RACTOPAMINE ELISA KIT,specification,price,image-Bio-Equip in China

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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%)

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

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

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.

SAMPLE PREPARATION
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.

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.

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.

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)