EIA PROCEDURE GUIDE

  SMART & PRIMATE-ONE ASSAYS

 

INITIAL SETUP

¨         Bring one bottle of the 20x Wash Solution to 500ml with distilled water. MIX WELL

¨         Prepare the plate map. The columns alternate between black-ringed Antigen wells and red-ringed Control-Antigen wells . Each Control, Calibrator and specimen requires two wells: an Antigen coated well and a Control-Antigen coated well.

 

Well Location        Identification                     

A1 & A2         Reactive Control                       

B1 & B2         Negative Control

C1 & C2         Calibrator    

D1 & D2         Specimen #1

E1 & E2         Specimen #2                    

F1 & F2         Specimen #3                    

G1 &G2         Etc.                 

   

¨         Controls are ready to use, do not dilute

¨         Set up the calibrator and specimen dilution tubes according to the plate map.

¨         Prepare a 1:51 dilution of the calibrator and specimens as follows:

Add 250ul of Dilution Buffer to all dilution tubes

Add 5ul of each specimen or calibrator to appropriate tube. MIX WELL

   

SERUM INCUBATION STAGE

¨         Break-off the required number of Antigen and Control-Antigen wells and place into the plate holder. (Unused wells must be kept sealed in a dry environment)

¨         Add 50ul of the pos and neg controls to the appropriate wells.

¨         Transfer 50ul of each specimen and the calibrator dilution tube to the appropriate wells.

¨         Incubate at room temperature for 30 MINUTES.

 

WASH STAGE

¨         After 30 minutes, dump the tray. Refill each well to the top with wash buffer and dump.

¨         Repeat the above step two more times for a total of 5 WASHES.

¨         After the last wash, dump the tray and slap wells hard 3-4 times against a paper towel to remove excess buffer.

 

CONJUGATE INCUBATION STAGE

¨         Add 50ul (1 drop) of conjugate to each well.

¨         Incubate at room temperature for 30 MINUTES.

 

WASH STAGE

¨         After 30 minutes, dump the tray. Refill each well to the top with wash buffer and dump.

¨         Repeat the above step two more times for a total of 5 WASHES.

¨         After the last wash, dump the tray and slap wells hard 3-4 times against a paper towel to remove excess buffer.

 

SUBSTRATE STAGE

¨         Add 50ul (1 drop) of Chromogen to each well.

¨         Incubate at room temperature for 10 MINUTES.

¨         DO NOT DUMP AFTER THIS INCUBATION PERIOD.

 

STOP STAGE

¨         Add 50ul (1 drop) of STOP SOLUTION to each well.

¨         Brace the plate with one hand and gently tap along the opposite side of the plate with the other to evenly distribute the Stop Solution in each well.

¨         Read the plate at 450/620-650nm wavelengths (or at 450nm if using a single wavelength reader) within 30 minutes of adding the Stop Solution.

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