Be0291

A 10-fold molar excess of Dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (NHS-PEG4-DBCO, A134–10, Click Chemistry Tools, USA) was added to anti-CD3 (Bio X Cell BE0001–2, clone OKT-3, 2 μg/μL) or anti-CD28 antibodies (Bio X Cell BE0291, clone CD28.2, 2 μg/μL) and incubated at room temperature for 1 h. Then the solution was purified by Amicon centrifugation (MWCO 10 kDa), 10000 g, 10 mins until the flow through was free from NHS-PEG4-DBCO (measured by characteristic absorbance of DBCO moiety at 309 nm using Nanodrop). The degree of DBCO incorporation (i.e. the number of DBCO per antibody) was determined from the absorbance scan of the purified conjugate (235‐400 nm) using the following equation.
(Molarity of DBCO) / (Molarity of antibody) = (A₃₀₉ DBCO x ε₂₈₀ Ab) / (ε₃₀₉ DBCO x A_280c Ab)
A₃₀₉ DBCO = DBCO-Ab conjugate’s absorbance at 309 nm
ε₂₈₀ Ab = 210,000M⁻¹cm⁻¹A_280C Ab = conjugate’s corrected absorbance at 280 nm = A₂₈₀ - (A₃₀₉ x CF DBCO)
ε₃₀₉ DBCO = 12000M-1cm-1
A₂₈₀Ab = DBCO-Ab conjugate’s absorbance at 280 nm
CF DBCO = DBCO correction factor at 280 nm = 1.089