Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

Abstract

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses 14C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten–BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r2 = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten–protein conjugations, and choosing the most effective epitope densities for conjugate vaccines.

Optimization of a methamphetamine conjugate vaccine for antibody production in mice

“…The procedure was similar to the method of Peterson et al [21]. In brief, IC KLH was activated by the addition of Sulfo-SMCC crosslinker, then desalted to remove unused linker.…”

“…Separate conjugations resulted in three lots of vaccine with hapten densities of 13, 16, and 23 SMO9 molecules per KLH monomer as measured by the method reported in Peterson et al [21]. These are labeled IC KLH -SMO9 13 , IC KLH -SMO9 16 , and IC KLH -SMO9 23 , respectively.…”

Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response

“…The reaction mixture was stirred and warmed to 50 °C and kept there for 4 h. The mixture was cooled, filtered through a pad of celite, washed with MeOH and concentrated in vacuo. The resulting oil was dissolved in CH 2 Cl 2 , filtered and concentrated to give an oil which was chromatographed on silica gel, eluting with 50CMA80/EtOAc, to yield 3.24 g (77%) of 6 as a yellow oil: [a] 22 D +7.4 (c = 0.645, MeOH). 1 H NMR (300 MHz, CDCl 3 ) δ 7.10–7.27 (m, 2H), 6.82–6.93 (m, 2H), 3.82 (s, 3H), 2.95–3.10 (m, 2H), 2.60–2.70 (m, 1H), 2.54 (s, 3H), 1.17 (d, J = 6.5 Hz, 3H).…”

“…The mixture was extracted with CH 2 Cl 2 to afford 3.00 g of a light yellow oil. The oil was chromatographed on silica gel, eluting with 50 hexane/EtOAc, to afford 2.0 g (59%) of 10 as a light yellow oil: [a] 22 D = +53.4 (c = 0.545, MeOH). 1 H NMR (300 MHz, CDCl 3 ) δ 7.07 (m, 1H), 6.98 (bd, 1H), 6.71–6.79 (m, 2H), 3.91 (t, J = 6.3 Hz, 2H), 2.55–2.75 (bm, 6H), 2.33 (t, J = 7.6 Hz, 2H), 1.68–1.90 (m, 4H), 1.50–1.65 (m, 2H), 1.28 (bs, 9H), 1.10 (d, J = 6.8 Hz, 3H).…”

“…Before administration, the MCV was thawed, diluted in administration buffer, and mixed with Sigma Adjuvant System. The IC KLH -SMO9 conjugation procedure is similar and is described in detail in the supplemental materials of Peterson et al 22 …”