How do I calculate the conjugation ratios (loading) for immunochemicals?
Conjugation ratio is the molar ratio of hapten to protein. We recommend using Beer’s Law to calculate the conjugation ratio:
A = ε * L * C
Below is an example conjugation ratio calculation for NP-BSA (4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Bovine Serum Albumin).
ANP (Measured absorbance of the hapten (NP) at 430 nm, pH 8.5): for this example we will use OD430 = 1
εNP (Extinction coefficient of the hapten (NP) at 430 nm): 4230 M-1 cm-1
L (Path length): typically this value is assumed to be 1 cm
Calculate CNP (Molar concentration of the hapten (NP))
CNP = ANP / (εNP * L)
CNP = OD430 (no units) / (εNP (M-1 cm-1) * 1 cm)
CNP = 1 / 4230 M-1
CNP = 0.000236 Molar or 236 uM
Calculate CBSA (Molar concentration of BSA)
If provided a mass concentration, make sure to convert to molar concentration by dividing the mass concentration by molecular weight. For example, if NP-BSA is dissolved in 0.1 M NaHCO3 (pH 8.5) to 1 mg / mL (as recommended per the use instructions found under the Technical Specs tab of the product page ), then BSA has a mass concentration of 1 mg / mL or 1 g / liter.
Knowing that BSA has an approximate molecular weight (MW) of 60,000 g / mol, the molar concentration of BSA is then:
CBSA = Mass concentration / MW
CBSA = (1 g / liter) / (60,000 g / mol)
CBSA = 0.0000167 mol / liter or 16.7 uM
Calculate conjugation ratio (molar ratio (n) of the hapten (NP) to (BSA))
n = CNP/CBSA
n = 236 uM / 16.7 uM
n ≈ 14 NP haptens conjugated to each BSA molecule (NP14-BSA)