ABSTRACT
Se realizó el estudio voltamétrico de la inmovilización de anticuerpos anti-Escherichia coli ATCC 25922 en electrodos de oro desnudos y electrodos de oro modificados con tiourea. Las cepas de Escherichia coli fueron cultivadas durante 24 horas en medio tripticasa de soya, provenientes del Laboratorio de Microbiología del Agua de la Facultad de Farmacia y Bioanálisis de la Universidad de Los Andes en Mérida-Venezuela. Los resultados obtenidos muestran que ocurre la inmovilización de los anticuerpos anti-E. coli tanto en la superficie de los electrodos de oro desnudos, así como en los modificados con tiourea, ya que en ambos casos ocurre la detección de Escherichia coli. Al comparar ambos resultados, podemos decir que en oro desnudo el potencial de pico anódico es menor que en oro modificado con tiourea, +0,158V y +0,251V respectivamente; igual comportamiento ocurre con las corrientes de pico anódicas, 0,127x10-4A y 0,156x10-4A respectivamente. Un mayor potencial implica que la presencia de la monocapa de tiourea en el electrodo, hace que se dificulte la transferencia de electrones desde el anticuerpo al electrodo. Así mismo, los resultados obtenidos permiten sugerir un método para la inmovilización de moléculas biológicas en superficies de oro modificadas. De igual forma, el método utilizado permitió demostrar la especificidad de la unión anticuerpo-antígeno (anticuerpo-E.coli), al agregar volúmenes de Klebsiella pneumoneae, demostrando que el inmunosensor tiene la capacidad de reconocer la presencia o ausencia de E. coli en un medio, así como conocer si un anticuerpo es específico o no para un determinado antígeno
The voltammetric study of immobilization of anti-Escherichia coli antibodies ATCC 25922 was carried out on naked gold electrodes and gold electrodes modified with thiourea. The strains of Escherichia coli were cultivated for 24 hours in trypticase soybean medium, from the Laboratory of Microbiology of Water of the Faculty of Pharmacy and Bioanalysis of the University of Los Andes in Merida-Venezuela. The results obtained show that the immobilization of the anti-E. coli antibodies occurs on both the surface of the naked gold electrodes as well as those modified with thiourea, since in both cases the detection of Escherichia coli occurs. When comparing both results, we can say that in naked gold the anodic peak potential is lower than in gold modified with thiourea, +0.158V and +0.251V respectively; similar behavior occurs with the anodic peak currents, 0.127x10-4A and 0.156x10-4A respectively. Higher potential implies that the presence of the thiourea monolayer in the electrode makes it difficult to transfer electrons from the antibody to the electrode. Likewise, the results obtained suggest a method for the immobilization of biological molecules on modified gold surfaces. Likewise, the method used demonstrated the specificity of antibody-antigen (antibody-E.coli) binding, by adding volumes of Klebsiella pneumoneae, demonstrating that the immunosensor has the ability to recognize the presence or absence of E. coli in a medium, as well as to know if an antibody is specific or not for a certain antigen
Subject(s)
Humans , Male , Female , Biosensing Techniques , Electrochemistry , Escherichia coli , Antibodies, Immobilized , Bacteria , Water Microbiology , Public Health , Foodborne Diseases , Antibody Formation , AntigensABSTRACT
Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein. Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (K D = 7.39e-10 M) than monoclonal antibody (mAb) (K D = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run. Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment.
Subject(s)
Humans , Surface Plasmon Resonance , Epidermal Growth Factor/analysis , Epidermal Growth Factor/genetics , Kinetics , Biosensing Techniques , Sensitivity and Specificity , Antibodies, Immobilized , Antibodies/analysisABSTRACT
There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.
Subject(s)
Animals , African Horse Sickness Virus/isolation & purification , Antibodies, Immobilized , Antibodies, Viral/immunology , Chlorocebus aethiops , Chickens , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G , Peptide Library , Serologic Tests/methods , Serotyping , Single-Chain Antibodies/immunology , Vero CellsABSTRACT
In order to immobilize myoglobin (Mb) monoclonal antibody on gold film solid-phase carrier, we grew a mixed self-assembled monolayers (SAMs) of acid thiol and mercapto ethanol on gold film. Then we analyzed the property of the sample by atomic force microscopy and X-ray photoelectron spectroscopy. Then, we used 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride as catalyst to couple SAMs with amino of antibody so that we immobilized antibody on surface of gold film, followed by detecting myoglobin antigen. Results showed that, by optimizing experimental conditions, when we treated gold film by a mixture of mercapto hexadecanoic acid and mercapto undecanol ethanol solution of concentration of 50 mmol/L at temperature of 60 degrees C for 3 hours, and Mb monoclonal antibody of concentration of 40 mg/L for 3 hours, respectively, antibody had high immobilization efficiency and the MbAg was detected to 30 microg/L. The method provided a theoretical and practical basis for using magnetoresistence biosensors to diagnosis myocardial infarction.
Subject(s)
Humans , Antibodies, Immobilized , Antibodies, Monoclonal , Allergy and Immunology , Biosensing Techniques , Methods , Gold , Chemistry , Membranes, Artificial , Microscopy, Atomic Force , Myocardial Infarction , Diagnosis , Myoglobin , Blood , Allergy and Immunology , Photoelectron Spectroscopy , Sulfhydryl Compounds , ChemistryABSTRACT
In order to optimize the fabrication of SiO2 tubes immobilized with antibody for hepatitis C virus antigen (HCAg) detection, we formed the activated amino on the surface of SiO2 tubes by using the activation of aminosilane. Then we immobilized the hepatitis C virus (HCV) monoclonal antibody on the surface of SiO2 tubes by using glutaraldehyde as a chemical cross-linker, followed by detecting HCAg. Sequence tests showed that when the SiO2 tubes were treated in 10% (V/V) aminosilane solution and 3% (V/V) glutaraldehyde solution for 3 hours and 2 hours, respectively, the HCV monoclonal antibody had high immobilization efficiency and low nonspecificity, and the HCAg was detected to 1 ng/mL. This experiment can provide principle and experimental data for establishment of HCAg magnetic immunoassay system.
Subject(s)
Humans , Antibodies, Immobilized , Allergy and Immunology , Antibodies, Monoclonal , Chemistry , Allergy and Immunology , Hepatitis C Antibodies , Chemistry , Allergy and Immunology , Hepatitis C Antigens , Allergy and Immunology , Silicon Dioxide , ChemistryABSTRACT
Rapid re-endothelialization of implanted coronary artery stent is a new way for preventing restenosis. In this paper, adhesive polypeptide mimics of marine organism and antibodies potentially selective targeting with CD34 antigens on endothelial cells (ECs) were immobilized onto ethylene-vinyl acetate copolymer (EVA) coatings for improving the re-endothelialization process. The results showed that the attachments, growths, viabilities, as well as cell retentions of ECs on EVA were improved after modification. The immobilized polypeptide promoted the attachments of ECs, but overmuch polypeptide would restrict the attachments. With the increase of antibody concentration, the immobilized antibodies and cell attachments were improved.