RESUMO
A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.
Assuntos
Creatina Quinase Forma MB/química , Impressão Molecular/métodos , Sequência de Aminoácidos , Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Peptídeos/química , Polímeros/química , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Técnicas de Microbalança de Cristal de QuartzoRESUMO
A molecularly imprinted film was fabricated, in the presence of epitope-peptides, onto a quartz crystal microbalance (QCM) chip. These five peptides are known linear or conformational epitopes of the anthrax protective antigen PA(83). Imprinting resulted in an epitope-cavity with affinity for the corresponding template. With the use of a basic monomer, the binding-effect was further enhanced increasing the affinity to nanomolar levels. The affinities of the peptide to their corresponding molecularly induced polymers (MIPs) were more closely related to the molecular weight of the analyte than to the number of residues. All epitope-cavities differentiated their epitope region on the protective antigen PA(83) as well as the corresponding furin cleavage fragments PA(63) and PA(20). The QCM chip differential response to the protective antigen fragment was observed in the picomolar range, thus demonstrating a method to manipulate protein on the surface with defined orientation.
Assuntos
Antígenos de Bactérias/análise , Toxinas Bacterianas/análise , Técnicas Biossensoriais/métodos , Epitopos/química , Impressão Molecular , Peptídeos/química , Sequência de Aminoácidos , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Epitopos/imunologia , Peptídeos/imunologia , Quartzo/química , Sensibilidade e EspecificidadeRESUMO
Serum is a readily available source for noninvasive studies in clinical research, but it contains abundant proteins such as albumin and immunoglobulin G that can hinder the presence of low-abundant proteins as well as decrease sample loading capacity of analytical methods. Therefore, depletion of these two proteins is required to observe low-abundance serum proteins. Molecularly imprinted polymers are template-induced artificial antibodies with the ability to recognize and selectively bind the target molecule. In this study, artificial albumin and immunoglobulin G antibodies were developed by using two epitopes of human serum albumin and immunoglobulin G as templates. Acrylic acid, acrylamide, and N-acryl tyramine were the corresponding monomers; N,N'-ethylene bisacrylamide served as a cross-linker, and cellulosic fibers were used as a supporting matrix. The adsorption capacity of these artificial antibodies was 15.2 mg, 10 mg, and 15 µL per gram for human serum albumin, immunoglobulin G, and human serum, respectively. The dissociation constant (Kd ) of these artificial antibodies toward the human serum albumin and immunoglobulin G was 1 µM and 0.6 µM, respectively. The biomimetic properties of these artificial antibodies, coupled with their economical and rapid production, high specificity and their reusability, make them attractive for protein separation and analysis.