Malachite Green ELISA Kit

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.

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

Homogenized, centrifuged, evaporated, diluted for assay

fish/shrimp and water

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.