实验动物总则

The polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two regions of known sequence (1–3). It requires two oligonucleotide primers that flank the DNA fragment to be amplified and employs repeated cycles of heat denaturation of the DNA, annealing of the primers to th ...

This chapter focuses on methods for epitope mapping on novel viral polypeptrdes and on fine tuning sensitivity and spectficity of the identified peptide antigens for application in virus diagnosis. Because of the development of efficient methods of multiple peptrde synthesis (1–3), a ...

The polymerase chain reaction (PCR), originally introduced by Satki et al. (1) and subsequently automated by Mullis and Faloona (2), has emerged as a powerful tool in molecular genetics for the exponential in vitro amplification of specific sequences of Interest from minute quantrties of DNA ...

Accurate and early diagnosis of a disease state such as a viral infection, or in a more complicated situation cancer, means live saving because proper medical interventions can be applied in a timely manner before it is too late to treat the disease. Thus it is crucial that good diagnostic markers for any ...

At present the protein expression systems used commonly by researchers incorporate an affinity tail fused to the protein of interest. These affinity tails provide a convenient and efficient method for the purification of the expressed fusion protein using affinity chromatography. ...

An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the applicat ...

One of the continuing objectives of molecular biology research is to characterize the functronal domains of proteins. Many proteins contam domains capable of binding specific ligands, such as cofactors, substrates, and domains of other proteins, that form the basis for interactions t ...

Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA’s complete sequence. The approach described here to clone the missing sequence (cDNA ends) employs polymerase chain reaction (PCR). Since the initial reports of rapid amp ...

Control of gene transcription, the process in which a gene’s DNA sequence serves as a template for mRNA synthesis, plays a critical role in the multistep process that regulates gene expression. Gene transcrtption levels within a cell change in response to a wide variety of signals that occur during ...

Recombinant proteins can be detected and characterized via several immunodetection schemes. Native proteins are typically detected by applying them to a membrane manually or using a vacuum manifold in a dot-or slot-blot format. This approach is suited to screening a number of samples for t ...

Enzyme-linked immunosorbent assays (ELISAs) can be designed to detect either antigens or antibodies. Nearly all ELISA formats require the separation of reactants from the products of the immunoassay. The product of the assay is an immune complex consisting of target ligand, the analyte, a ...

The arm of this chapter is to provide a thorough yet terse treatment of Western blotting with multiple-channel immunodetection. Principles and practical aspects of transverse electrophoresrs will be presented, and the advantages and practice of multiple-channel antibody-bas ...

Two basic methods can be used for the identification of specific antigens by their corresponding antibodies: immunoprecipitation and immunoblotting (often referred to as Western blotting) (1,2). Each has its advantages. Immunoprecipitation is likely to permit the detection of bo ...

The use of recombinant antigens and chemically synthesized peptides are the new approaches for the construction of reliable and sensrtive diagnostic assays. Moreover, in the field of virology, the use of recombinant antigens eliminates the need to handle highly hazardous material in t ...

Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of hum ...

Despite the multitude of different parameters currently measured in the clinical laboratory, only a minor part of these are measured by means of biosensor-based methods. Within the group of biochemical sensors, enzyme or metabolic sensors as integrated devices in clinical analyzers a ...

This chapter demonstrates a preliminary experimental approach to detect antiviral antibodies by means of a quartz crystal immunosensor. As an example of the large numbers of immunoassays currently applied in the clinical laboratory, the screening for human immunodeficiency vir ...

One of the most important classes of reagents for clinical diagnosis is antibodies, either in polyclonal preparations, as monoclonal antibodies (MAbs), or as customized reagents prepared by genetic engineering. The enormous range of antibodies produced by hybridoma and recombin ...

Rapid growth in the field of antibody engineering occurred after it was shown that functional antibody fragments could be secreted into the periplasmic space and even into the medium of Escherichia coli by fusing a bacterial signal peptide to the antibody’s N-terminus (1,2). These findings a ...

For cloning and expressing the antigen-binding variable (Fv) portion of an antibody in Escherzchia coli, vectors have been constructed that combine the two variable regions (VH and VL) with a peptide linker (1–3). The genetic information for VH and VL is generally amplified from hybridoma cel ...