Preparation
     In this example, 1 blank (which is simply the sample diluent), 7 standards, 3 controls, and 37 unknown samples are being tested in duplicate in 1 microtiter plate MoAb-PoAb sandwich immunoassay.  

Standards

     As each kit typically comes with only 1 vial of a high-concentrate standard which is lyophilized (freeze dried into a powder), it must be reconstituted, and then further diluted with sample diluent.  If the lyophilized standard was at 5000pg, when reconstituted with 5mL, it would have a concentration of 5000pg/5mL or 1000pg/mL.  To generate a standard curve, it should be serially diluted 1:2  (500uL standard with 500uL sample diluent) to create standards at 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, and 15.13pg/mL.

Controls
     The controls may or may not come with a kit, but are often lyophilized and must also be reconstituted, typically with 2mL sample diluent.  Controls are usually not diluted further.

Unknown Samples
    
Any samples thought to have a value higher than the reconstituted standard (at 1000pg/mL) should be diluted with the sample diluent so that it falls between the high standard (at 1000pg/mL) and the low standard (at 15.13 pg/mL).   If they do not need to be diluted, samples often can be run neat.  However, if the samples have some sort of interfering substance, such as rheumatoid factors, or complement, they may need to be treated prior to running in the assay.  If you do not know about your samples, simply run them in the assay once and see where they fall (neat and diluted samples can be run at the same time).  If the values are too high, just dilute, and/or treat the samples and run the assay again.  Each investigator must determine their own protocols for sample dilutions and pre-assay treatments.

Immunoassay Procedure
Step                                                  Discription
1 Add 50 uL per well of assay diluent into every well of the plate.
2 Add 200 uL per well of your blanks, standards, controls, or samples onto the plate.  
Each item should be tested in duplicate (in 2 wells).
3 Cover with plate sealer and incubate 2 hours at room temperature.
4 Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4  cycles.  Blot on paper towels.
5 Add 200uL per well of PoAb-HRP conjugate solution into every well of the plate.
6 Cover with plate sealer and incubate 2 hours at room temperature.
7 Wash plate by filling wells with 400 uL wash buffer and dumping. Wash for a total of 4  cycles.  Blot on paper towels.
8 Add 200uL per well of TMB substrate solution into every well of the plate.
9 Incubate 20 minutes at room temperature.
10 Add 50 uL per well 2N HCl stop solution into every well of the plate
11 Read plate at 450nm while subtracting a reference wavelength of 540nm.
12 Calculate data based on OD values of the unknown samples compared to the
known values of the standard curve.
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