Quick and simple method for conjugating mAbs

Note 1: as the method for coupling both FITC and Biotin are very similar we usually make both conjugates at the same time (unless antibody is in short supply or bought commercially at great expense!).

Note 2: small amounts of antibody (ie. 100mcg to 1mg) can be conjugated directly in a "Slide-A-Lyzer 10K" dialysis cassette (Pierce).

1) Take 1 mg of purified, carrier-free antibody and dialyse against bicarbonate buffer (17.3 g/l NaHCO3 + 8.6 g/l Na2CO3) for at least 1 hour (if antibody contained azide or Tris then dialyse twice). See here for methods of purification.

2) Measure OD 280 and dilute to 1mg/ml (OD=1.4). If antibody is already more dilute than this treat it as 1mg/ml when calculating amounts of FITC or biotin to add as excess will compensate for poor efficiency at lower concentrations.

3a) FITC: Make up FITC (FITC Isomer I from Sigma F4274: note must be kept 4degC and dessicated) just before use to 1mg/ml in DMSO, using a glass bottle (FITC solution should be a very pale yellow if DMSO has not absorbed water), and add 45 mcl per milligram of protein (or per ml where less than 1mg/ml: at this point the solution should turn bright yellow/green). This should generate a molar ratio of around 5 to 1 (which is ideal for FACS staining). Mix and incubate in the dark at room temperature for at least 2 hours (or 4degC overnight) with occasional mixing.

3b) Biotin: Make up Sulfo-NHS-LC-Biotin (EZ-Link from Pierce Cat No: 21335, which must be stored frozen and dessicated and must therefore be allowed to warm up before opening to avoid condensation) just before use to 1mg/ml in pure water (ordinary de-ionized is not ideal due to possible contaminating amines), using a glass bottle, and immediately add 37 mcl per milligram of protein (or per ml where less than 1mg/ml). Mix and incubate at room temperature for at least 2 hours (or 4degC overnight) with occasional mixing.

4) Dialyse back into phosphate buffered saline (PBS) to remove unconjugated reagents, for 2 hours at room temperature or 4 degC overnight.

5) For FITC conjugates it is worth measuring the OD280 and OD493, which should have a ratio of around 1.1 for a molar coupling ratio of 5.

6) We usually add BSA to 1% and 0.1% NaN3 to stabilize and preserve the conjugates, which can generally be stored either at 4 degC or frozen in aliquots at -20 degC. However, it is worth noting that some FITC conjugates in particular seem to be less stable than the original antibody (it seems unpredictable but a function of the individual antibody), and we have found that storage at -70 degC, or changing the pH of the buffer (eg 0.1M NaHCO3 at pH8.3) can help.

7) All conjugates need to be tested by titrating in the chosen application - usually for FACS staining - and should be used at 2-4 times the concentration that gives saturation without background. For most well behaved antibodies this should be in the range of 5-10 mcg/ml.

Steve Cobbold - April 1997