ELISA - Standard Sandwich Protocol

ELISA
ELISA Plate
   Dynex Immulon 4HBX 96-well plate (ISC BioExpress)
   or
   Corning EIA/RIA 96-well plate High Binding Certified Catalog #9018 (Fisher)
Coat Buffer:
   0.1M Na2HPO4 7H₂O (FW 268.07)
   pH9.0
   Filter with cell culture bottle filter
10X PBS (1L)
   2.57g NaH₂PO₄ H₂O (FW 137.99)
   22.49g Na₂HPO₄ 7H₂O (FW 268.07)
   87.65g NaCl
   pH7.4
Blocking Buffer
   1X PBS
   1% BSA (10mg/mL)
   Make 500mL
   Filter using cell culture bottle filter (contaminates quickly)
   Azide can be used for final 0.3%, however, avoid it in any HRP-labeled step
Freeze 250mL at -20C (45mL aliquots into 50mL falcon tubes)
   Store 250mL at 4C
Wash Buffer
   1XPBS
   0.0375%-0.05% Tween-20 (375-500ul of Tween-20 in 1L 1X PBS)

Streptavidin-HRP
   BD Pharmingen Catalog# 554066
Substrate Solution
   1XTMB Substrate Solution eBioscience Catalog# 00-4201-56
Stop solution
   3M Sulfuric Acid

Prepare coat buffer/Ab mixture:
Need 50ul per well
Antibody concentration needs to be 4ug/mL, user determines optimal amount
For example: If you are doing 12 wells
      12 X 50ul/well = 600ul coat buffer
      4ug/mL X 0.6mL = 2.4ug
      Antibody concentration is 1.5ug/ul : 1ul/1.5ug X 2.4ug = 1.6ul
   Add 1.6ul antibody to 600ul of coat buffer and mix

1-   Add 50ul per well Antibody/Coat buffer mixture into each well
Tap to cover wells evenly
Place parafilm over top of wells
Incubate at Room Temperature (RT) for at least 2 hours (or all day or at 4C overnight)
2-   Add 100ul per well Blocking buffer to the Antibody/Coat buffer mixture
Incubate at RT for 1 hour
3-   Remove all the buffer in the wells by dumping it into waste (or sink) , then pound plate on paper towels to remove as much buffer as possible
Wash 1 time with 150ul per well of wash buffer
Remove the wash buffer as previously
4-   Add cell lysates to wells
Add 25-100ul total volume of cell lysates (usually 50ul); do each lysate in duplicates or triplicates (best)
Incubate at 4C overnight or 2-4 hours RT
5-   Remove lysates from all the wells by dumping it into waste, then pound plate on paper towels. Be careful not to cross contaminate. Be sure to NOT get any lysates on the top of the plate or from one well into another.
6-   Add 150ul per well Wash buffer and then remove as previously.
Repeat wash for a total of 3 times.
On last wash after removing buffer be sure to smack plate against paper towels numerous times to be sure that it is very dry.
7-   Add 50ul per well of Secondary antibody diluted in blocking buffer
Incubate for 1 hour at RT
Use secondary antibody at a similar concentration as the primary antibody.
Therefore we need a concentration of 4ug/mL.
Secondary antibody = 9E10-biotinylated at 1000ug/mL so dilute it 1:250
Add 4ul of 9E10 in 1mL of Blocking buffer
(When adding secondary antibody do not touch the tip in the wells you do not want to cross-contaminate. Therefore it is best to set the pipette to 55ul, hold tip above well and only release to the first stop on the pipette.)
8-   Remove secondary antibody by dumping it into waste and then hitting plate onto paper towels.
9-   Add 150ul per well Wash buffer and then remove the buffer. Repeat wash for a total of 3 times.
10-   Add 50ul per well streptavidin-HRP (buy from BD Bioscience or E-Bio, ELISA grade this is best)
Incubate 45min-1.5 hour at RT (Never go longer than 1.5 hours)
11-   Remove streptavidin-HRP by dumping it into waste and then hitting plates on paper towel.
12-   Add 150ul per well Wash buffer and then remove as previously. Repeat for a total of 4 times.
13-   Add 50ul per well Substrate solution. Gently tap plate to mix.
14-   Put plate on white sheet of paper and wait for blue color to develop (length of time will vary 30sec-10min depending on samples) (Ideal time is when noise is just noticeable)
15-   Then add 50ul of Stop solution (e.g. sulfuric acid). Gently tap plate to mix.
Color will then turn yellow instantly.
16-   Read plate on plate reader at 450nm.