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Protocols - ELISA: Direct

STANDARD AND ANTIBODY
PeproTech’s Recombinant Protein1
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); 
Avidin-HRP conjugate (Sigma Cat. # A-7419)1
ABTS Liquid Substrate Solution (Sigma Cat. # A3219)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 (Cat. # 900-K00)

PLATE PREPARATION 
  1. Standard/Sample: Serial dilute standard from 0.1μg/ml to zero in PBS. Add 100μl of standard or sample to each well in triplicate. Incubate at room temperature overnight.  
  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 2 hour at R.T. 
  4. Aspirate and wash plate 4 times (as in step 2). 

ELISA PROTOCOL

Detection:
Wash plate four times. Dilute detection antibody (biotinylated) in diluent to a concentration of 1 μg/ml. Immediately add 100μl per well. Incubate at room temperature for 2 hours. 

Avidin-HRP Conjugate: Aspirate and wash plate 4 times. Dilute Avidin-HRP conjugate 1:2000 in diluent. 
Add 100μl per well. Incubate 30 min at room temperature. 

Substrate: 
Aspirate and wash plate 4 times. Add 100μl of substrate solution to each well. Incubate at room temperature for color development. Monitor color development with an ELISA plate reader at 405 nm with wavelength correction set at 650 nm. 

NOTE: Reliable standard curves are obtained when O.D. readings do not exceed 0.2 units for the zero standard concentrations, or 1.2 units for the highest standard concentration. The plate should be monitored at 5 minute intervals until desired O.D. readings are obtained. The typical range is 5-40 minutes. O.D. readings may vary. 

Assay sensitivity may be increased with additional washings.