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Protocols - ELISA: Sandwich TMB

STANDARD AND ANTIBODIES
PeproTech’s Recombinant Protein1
PeproTech’s Antigen Affinity Purified Polyclonal or Monoclonal Antibody1
PeproTech’s Biotinylated Antigen Affinity Purified Polyclonal Antibody1
**PeproTech’s Standards and Antibodies should be reconstituted according to the data sheets which accompany each product.

RECOMMENDED MATERIALS 
ELISA microplates (Nunc MaxiSorp Prod. # 439454, or Corning Prod. # 3590)2;
Tween-20 (Sigma Cat. # P-7949); 
BSA (Sigma Cat. # A-7030);
Streptavidin-HRP conjugate (Pierce Cat. # 31032)1;
TMB Liquid Substrate Solution (KPL Cat. # 52-00-02)2
Dulbecco’s PBS [10x] (Gibco BRL Cat. #14200-075)
Sealing Film2.

RECOMMENDED SOLUTIONS
All solutions should be at ambient temperature prior to use.
PBS: Dilute 10xPBS to 1xPBS, pH 7.20 in sterile water2.
Wash Buffer: 0.05% Tween-20 in PBS2                 
Block Buffer: 1% BSA in PBS*2           
Diluent: 0.05% Tween-20, 0.1% BSA in PBS*2                                                     

*Sterile filter and store at 4°C for up to 1 week.   

1Included in Standard and Mini ELISA Development Kits
2Included in ELISA Buffer Kit (900-T00)                                                 

PLATE PREPARATION 
  1. Dilute capture antibody (polyclonal) with PBS to a concentration of 1μg/ml. Immediately, add 100μl to each ELISA plate well. Seal the plate and incubate overnight at room temperature. (Monoclonal Antibody – at least 2 μg/ml).
  2. Aspirate the wells to remove liquid and wash plates 4 times. Each wash consists of adding 300μl wash buffer per well, followed by aspiration. After the last wash invert plate to remove residual buffer and blot on paper towel. 
  3. Add 300μl blocking buffer to each well. Incubate 1 hour at R.T. 
  4. Aspirate and wash plate 4 times (as in step 2). 

ELISA PROTOCOL
Standard/Sample: Serial dilute standard from 0.01μg/ml to zero in diluent. Add 100μl of standard or sample to each well in triplicate. Incubate at room temperature for at least 2 hours.

Detection: Wash plate four times. Dilute detection antibody (biotinylated) in diluent to a concentration of 0.5μg/ml (500ng/ml). Immediately add 100μl per well. Incubate at room temperature for 2 hours. 

Streptavidin-HRP Conjugate: Aspirate and wash plate 4 times. Dilute Streptavidin-HRP conjugate 1:20,000 in diluent. 
Add 100μl per well. Incubate 30 min at room temperature. (Dilutions can also be tried at 1:10,000 and 1:40,000, though a dilution of 1:20,000 is most commonly used.) 

TMB Liquid Substrate: Aspirate and wash plate 4 times.  Add 100μl of substrate solution to each well.  
Incubate at room temperature for 20 minutes. Add 100μl 1M HCl stop solution to each well. Monitor color development with an ELISA plate reader at 450 nm with wavelength correction set at 620 nm. 

NOTE: Reliable standard curves are obtained when O.D. readings do not exceed 0.150 units for the zero standard concentrations. Development time and O.D. readings may vary.