DETECTION OF RECOMBINANT PROTEINS BY ELISA

Refer to the Western Blot Protocol for preparation of bacterially expressed protein. From a 10 ml bacterial culture, 2-4 plates can be coated for a direct ELISA.

Direct ELISA:

2. Lyse and sonicate the bacteria that express the protein of interest. If the recombinant protein is not soluble in H₂ O, dissolve the insoluble pellet in a volume of 7 M urea equal to 1/10 of the bacterial culture volume. Vortex the samples at a medium speed for about 30 minutes (use a vortex setting of 4.5 on the Fisher Vortex Genie 2). Monitor the solubility of the protein visually. There may be some debris left that may typically be removed by binding to the tip of a plastic pipet tip.
   
   
4. Increase the volume of the antigen to 1 - 2 times the original culture volume with carbonate coating buffer (pH 9.6) to coat the ELISA plate. Two plates can be coated with the 1 times the volume, 4 plates with 2 times the volume.
   
   
6. Label 96 well Costar (#2797) plates as needed. (See attached example of a 96 well plate which we use for bacterially expressed recombinant proteins). Controls will include no antigen, and extracts from bacteria transformed with the appropriate expression vector which lack the insert. Add 50µl per well of the appropriate solution in coating buffer. Wait 2 hours at room temperature or overnight at 4°C. Plates coated with the 52kD Ro/SSA recombinant protein can be stored at -70°C for 2 months. The stability of other molecules may vary.
   
   
8. Rinse the plates 5 times with PBS-Tween washing solution using the ELISA plate washer. Add 200 µl per well of 1% BSA in PBS to block the plates. Incubate for 30 minutes at room temperature or overnight at 4°C.
   
   
10. Rinse the plates again 5 times with PBS-Tween. Add 50 µl per well of serum diluted in ELISA diluent (serial 10-fold dilutions from 10 -2 to 10 -5 are useful for initial screenings). Incubate for 2 hours at room temperature, or for 1 hour at 37°C. Antibodies may be blocked with an oligopeptide by incubating them together overnight at 4°C, or for 2 hours at 37°C.
    
    
12. Rinse plates 5 times with PBS-Tween. Add 50 µl per well of the alkaline phosphatase-conjugated secondary antibody diluted 1:2000 in ELISA conjugate diluent. Incubate for 2 hours at room temperature, or 1 hour at 37°C.
    
    
14. Rinse plates 5 times with PBS-Tween. Add 50 µl per well of 1 mg/ml p-nitrophenyl phosphate substrate prepared in substrate buffer. Incubate about 1 hour to develop - WATCH CLOSELY
    
    
16. Calibrate the ELISA plate reader so that a 10 -3 dilution of a "reference serum gives an OD of 1.0 at 405 nm. Continue to read the remainder of the plate. If the ELISA plate reader is not available, stop the reaction by adding 10 µl of 30% NaOH per well. Store at 4°C overnight, and read the next day. If no color develops after 1 hour, plates can be wrapped in foil and kept at 4°C overnight.
    

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• • Carbonate Coating Buffer, pH 9.6 (0.15 M sodium carbonate, 0.35 M sodium bicarbonate, 0.03 M sodium azide)

    3.18 g Na₂ CO 3

    5.86 g NaHCO

    3

    0.4 g NaN

    3

    Adjust pH to 9.6 with HCl. QS to 200 ml with H

    2O. Millipore filter (optional).
    
    

    10x Phosphate Buffered Saline, pH 7.4 (0.2 M phosphate, 1.5 M NaCl)

    2.28 g NaH

    2PO 4 (mw=120); 0.038M)

    -or- 2.62 g NaH

    2PO 4(H 2O) (mw=137.99; 0.038M)

    11.5 g Na

    2HPO 4 (mw=141.96; 0.162 M)

    43.84 g NaCl

    pH to 7.4. QS to 500 ml with water. Dilute 1:10 for final concentration.
    
    

    PBS-Tween (washing solution) PBS plus 0.05% Tween-20, 0.02% sodium azide)

    400 ml 10x PBS

    8 ml 10% sodium azide

    2 ml Tween-20

    QS with to 4 L with H

    2O.
    
