Antibody Labeling
Immunoassay designs rely on the great specificity of antibodies and a suitable label which facilitates the generation of a quantitative signal. Antibody labeling techniques are involved in the interaction of reactive groups between enzyme/chemicals and proteins. It is very important to retain all the immunoreactivity of antibody and all the signal-generating capacity of the label. Here is a summary of protein/enzyme with broad estimation.
Protein Source MW(kDa) Number of-NH2 Number of-SH Number of-COOH
HRP Horseradish 44 6 none 28
AP E. Coli 94 56 none 98
AP Bovine 125 42 none 106
b-Galactosidase E. Coli 465 80 64 508
BSA Bovine 68 59 35 98
Antibodies can be labeled by covalent coupling to enzymes, biotin, fluorochromes, etc. The choice of the label depends on the technique or immunoassay methods in different applications. Horseradish peroxidase (HRP), alkaline phosphatase (AP), b-galactosidase are also used for labeling antibodies.
Biotinylation of Purified Antibody Protocol
Biotinylation of primary antibodies is the most commonly used method. This reaction does not inactivate the antibody and comprise simple techniques. Biotinylated primary antibodies can be detected using the biotin-binding protein streptavidin.
Materials:
1 M NH4Cl
N-hydroxysuccinimidate biotin
Dimethyl sulfoxide (DMSO)
0.1 M NaBH4, pH.8.8
Primary antibody
Procedure:
Dialyze antibody against 0.1 M NaHCO3 at 4°C overnight.
Prepare a solution of N-hydroxysuccinimidate biotin at 10 mg/mL in DMSO.
Mix the antibody solution with biotin ester at a ratio of 25-250 mg of ester per mg of antibody; stir the mixture for 4 h at room temperature
Add 20 mL 1 M NH4Cl per 250 mg ester; incubate for 10 min at room temperature
Dialyze the mixture against PBS, pH 7.4.
Adjust the pH of the antibody solution and add stabilizer for long-term storage.
Labeling Antibodies with Horseradish Peroxidase (HRP) Protocol
Materials
Horseradish Peroxidase (HRP) 0.1 M Sodium Periodate
0.1 M NaHCO3, pH 9.5
NaBH4 solution (4 mg/mL)
Procedure
Antibody preparation: Dialyze purified antibody against 0.1 M NaHCO3, pH 9.5 at 4°C overnight.
HRP preparation: Dissolve 10 mg HRP in 2 mL HPLC-grade DI water. Add sodium periodate to final concentration of 5 mM and incubate for 20 min at room temperature.
Combine HRP with antibody and incubate for 3 h at room temperature.
Add 100 mL NaBH4 solution (4 mg/mL) and incubate for 30 min at room temperature.
Dialyze the mixture against PBS, pH 7.4 at 4°C overnight.
Add conjugate stabilizer and aliquot for long-term storage.
Labeling Antibodies with Alkaline Phosphatase (AP) Protocol
Materials
Alkaline Phosphatase (AP) 0.1 M SPBS
25% Glutaraldehyde
1 M Ethanolamine
Procedure
Mix 10 mg antibody with 5 mg AP in a final volume of 1mL.
Dialyze the mixture against four changes of sodium phosphate buffer at 4°C overnight.
Place the mixture in a small container, add small stir bar.
Add 50 mL 1% EM grade glutaraldehyde; stir for 5 min, then incubate for 3 h at room temperature.
Add 0.1 mL of 1 M ethanolamine, pH 7.0 and incubate for 2 h.
Dialyze overnight at 4°C against 3 changes of PBS.
Spin the mixture at 40,000g for 20 min and store the supernatant at 4°C in the presence of conjugate stabilizer.
Gluteraldehyde conjugation of oligopeptides to proteins for ELISA
Dissolve 30 mg of the carrier protein (ex. KLH) in 3 ml of phosphate buffered saline (PBS) The amount of protein should be about 4 times more than you expect to use. Dialyze for 24 hours in PBS. Centrifuge at about 12,000 xg (use 10,000 rpm in an SS-34 rotor) for 20 minutes to remove insoluble material.
Measure the absorbance of the protein solution at A280 after calibrating a spectrophotometer with PBS. For KLH, O.D. = 2.02 for a 1 mg/ml solution.
In the following experiments, we were conjugating a 15 amino acid peptide to KLH. The mass ratio of peptide to carrier protein was 1:2. Conjugation was performed in a 3 ml volume. Add PBS to the KLH, add the peptide to the carrier protein, and then add the gluteraldehyde as a 1:100 dilution in PBS while stirring. Two examples are given below.
Example KLH PBS Peptide Gluteraldehyde 1 M Lysine (in H2O)
1 20 mg 2 ml 10 mg 1 ml 120µl
2 3.38 mg in 1.2 ml 0.7 ml 1.69 mg in 0.1 ml 1 ml 120µl
Stir at room temperature for 1-6 hours. Check A405 about every 15 minutes to check for a plateau. Add lysine when the OD plateaus to stop the reaction. Continue to stir for another hour.
Dialyze in PBS overnight at 4°C.
Make dilutions of peptide to 100µg/ml, 50µg/ml, 10µg/ml, and 1µg/ml in carbonate coating buffer. For the second conjugation above, 1.69mg/3.12ml = 542µg/ml. Coat ELISA plate with 50µl/well for 2 hours at room temperature.