INTRODUCTIONELISA, also called Enzyme Linked Immunosorbent Assay, employsantigens or antobodies conjugated to enzymes in such a way thatthe immunological and enzymatic activity of each component ismaintained. These assays are very sensitive and give accurateresults. The estimation of results can be made either visually orspectrophotometrically. Various formats of ELISA are available.Depending on the need and availability of reagents differentreaction format can be used in Dot ELISA.PRINCIPLEIn DOT ELISA antigen is coated on a nitrocellulose membrane(Instead of an ELISA plate). After incubation unadsorbed antigensare washed out, then rest of the reaction sites are blocked on themembrane. Specific antibody bind with antigen in the NCmembrane, unbound antibodies get washed out. The antibodieswhich are bound then detected by the addition of enzyme labelledwith secondary antibody. This complex appears as clear brown dotafter addition of specific substrate.
(Enough to perform 15 Experiments) S.No. Name of Items Quantity Storage 1. Antibody 200µl -20°C 2. Precoated (Antigen) Strips 3x15 Nos 4°C 3. Blocker (Dissolve 100 mg in 5ml 1X PBS) 1.5g 4°C 4. Substrate 30mg 4°C (Always prepare substrate freshly before each test) 5. Conjugate 10ml 4°C 6. Hydrogen peroxide 500µl 4°C 7. 1X PBS 100ml RT 8. Wash Buffer 100ml x 2 RT 9. 96 well Microtiter Plate 1 No. RTImportant Note : All Kit components are recommended to bestored at respective storage conditions as mentioned above uponreceiving this kit. Repeated thawing of antigen and antibody is notrecommemnded.MATERIALS NEEDED BUT NOT PROVIDED1. Triple Distilled water2. Glasswares (Conical Flask, Measuring cylinder, Pipette)3. Micropipette & Pipette Tips
WORKING SOLUTION PREPARATION1. BLOCKING SOLUTIONTo prepare 2% blocking solution take of 1X PBS and add 100mg ofblocker provided and mix well.Note: Prepare freshly everytime before each experiment.2. SUBSTRATE - Stock Solution PreparationWith the given total quantity of substrate bottle add 1ml of tripledistilled water and mix well by repeat pipetting. Transfer this 1ml to29ml of triple distilled water. Aliquot this 30 ml stock solution into 3separate 10 ml storage tubes and wrap it with aluminium foil andstore at -20°C for subsequent usage. This will avoid the loss ofeffectiveness of the substrate stock solution at the time of thawingfor the subsequent usage of each test.SUBSTRATE - Working Standard PreparationTake 1ml of the substrate stock solution and mix with 1µl ofhydrogen peroxide. The working Standard from substrate stockthus prepared is sufficient for one experiment. (Prepare this stepfreshly before each test.)PROCEDURENOTE : All the micropipettes should be well calibrated and thepipetting should be accurate to get optimum results. Any smallvariations would lead to major differences in the optical densityvalues.PRECOATED (ANTIGEN) STRIPSPrecoated Antigen strips are provided in the kit as given in the figure
Bring the strips to room temperature and incubate at 37°C for10-15min. Please note that these precoated strips are usedthroughout the procedure of following steps.
DOT ELISA Incubate the strips at 37° C for 10 - 15 mins.BLOCKING Place the strips in 3 wells of microtitre plate Add 300 µl of Blocker solution Incubate at 37° C for 45 mins.WASHING Immerse the strips in 300 µl of wash buffer Shake vigourouslyANTIBODY INCUBATION Take 200 µl of diluted antibody in three wells Dip the strips and incubate at 37° C for 45 mins. Repeat Washing step
CONJUGATE Dip the strips in 200 µl of conjugate in three wells Incubate at 37° C for 45 mins. Repeat Washing StepSUBSTRATE Dip the strips in 200 µl of substrate in three wells Incubate at 37° C for 10 mins. Stop the reaction using tap water Air dry for few mins. Observe the Result
RESULT AND INTERPRETATIONThe appearance of brown dot indicates the presence of antigenwhereas nothing appears in negative control (NC). No colour isappeared in the (NC)membrane due to presence of non-specificantigen in the strip and hence no reaction took place.