Characterization of hapten binding to immunoconjugates by electrospray ionization mass spectrometry

Straub, Kenneth; Levy, Mark J.

doi:10.1021/bc00027a003

Cited by 16 publications

(7 citation statements)

References 11 publications

(17 reference statements)

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“…Thus, ESI-MS is becoming a common tool for molecular weight determination of proteins, DNA/RNA (20)(21)(22), peptides, and other macromolecules. However, there have been limited reports on characterization of conjugates using ESI-MS. A recent report detailed the characterization of haptenenzyme conjugates and concluded that for proteins in the mass range of 55 kDa, only conjugates with a hapten density of eight or less could be analyzed (23).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

1996

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Fifteen hapten-bovine serum albumin (BSA) conjugates were prepared from five commercially available activated haptens. Each hapten was coupled to BSA at three different ratios. The conjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two mass spectrometry (MS) methods: matrix-assisted laser desorption ionization (MALDI) and liquid chromatography-electrospray ionization (LC-ESI). SDS-PAGE was useful in detecting protein cross-linking, but not assessing hapten density. MALDI-MS and LC-ESI-MS gave comparable qualitative results, but LC-ESI-MS provided a clearer representation of the distribution of hapten-protein species present in the conjugates. Conjugate species substitute with up to 25 haptens per BSA were recorded by LC-ESI-MS.

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Section: Introductionmentioning

confidence: 99%

Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

1996

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“…However, this technique is not advocated for photochemical coupling due to non specific (“dark”) labeling or photodeiodination ( , ). Mass spectrometry, particularly electrospray and matrix-assisted laser desorption ionization techniques, have become increasingly useful for the characterization of conjugates which are soluble at sufficient (approximate micromolar) concentration ( − ).…”

Section: Resultsmentioning

confidence: 99%

Optimization and Immunological Characterization of a Photochemically Coupled Lysergic Acid Diethylamide (LSD) Immunogen

Kerrigan

Brooks

1998

Bioconjugate Chem.

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A photoreactive heterobifunctional linker was used to prepare an immunogen in which lysergic acid diethylamide was indirectly coupled to keyhole limpet hemocyanin at multiple sites on the drug. It was possible to attach approximately 35 drug molecules to each protein using approximately equal amounts of both species during the reaction. The presence of buffer components or water severely compromised reaction efficiency, as estimated from the molar substitution ratio. Factors such as excess linker, pH, irradiation of dry matrix in the absence of buffer, and the drug/protein ratio used during photolysis were shown to have pronounced effects on reaction efficiency. Structural insights regarding immunogen coupling were obtained by determining the specificities of antibodies which were raised against the immunogen. Cross-reactivity data indicated that haptenation of protein likely occurred at positions N1 and N6 of lysergic acid diethylamide, which is plausible given the electrophilicity of the photogenerated aryl nitrene.

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“…Normalmente, para se obter uma boa produção de anticorpos, são necessários entre 10 e 30 grupos haptênicos por cada 100 KDa de proteína 32 , apesar de já terem sido obtidos resultados satisfatórios com relações mais altas ou mais baixas. Existem vários procedimentos -geralmente por espectrometria UV-vis -para se estimar quantitativamente o número de conjugados de hapteno unidos à proteína [33][34][35] , embora nos últimos anos tenham surgido outros métodos, como dessorção da matriz por meio de laser-espectrometria de massas (MALDI-MS) e a ionização "electrospray"-espectrometria de massas (ESI-MS) [36][37][38] .…”

Section: Preparação Dos Imunógenosunclassified

Métodos imunoquímicos para análise de contaminantes ambientais: conceitos, estado da arte e perspectivas

Nunes¹

2005

Quím. Nova

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Recebido em 6/2/04; aceito em 20/10/04; publicado na web em 28/2/05 IMMUNOCHEMICAL METHODS FOR ANALYSIS OF ENVIRONMENTAL CONTAMINANTS: CONCEPTS, STATE OF THE ART AND PERPECTIVES. Immunoassay techniques provide simple, powerful and inexpensive methods for analysis of environmental contaminants. However, the acceptance of immunoassays is dependent on the clear demonstration of quality and validity compared to more traditional techniques. In this review, primarily, the understanding and the fundamentals of immunoassay methods are given in order to make good use of immunoassays, especially of EIA tests. Special attention is given to the concepts related to the enzymelinked immunosorbent assay (ELISA) formats, such as inhibition concentration at 50% (IC 50 ), detection limit (LOD), cross-reactivity (CR %). It is also explained why some immunoassays are quantitative methods whereas others can only be used as screening methods. A list of main commercial kits for detection of priority pollutants is given in order to help analysts. Others formats, such as flowinjection immunoassay analysis (FIIA), immunoassay chromatography and immunosensors are also cited.Keywords: immunoassays; environmental contaminants; monitoring. INTRODUÇÃOA origem dos métodos imunológicos situa-se no final da déca-da de 50, quando Yalow e Berson 1 descreveram o primeiro ensaio imunológico para detectar insulina humana, o que lhes rendeu o Prêmio Nobel em 1977. A partir de então, as técnicas imunoquí-micas foram desenvolvidas como métodos de análise de macromoléculas (enzimas, hormônios), e posteriormente para moléculas orgânicas pequenas (drogas, fármacos), tendo seu maior êxito no campo da química clínica 2-4 . Em meados da década de 70, foi desenvolvido o primeiro ensaio biológico para pesticidas (aldrin e dieldrin) 5 . A este ensaio, seguiram-se outros, também baseados na marcação radioativa, chamados ensaios radioimunológicos ("radioimmunoassay, RIA"). Os ensaios imunoenzimáticos ("enzyme immunoassay, EIA") surgiram na década de 60 para identificação e localização de antígenos em preparações histológicas 2,6,7 , tendo sido a sua utilidade reconhecida posteriormente em outros campos distintos [8][9][10][11][12][13] . No início dos anos 80, começou-se a discutir as possibilidades que estes tipos de métodos analíticos podiam oferecer na detecção de contaminantes, tanto em amostras ambientais como de alimentos 12,14-16 , o que fomentou a sua aplicação no campo da química ambiental. A partir desse momento, começou-se a utilizar a marcação enzimática, que proporcionava menores riscos e apresentava sensibilidade similar aos métodos convencionais de análise 12,[15][16][17][18][19] . Inúmeras vantagens podem ser obtidas com a utilização dos métodos imunoquímicos e, paulatinamente, vão sendo disponibilizadas informações sobre diversos aspectos analíticos, tais como i) interpretação dos dados obtidos no desenvolvimento dos métodos; ii) estratégias utilizadas para a garantia da qualidade dos procedimentos operacionais; iii) análise dos dados e das informa...

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Characterization of hapten binding to immunoconjugates by electrospray ionization mass spectrometry

Cited by 16 publications

References 11 publications

Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

Characterization of Protein−Hapten Conjugates. 2. Electrospray Mass Spectrometry of Bovine Serum Albumin−Hapten Conjugates

Optimization and Immunological Characterization of a Photochemically Coupled Lysergic Acid Diethylamide (LSD) Immunogen

Métodos imunoquímicos para análise de contaminantes ambientais: conceitos, estado da arte e perspectivas

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