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Malachite Green ELISA Kit

Malachite Green ELISA Kit

Hersteller	Creative Diagnostics
Kategorie	Food Safety Antibiotics Fungicides ELISA ELISA & Immunoassays ELISA Kits Immunoassay
Typ	Elisa-Kit
Specific against	other
Menge	96T
ArtNr	DEIA057
eClass 6.1	32160605
eClass 9.0	32160605
Lieferbar
Abbr	Malachite Green Kit
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-labeledMalachite green 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 a direct competitive ELISA assay. The anti-mouse secondaryantibody of interest has been coated in the plate wells. During the analysis, sample is added along with the HRP labeled Malachite green for theanti-Malachite green monoclonal antibody. If the target is present in thesample, it will compete for the antibody, thereby preventing the antibodyfrom binding to the anti-mouse secondary antibody attached to the well. Theresulting color intensity, after addition of substrate, has an inverserelationship with the target concentration in the sample.
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 10× 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	fish/shrimp and water
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 color of themixture turns blue within few minutes, do not continue carrying out thetesting and replace the reagents with fresh ones.
Assay Procedure	1.Add 50ul anti-Malachite green antibody, and then add 20ul of the standardsolutions or samples (sample extracts) into the wells of the test stripsaccording to the working scheme given. We recommend using duplicates ortriplicates. 2.Add 30ul of HRP-labeled Malachite green to the individual wells successivelyusing a multi-channel pipette or a stepping pipette. 3.Cover the wells with parafilm or tape and mix the contents by moving thestrip holder in a circular motion on the benchtop for about 30 seconds. Be carefulnot 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.Cover the wells with parafilm or tape and mix the contents by moving thestrip holder in a circular motion on the benchtop for about 30 seconds.Incubate the strips for 15-20 minutes at room temperature. Protect the stripsfrom direct 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 each standard by dividing the mean absorbance value for eachstandard by the Zero Standard (Standard 0) mean absorbance. Construct astandard curve by plotting the %B/B0 for each standard on the vertical linear(y) axis versus the corresponding Enrofloxacin concentration on the horizontallogarithmic (x) axis on graph paper. %B/B0 for samples will then yield levelsin ppb of a Enrofloxacin by interpolation using the standard curve. Samplesshowing lower concentrations of Enrofloxacin compared to Standard 1 (0.15ng/mL) are considered as negative. Samples showing a higher concentrationthan Standard 5 (4.5 ng/mL) must be diluted further to obtain accurateresults.

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