Back to Search
Start Over
A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse
- Source :
- Analytical and Bioanalytical Chemistry
- Publication Year :
- 2015
- Publisher :
- Springer Berlin Heidelberg, 2015.
-
Abstract
- The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (Kd) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with Kd in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of Kd as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure Kd of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave Kd values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50 % inhibition concentration (IC50) values in the micromolar range. Despite the differences in Kd and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not bind or poorly bound to heroin, 6-AM, and morphine. This simple and nonradioactive method can be extended to other platforms, such as oxycodone, cocaine, nicotine, and methamphetamine for the selection of the lead hapten design during substance abuse vaccine development. Graphical Abstract Determination of the antibody affinities using competition equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-9223-z) contains supplementary material, which is available to authorized users.
- Subjects :
- 0301 basic medicine
Heroin hapten
Antibody Affinity
Apparent dissociation constant (Kd)
UPLC/MS/MS
Enzyme-Linked Immunosorbent Assay
Chemistry Techniques, Synthetic
Tandem mass spectrometry
Biochemistry
Antibodies
Analytical Chemistry
03 medical and health sciences
Mice
Drug Stability
Tandem Mass Spectrometry
mental disorders
medicine
Animals
Chromatography, High Pressure Liquid
Morphine Derivatives
Chromatography
biology
Morphine
Chemistry
Toxoid
Deuterium
3. Good health
Competition ELISA
Substance Abuse Detection
030104 developmental biology
Equilibrium dialysis
Polyclonal antibodies
biology.protein
Antibody
Esterase inhibitor
Hapten
Oxycodone
Haptens
medicine.drug
Research Paper
Subjects
Details
- Language :
- English
- ISSN :
- 16182650 and 16182642
- Volume :
- 408
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....726961eb038d1cc431e3c68d1316d6cc