1. INTENDED USE

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

Sensitivity:  0.05 ppb

Detection Limit:    Animal’s tissue and pork live     0.05 ppb / 0.2 ppb

Urine                                          0.05 ppb

Feed                                           2.5 ppb

Recovery Rate: Urine and Tissue   90% ±　15%

                           Pork’s live and feed   80% ±　15%                      

2. 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, 1.35 ng/ mL, 4.05 ng/ mL
• 1 X Salbutamol 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

3. 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

4. PREPARATION OF WORKING SOLUTIONS

• Washing buffer: dilute 10X concentrate with distilled water (i.e. 1 mL + 9 mL).
• Extract solution: mix 4% (m/v) NaCl Solution with 0.1 M HCl at the volumetric ratio 4:1.
• Standard solution, Enzyme-linked conjugate, substrate A, substrate B and stopping solution are ready-to-use.

5. SAMPLE PREPARATION

1. Animal’s tissue and pork live

Method 1 (dilution factor: 4):

• Homogenize the sample (free of fat) for 1 min at 10,000 r/min.
• Weigh out 1.0 g of homogenate into a 15 mL centrifugal tube. Add 3 mL of extract solution and mix well and shake for 5 mins.
•  Do a centrifugation at 5000 r/min at 20-25 °C for 5 mins.
• Take 1 mL of supernatant and mix it with 15μL of 1 M NaOH into  polystyrene centrifugal tube. 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 50 mL centrifugal tube. Add 6 mL of Methyl and shake for 5 mins.
• Do a centrifugation at 5000 r/min for 5 mins.
• 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 distilled water and add 0.5 mL of Hexane. Shake to mix well.
• Get rid of the upper layer and take 50 μL liquid for assay.

 2. Urine (dilution factor 1)

• Take 50 μL of clear urine sample directly for assay(Do centrifuge if urine is not clear). Make dilution by distilled water before assay if the concentration of Salbutamol is high.

 3. 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. Shake to mix well.
• Do a centrifugation at 5000 r/min for 5 mins.
• 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.

6. 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 Salbutamol 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)