    

    ELISA Diluent (1x PBS, 0.05% Tween-20, 0.1% BSA, 0.02% azide)

    50 ml 10x PBS

    1 ml 10% sodium azide

    0.25 ml Tween-20

    0.5 g BSA

    QS to 500 ml with H

    2O.
    
    

    ELISA Conjugate Diluent

    PBS, 0.05% Tween-20, 0.1% BSA, 2 µg/ml IgG)

    Make above ELISA diluent and add 1 mg/ml Bovine Cohn Fraction II IgG

    QS to 500 ml with H

    2O.
    
    

    Blocking Solution (1x PBS with 1% BSA, 0.02% azide)

    50 ml 10x PBS

    1 ml 10% sodium azide

    5 g BSA

    QS to 500 ml with H

    2O
    
    

    Substrate Buffer, pH 8.6

    1.4 g NaHCO

    3 (0.167M)

    0.13 g Na

    2CO 3 (0.012M)

    0.02 g MgCl

    2 (100 µl 1 M stock; 1 mM)

    200 µl 10% sodium azide

    Add Na

    2CO 3 slowly while stirring. Adjust pH to 8.6 and QS to 100 ml with H 2O.
    
    

    Substrate Solution (1 mg/ml paranitrophenyl phosphate PNPP)

    100 mg capsule of PNPP (Sigma 104-100)

    100 ml substrate buffer

    Store at -20°C in 10 ml aliquots. Keep protected from light
    
    

    Sodium Azide, 10%

    For 20 ml:

    2g sodium azide

    QS to 20 ml with water
• • Carbonate Coating Buffer, pH 9.6 (0.15 M sodium carbonate, 0.35 M sodium bicarbonate, 0.03 M sodium azide)

    3.18 g Na₂ CO 3

    5.86 g NaHCO

    3

    0.4 g NaN

    3

    Adjust pH to 9.6 with HCl. QS to 200 ml with H

    2O. Millipore filter (optional).
    
    

    10x Phosphate Buffered Saline, pH 7.4 (0.2 M phosphate, 1.5 M NaCl)

    2.28 g NaH

    2PO 4 (mw=120); 0.038M)

    -or- 2.62 g NaH

    2PO 4(H 2O) (mw=137.99; 0.038M)

    11.5 g Na

    2HPO 4 (mw=141.96; 0.162 M)

    43.84 g NaCl

    pH to 7.4. QS to 500 ml with water. Dilute 1:10 for final concentration.
    
    

    PBS-Tween (washing solution) PBS plus 0.05% Tween-20, 0.02% sodium azide)

    400 ml 10x PBS

    8 ml 10% sodium azide

    2 ml Tween-20

    QS with to 4 L with H

    2O.
    
    

    ELISA Diluent (1x PBS, 0.05% Tween-20, 0.1% BSA, 0.02% azide)

    50 ml 10x PBS

    1 ml 10% sodium azide

    0.25 ml Tween-20

    0.5 g BSA

    QS to 500 ml with H

    2O.
    
    

    ELISA Conjugate Diluent

    PBS, 0.05% Tween-20, 0.1% BSA, 2 µg/ml IgG)

    Make above ELISA diluent and add 1 mg/ml Bovine Cohn Fraction II IgG

    QS to 500 ml with H

    2O.
    
    

    Blocking Solution (1x PBS with 1% BSA, 0.02% azide)

    50 ml 10x PBS

    1 ml 10% sodium azide

    5 g BSA

    QS to 500 ml with H

    2O
    
    

    Substrate Buffer, pH 8.6

    1.4 g NaHCO

    3 (0.167M)

    0.13 g Na

    2CO 3 (0.012M)

    0.02 g MgCl

    2 (100 µl 1 M stock; 1 mM)

    200 µl 10% sodium azide

    Add Na

    2CO 3 slowly while stirring. Adjust pH to 8.6 and QS to 100 ml with H 2O.
    
    

    Substrate Solution (1 mg/ml paranitrophenyl phosphate PNPP)

    100 mg capsule of PNPP (Sigma 104-100)

    100 ml substrate buffer

    Store at -20°C in 10 ml aliquots. Keep protected from light
    
    

    Sodium Azide, 10%

    For 20 ml:

    2g sodium azide

    QS to 20 ml with water

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