1. INTENDED USE

Ractopamine ELISA Kit is an indirect enzyme-linked immunosorbent assay for the quantitative detect the presence of ractopamine in samples of animal’s tissue, urine and feed.

Sensitivity:  0.025 ppb

Detection Limit:     Animal’s tissue            0.1 ppb / 0.025 ppb

                                Urine                            0.025 ppb

                                Feed                            1.25 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, 0.025 ng/ mL, 0.075 ng/ mL, 0.225 ng/ mL, 0.675 ng/ mL, 2.025 ng/ mL
• 1 X Ractopamine 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

• Washing buffer: dilute 10X concentrate with distilled water (i.e. 1 mL + 9 mL).
• 1 M HCl: dilute 8.33mL concentrated hydrochloric acid with distilled water and  make the constant volume to 100 mL, OR dilute 10X 1M HCl with distilled water (i.e. 1 mL + 9 mL).
• Extract solution: mix 8g NaCl with 100mL 1M HCl .
• Standard solution, Enzyme-linked conjugate, substrate A, substrate B and stopping solution are ready-to-use.

1. SAMPLE PREPARATION
2. Animal’s tissue

Method 1 (dilution factor: 4):

• Homogenize the sample (free of fat) for 1 min at 10,000 r/min.
• Weigh out 2.0 g of homogenate into a 15 mL centrifugal tube. Add 6 mL of extract solution and mix well and shake for 5 min.
• Do a centrifugation at 5000 rpm at 20-25 °C for 5 min.
• Take 1 mL of supernatant to polystyrene centrifuge tube and mix it with 20 μL of 1 M NaOH. Adjust the pH value to 7.0-8.0.
• 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 g 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 at 20-25 °C for 5 min.
• Take 1.5 mL of supernatant (organic layer) into a glass tube and dry it by 50 °C of Nitrogen flow.
• Dissolve the residue by 0.5 mL of sample dilution buffer and add 0.5 mL of n-Hexane. Shake to mix well.
• Transfer 1ml into centrifuge tube.
• Get rid of the upper layer and take 50 μL liquid for assay.

1. Urine (dilution factor 1)

• Take 50 μL of clear urine sample directly for assay. Make dilution by distilled water before assay if the concentration of ractopamine is high.

1. Feed (dilution factor 50)

• Weigh out 2 g of comminuted feed sample into a 50 mL centrifugal tube. Add 2 mL of 1 M HCl and 16 mL of distilled water. Add 1.6g NACL, mix well.
• Do a centrifugation at 5000 rpm at 20-25 °C for 5 min.
• Take 200 μL of supernatant and mix well with 800 μL of sample dilution buffer. Shake to mix well.
• Adjust the pH value to 7.0-8.0 by 1 M NaOH.
• Take 50 μL 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 Ractopamine antibody into each well
• Mix gently by rocking the plate manually.
• Incubate 40 min at room temperature (20-25 °C). Tapping the microwells holder in incubation can decrease the inner errors between the duplicate wells.
• Pour the liquid out of the wells and tap the microwells holder upside down against absorbent laboratory paper to ensure complete liquid removal.
• Fill completely all the wells with washing buffer (approx 250 μL/well). Repeat the washing step 4 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)