title: Synthesis of Hapten-Protein Conjugate Vaccines with Reproducible Hapten Densities.
abstract: The ability to prepare hapten-carrier conjugates reproducibly with consistent lot-to-lot hapten densities and protein yields is a critical component of hapten vaccine development. This entails the development of appropriate coupling chemistries that do not cause protein precipitation and the development of methods to quantify hapten density. Recently, extensive efforts have been devoted to design vaccines against drugs of abuse. We describe, herein, a method for conjugation of a morphine-like hapten (MorHap) to tetanus toxoid (TT), which involves conjugation of MorHap to the surface lysines of TT through the N-hydroxysuccinimide portion of a heterobifunctional linker and the subsequent attachment of the thiol on MorHap to the maleimide portion of the cross-linker. Methods are described for the analytical quantification of the hapten density of the conjugates using modified Ellman's test, trinitrobenzenesulfonic acid (TNBS) assay, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.|US Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD, 20817, USA.
authors.forename: Oscar B
authors.initials: OB
authors.lastname: Torres
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.
authors.forename: Carl R
authors.initials: CR
authors.lastname: Alving
authors.affiliation: Laboratory of Adjuvant and Antigen Research, US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA. gmatyas@hivresearch.org.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Matyas
mesh_terms: D060326:Chemistry Techniques, Synthetic / Q000379:methods*; D003956:Dialysis; D006241:Haptens / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D009020:Morphine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013744:Tetanus Toxin / Q000737:chemistry; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry; D018074:Vaccines, Conjugate / Q000737:chemistry*
chemical_list: D006241:Haptens; D008301:Maleimides; D011506:Proteins; D013744:Tetanus Toxin; D018074:Vaccines, Conjugate; C043592:maleimide; D009020:Morphine; D014302:Trinitrobenzenesulfonic Acid
keywords: Drugs of abuse; Ellman’s test; Hapten density; Hapten-protein conjugates; Heroin vaccine; MALDI-TOF MS; MorHap; Tetanus toxoid; Thiol-maleimide chemistry; Trinitrobenzenesulfonic acid assay