Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis

Kamps‐Holtzapple, Carol; Carlin, Robert J.; Sheffield, Cynthia L.; Kubena, L. F.; Stanker, Larry H.; DeLoach, John R.

doi:10.1016/0022-1759(93)90317-z

Cited by 39 publications

(15 citation statements)

References 16 publications

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“…Previous studies have reported positive effects of increased linker arm length on the specificity and the sensitivity of the resulting antibodies (Vallejo et al, 1982;Wei & Hammock, 1984;Harrison et al, 1991;Sheth & Sporns, 1991;KampsHoltzapple et al, 1994). Extending small haptens away from the protein mass generally has been shown to be helpful, or indeed even critical, in the production of high-affinity antibodies (Hastings et al, 1988;Kamps-Holtzapple et al, 1994). This fumonisin Bi-protein conjugate (FBj-OVA) was used to immunize Balb/c mice.…”

Section: Modeling Of Fb! Fb 2 Fb 3 and Hb-fbimentioning

confidence: 99%

“…This fumonisin Bi-protein conjugate (FBj-OVA) was used to immunize Balb/c mice. Immunization schedules using repetitive injections and waiting periods were incorporated to promote the occurrence of affinity maturation, thus potentially increasing the production of high-affinity antibodies (Siskind & Benacerraf, 1969;Golub, 1981;Kamps-Holtzapple et al, 1994). Spleen cells from these mice were fused with SP2/O myeloma cells and produced hybridomas which secreted a variety of antibodies.…”

Section: Modeling Of Fb! Fb 2 Fb 3 and Hb-fbimentioning

confidence: 99%

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Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits

Elissalde

Kamps‐Holtzapple

Beier

et al. 1995

Food and Agricultural Immunology

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1995) Development of an improved monoclonal antibody-based Elisa for fumonisin b 1-3 and the use of molecular modeling to explain observed detection limits, Food and Agricultural Immunology, 7:2, 109-122, Monoclonal antibodies were prepared against the fumonisins, a group of mycotoxins produced by the plant pathogen, Fusarium moniliforme. Splenic lymphocytes, from Balb/c mice immunized with fumonisin B 1 -ovalbumin conjugate, were fused with SP2/O myeloma cells, and 14 hybridomas were selected. In a competitive enzyme-linked immunosorbent assay, fumonisin B 1 -bovine serum albumin and free fumonisin B 1 (FB 1 ) competed for the monoclonal antibody. The concentrations of FB 1 required to inhibit 50% antibody binding (IC 50 ) ranged from 300 to 670 ppb. Antibodies also cross-reacted with fumonisins B 2 and B 3 (FB 2 , FB 3 ), and the hydrolyzed backbone of fumonisin B 1 (HB-FB 1 ). None of the 14 monoclonal antibodies recognized the sphingolipids, sphingosine and sphinganine, that are structurally similar to the backbone of the fumonisins. Three-dimensional computer models of FB 1 , FB 2 and FB 3 show the amine backbone folding with the two esterified trimethylpropane-1,2,3-tricarboxylic acid side-chains to form a cage into which the hydroxyl and acid groups of these fumonisins extend. The HB-FB 1 molecule, with the two trimethylpropane-1,2,3-tricarboxylic acid esterified moieties at carbons 14 and 15 removed, does not possess two of the three branches which are folded together with inter-hydrogen bonding to formulate the three-dimensional structure that makes up the cage feature of FB 1 , FB 2 and FB 3 . Because of the unexpected folding of the three arms to make a cage of FB 1 , attachment of the protein to the amine group, which is close to, or appears to be pari of, the epitope, may have allowed the immune system of the mouse to produce antibodies more specific for the FB 1 -protein conjugate than to free FB 1 .

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Section: Modeling Of Fb! Fb 2 Fb 3 and Hb-fbimentioning

confidence: 99%

Section: Modeling Of Fb! Fb 2 Fb 3 and Hb-fbimentioning

confidence: 99%

Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits

Elissalde

Kamps‐Holtzapple

Beier

et al. 1995

Food and Agricultural Immunology

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“…SDS-PAGE, which separates proteins on the basis of molecular size, has been useful when small proteins (<20 000 Da) are conjugated with larger haptens (>800 Da) (14). More recently reported is the use of isoelectric focusing electrophoresis of protein-ligand conjugates where the separation method is based on charge (pi) which will change when amines or carboxylic acids of the polypeptide are modified by conjugation with hapten (9,15).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Protein-Hapten Conjugates. 1. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Immuno BSA-Hapten Conjugates and Comparison with Other Characterization Methods

Adamczyk

Buko²,

Chen³

et al. 1994

Bioconjugate Chem.

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Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA. The results from mass spectrometry were compared with results obtained from other more traditional methods of immunogen characterization (UV analysis, trinitrobenzenesulfonic acid titrations, and gel electrophoresis). In each case we were able to calculate the average number of hapten covalently bound to BSA for each synthetically prepared immunogen using matrix-assisted laser desorption ionization mass spectrometry. The other methods presented limitations in certain cases.

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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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Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis

Cited by 39 publications

References 16 publications

Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits

Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits

Characterization of Protein-Hapten Conjugates. 1. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Immuno BSA-Hapten Conjugates and Comparison with Other Characterization Methods

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

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Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis

Cited by 39 publications

References 16 publications

Development of an improved monoclonal antibody‐based Elisa for fumonisin b1–3and the use of molecular modeling to explain observed detection limits

Development of an improved monoclonal antibody‐based Elisa for fumonisin b1–3and the use of molecular modeling to explain observed detection limits

Characterization of Protein-Hapten Conjugates. 1. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Immuno BSA-Hapten Conjugates and Comparison with Other Characterization Methods

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

Contact Info

Product

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Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits

Development of an improved monoclonal antibody‐based Elisa for fumonisin b_1–3and the use of molecular modeling to explain observed detection limits