s tissue extract solution: mix 2% NaCl solution with 0.1M HCl at the volumetric ratio 4:1.
• Standard solution, Enzyme-linked conjugate, substrate A, substrate B and stopping solution are ready-to-use.

1. SAMPLE PREPARATION

1.   Urine (dilution factor 1)

• Take 50 μL of clear urine sample directly for assay. (If urine sample is turbid, please filter or centrifuge it to clear, and use the supernatant.) Make dilution by distilled water before assay if the concentration of Clenbuterol is high.

1.  Animal’s tissue/liver

Method 1 (dilution factor: 4):

• Homogenize the sample (free of fat) for 1 min at 10,000 r/min.
• Weigh out 1.0±0.01g of homogenate into a 15 mL centrifugal tube. If animal tissue, please add 3 mL of extract solution and mix well; if liver, please add 3 mL 0.1M HCl, shake for for 5 min.
• Do a centrifugation at 5000 rpm at 20-25 °C for 5 min.
• Take 1 mL of supernatant and mix it with 2mL polystyrene to a centrifuge tube, then add 15μL of 1M NaOH and mix well. Adjust the pH value to 7 – 8.
• Take 50 μL liquid from water phase for assay.

Method 2 (dilution factor: 1):

• Homogenize the sample (free of fat) for 1 min at 10,000 r/min.
• Weigh out 2.0±0.01g of homogenate into a 15 mL centrifugal tube. Add 6 mL of methyl alcohol and shake for 5 min.
• Do a centrifugation at 5000 rpm for 5 min.
• Take 1.5 mL of supernatant (methyl alcohol layer) into a glass tube and dry it by 50 °C of Nitrogen flow.
• Totally dissolve the residue by 0.5 mL of Sample dilution buffer, then add 0.5 mL of n-Hexane. Shake to mix well.
• Get rid of the upper layer and take 50 μL liquid for assay.

1. Feed (dilution factor 50)

• Weigh out 2.0±0.01g of comminuted feed sample into a 50 mL centrifugal tube. Add 2 mL of 1M HCl and 16 mL distilled water. Shake to mix well.
•  Do a centrifugation at 5000 rpm for 5 min at room temperature.
• Take 200 μL of supernatant to a 1.5 mL centrifugal tube and mix well with 800 μL of Sample dilution buffer.
• Adjust the pH value to 7.0-8.0 by 15 uL 1M NaOH.
• Take 50 uL for assay.

1. ELISA TESTING PROTOCOL

6.1    Assay Preparation

• Bring all reagents to room temperature (20-25°C) before use.
• Take necessary strips for assay and the rest should be seal well again.
• Predispose a duplicate for each standard point and a duplicate for each sample.

6.2    Testing procedures

• Place 50 μL of each standard into the standard wells.
• Place 50 μL of each sample into the sample wells.
• Place 50 μL of HRP conjugated antibody into each well
• Place 50 uL of Clenbuterol antibody into each well
• Mix gently by rocking the plate manually.
• Incubate 40 min at room temperature (20-25 °C).
• Pour the liquid out of the wells, fill completely all the wells with washing buffer (approx 250 μL/well). Repeat the washing step 4-5 times. After the last washing step, tap the microwells holder upside down against absorbent paper to ensure complete liquid removal.
• Add 50 μL of substrate A solution into each well.
• Add 50 μL of substrate B solution into each well.
• Tap the microwells holder to make them mix thoroughly.
• Incubate 15 min at room temperature (20-25 °C).
• Add 50 μL of stopping solution into each well. Mix gently by rocking the plate manually.
• Read the absorbance at 450 nm filter with a Microplate reader within 5 min. (* very important)