Furaltadone ELISA Kit INQUIRY

Cat.No: EF-145
Prod. No. EF-145
Pkg. Size 96T
General Description The Furaltadone ELISA Kit is a competitive enzyme immunoassay for the quantitative analysis of Furaltadone in feed, fish, shrimp, meat (beef, chicken and pork), eggs, honey, milk, serum and urine. Furaltadone belongs to the class of 5-nitrofuran antibiotics and has been widely and effectively used for the prevention and treatment of gastrointestinal infections caused by Escherichia coli and Salmonella spp. in cattle, pigs and poultry. It was also used as a growth promoter in food-producing animals.
The unique features of the kit are:
1) Rapid extraction method for various samples with high recovery 90%±30%.
2) High sensitivity (0.5 ng/g or ppb) and low detection limit (0.3 ng/g or ppb for tissue).
3) A quick ELISA assay (less than 2 hours regardless of number of samples).
4) High reproducibility.
Principle Of The Test The method is based on an indirect competitive 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.
Reagents And Materials Provided Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with Furaltadone;
Furaltadone Standards (6): 0, 0.5, 0.15, 0.45, 1.8, 5.4ppb, 6 vials;
High concentration Furaltadone Standards: 1ppb, 1ml, 1 vial;
Antibody Solution: 7ml, 1 vial;
HRP Conjugate Antibody: 12ml, 1 vial;
Sample Diluent(4×): 30 ml. Use to dilute samples.
Wash Solution (20×) Concentrate: 40 ml, 1 vial;
TMB Solution A: 7ml, 1 vial;                                                       
TMB Solution B: 7ml, 1 vial;                                                       
TMB Stop Solution: 7ml, 1 vial;                                                       
Microtiter plate sealers
Plastic Sealable Bag
Materials Required But Not Supplied
1. Validated microplate reader.
2. Homogenizer
3. Electronic balance
4. Centrifuger
5. Shaker for microtiter plates (optional)
6. Organomation
7. Vortex genie
8. Validated adjustable micropipettes, single and multi-channel.
9. Timer
Storage Unopened Kit: Store at 2 - 8°C. Do not use past kit expiration date.
Opened/Reconstituted Reagents: TMB Solution A; TMB Solution B; TMB Stop Solution; Wash Buffer; HRP-conjugate antibody
The above mentioned reagents should be stored for up to 1 month at 2 - 8°C.
Microplate Wells: Return unused wells to the foil pouch containing the desiccant pack, reseal along entire edge of zip-seal. May be stored for up to 1 month at 2 - 8°C.
Precautions 1. The kit should be equilibrated to room temperature (20-23) before opening any vials and starting the assay. It is highly recommended that the solutions be used as soon as possible after rehydration.
2. Do not mix or substitute reagents with those from other lots or sources.
3. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
5. Crystals could appear in the 20X wash solution due to high salt concentration in the stock solutions. Crystals are readily dissolved at room temperature or at 37°C before dilution of the buffer solutions.
6. Keep TMB Substrate protected from light.
7. The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Specimen Treatment Homogenized, centrifuged, evaporated, diluted for assay.
Assay Procedure 1. Add 50μl of the standard solutions or samples (sample extracts) into the wells of the test strips according to the working scheme given. We recommend using duplicates or triplicates.
2. Add 50μl of enzyme conjugate solution to the individual wells successively using a multi-channel pipette or a stepping pipette.
3. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular motion on the benchtop for about 30 seconds. Be careful not to spill contents.
4. Incubate the strips for 40 minutes at room temperature.
5. After incubation, remove the covering and vigorously shake the contents of these wells into a sink. Wash the strips three times using the 1X washing buffer solution. Use at least a volume of 260 μl of washing buffer for each well and each washing step. Remaining buffer in the wells should be removed by patting the plate dry on a stack of paper towels.
6. Dispense 50 µl of TMB Solution A and 50 µl TMB Solution B into each well. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular motion on the benchtop for about 30 seconds. Incubate the strips for 15-20 minutes at room temperature. Protect the strips from direct sunlight.
7. Add 50 μl of stop solution to the wells in the same sequence as for the substrate solution.
8. Read the absorbance at 450 nm and 630 nm using a microplate ELISA photometer within 5 minutes after the addition of the stopping solution.
Evaluation The evaluation of the ELISA can be performed using commercial ELISA evaluation programs (4-Parameter (preferred) or Logit/Log. For manual evaluation, calculate the mean absorbance value for each of the standards. Calculate the %B/B0 for each standard by dividing the mean absorbance value for each standard by the Zero Standard (Standard 0) mean absorbance. Construct a standard curve by plotting the %B/B0 for each standard on the vertical linear (y) axis versus the corresponding Furaltadone concentration on the horizontal logarithmic (x) axis on graph paper. %B/B0 for samples will then yield levels in ppb of a Furaltadone by interpolation using the standard curve. Samples showing lower concentrations of Furaltadone compared to Standard 1 (0.5 ng/mL) are considered as negative. Samples showing a higher concentration than Standard 5 (5.4 ng/mL) must be diluted further to obtain accurate results.
Working Scheme The microtiter plate consists of 12 strips of 8 wells, which can be used individually for the test. The standards must be run with each test. Never use the values of standards which have been determined in a test performed previously.
Std 0-Std 5: Standards
0, 0.5, 0.15, 0.45, 1.8, 5.4ppb
Performance Data A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each reference standard against its concentration in ng/mL on a logarithmic curve.
A standard curve can be constructed by plotting the mean relative absorbance (%) obtained from each reference standard against its concentration in ng/mL on a logarithmic curve.
Relative absorbance (%) = absorbance standard (or sample) x 100/ absorbance zero standard.
Use the mean relative absorbance values for each sample to determine the corresponding concentration of the tested drug in ng/mL from the standard curve.
The ELISA sensitivity is 0.5 ppb, and the range of the standard curve is 0.5-5.4 ppb.
Test sensitivity:
Sample TYPE                  Detection Limit(ng/g or ppb)
Tissue                                  0.3
Specificity (Cross-Reactivity):
NP-SEM……………………………  <0.1%
Indications Of Instability Or Deterioration Of The Reagents 1. Values of the Positive or Negative controls, which are out of the indicated Quality control range, are indicator of possible deterioration of the reagents and/or operator or equipment errors. In such case, the results should be considered as invalid and the samples must be retested. In case of constant erroneous results classified as due to deterioration or instability of the reagents, immediately substitute the reagents with new ones, or contact our technical support for further assistance.
2. If after mixing of the TMB Solution  A and B into the wells, the color of the mixture turns blue within few minutes, do not continue carrying out the testing and replace the reagents with fresh ones.
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