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Diazepam ELISA Kit

Diazepam ELISA Kit

Manufacturer	Creative Diagnostics
Category	Food Safety Drugs ELISA ELISA & Immunoassays ELISA Kits Immunoassay
Type	Elisa-Kit
Specific against	other
Amount	96T
Item no.	DEIA053
eClass 6.1	32160605
eClass 9.0	32160605
Available
Abbr	Diazepam Kit
Full name	Diazepam
Storage	Unopened Kit: Store at 2 - 8oC. Donot use past kit expiration date. Opened/Reconstituted Reagents: TMBSolution A, TMB Solution B, TMB Stop Solution, Wash Buffer, HRP-conjugateantibody Theabove mentioned reagents should be stored for up to 1 month at 2 - 8oC. Microplate Wells: Return unused wellsto the foil pouch containing the desiccant pack, reseal along entire edge ofzip-seal. May be stored for up to 1 month at 2 - 8oC.
Principle Of The Test	Themethod is based on an indirect competitive ELISA assay. The drug of interesthas been coated in the plate wells. During the analysis, sample is addedalong with the primary antibody specific for the target drug. If the targetis present in the sample, it will compete for the antibody, therebypreventing the antibody from binding to the drug attached to the well. Thesecondary antibody, tagged with a peroxidase enzyme, targets the primaryantibody that is complexed to the drug coated on the plate wells. Theresulting color intensity, after addition of substrate, has an inverserelationship with the target concentration in the sample.
Reagents And Materials Provided	Microplate:96well polystyrene microplate (12 strips of 8 wells) coated with Diazepam, DiazepamStandards (6): 0, 0.1, 0.2, 0.4, 0.8, 1.6ppb, 6 vials, Highconcentration Diazepam Standards: 100ppb, 2ml, 1 vial, Antibody Solution: 7ml, 1 vial, HRPConjugate Antibody: 12ml, 1 vial, Sample Diluent(10×): 30 ml. Use to dilutesamples. Wash Solution (20×) Concentrate: 40 ml, 1vial, 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	Equipment: 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
Precautions	1.The kit should be equilibrated to room temperature (20-23C) before opening anyvials and starting the assay. It is highly recommended that the solutions beused 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 eachstandard 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 concentrationin the stock solutions. Crystals are readily dissolved at room temperature orat 37C 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.
Detection Method	Competitive Enzyme Immunoassay
Sample	tissue, feed and urine
Indications Of Instability Or Deterioration Of The Reagents	1.Values of the Positive or Negative controls, which are out of the indicatedQuality control range, are indicator of possible deterioration of thereagents and/or operator or equipment errors. In such case, the resultsshould be considered as invalid and the samples must be retested. In case ofconstant erroneous results classified as due to deterioration or instabilityof the reagents, immediately substitute the reagents with new ones, orcontact our technical support for further assistance. 2.If after mixing of the TMB Solution A and B into the wells, the colorof the mixture turns blue within few minutes, do not continue carrying outthe testing and replace the reagents with fresh ones.
Assay Procedure	1.Add 50ul of the standard solutions or samples (sample extracts) into thewells of the test strips according to the working scheme given. We recommendusing duplicates or triplicates. 2.Add 50ul of enzyme conjugate solution to the individual wells successivelyusing a multi-channel pipette or a stepping pipette. 3.Add 100ul of antibody solution to the individual wells successively using amulti-channel pipette or a stepping pipette. Cover the wells with parafilm ortape and mix the contents by moving the strip holder in a circular motion onthe 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 ofthese wells into a sink. Wash the strips three times using the 1X washingbuffer solution. Use at least a volume of 260 ul of washing buffer for eachwell and each washing step. Remaining buffer in the wells should be removedby patting the plate dry on a stack of paper towels. 6.Dispense 50 ul of TMB Solution A and 50 ul TMB Solution B into each well. Coverthe wells with parafilm or tape and mix the contents by moving the stripholder in a circular motion on the benchtop for about 30 seconds. Incubatethe strips for 15-20 minutes at room temperature. Protect the strips fromdirect sunlight. 7.Add 50 ul of stop solution to the wells in the same sequence as for thesubstrate solution. 8.Read the absorbance at 450 nm and 630 nm using a microplate ELISA photometerwithin 5 minutes after the addition of the stopping solution.
Evaluation	Theevaluation of the ELISA can be performed using commercial ELISA evaluationprograms (4-Parameter (preferred) or Logit/Log. For manual evaluation, calculate the mean absorbance value for each of the standards. Calculate the%B/B0 for eachstandard by dividing the mean absorbance value for each standard by the ZeroStandard (Standard 0) mean absorbance. Construct a standard curve by plottingthe %B/B0 for eachstandard on the vertical linear (y) axis versus the corresponding Diazepamconcentration on the horizontal logarithmic (x) axis on graph paper. %B/B0 forsamples will then yield levels in ppb of a Diazepam by interpolation usingthe standard curve. Samples showing lower concentrations of Diazepam comparedto Standard 1 (0.1 ng/mL) are considered as negative. Samples showing ahigher concentration than Standard 5 (1.6 ng/mL) must be diluted further toobtain accurate results.

Note: The presented information and documents (Manual, Product Datasheet, Safety Datasheet and Certificate of Analysis) correspond to our latest update and should serve for orientational purpose only. We do not guarantee the topicality. We would kindly ask you to make a request for specific requirements, if necessary.

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