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R Rapley

Publications and source records attributed to R Rapley.

11 recordsLinked to original sources
PubMed1995

The biotechnology and applications of antibody engineering.

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.

Animals↗
PubMed1994

Enhancing PCR amplification and sequencing using DNA-binding proteins.

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.

DNA-Binding Proteins↗
PubMed1993

Nucleotide sequence analysis of CDR3 elements of a panel of anti-peptide monoclonal antibodies recognizing parathyroid hormone-related protein.

Nucleotide sequences of heavy (VH) and light (VL) chain variable region complementarity determining regions have been determined from in vitro amplified mRNA isolated from a panel of monoclonal antibodies (mAb) raised to a synthetic 34mer peptide representing the N-terminal portion of human parathyroid hormone-related protein (PTHrP or parathyrin) reported to contain an immunodominant epitope. These mAb vary in affinity for the synthetic peptide and native PTHrP (Ka between 5.9 x 10(8) and 1.9 x 10(11)l/M). All 10 mAb studied were found were found to utilized restricted VH2, V kappa 2, JH4 and J kappa 1 family genes. Significant differences in the length and sequence of D elements were found; however 9/10 mAb utilize members of the DSP2 family. Significantly, two broad ranges of affinity could be determined based on the presence of Asp or Ala at residue 101 in JH.

Amino Acid Sequence↗
PubMed1992

Isolation and amplification of human IgE Fd encoding mRNA from human peripheral blood lymphocytes.

In order to establish the feasibility of applying recombinatorial library technologies to investigate human in vivo IgE responses, and as a pre-requisite of recombinatorial library construction, we have attempted to determine workable peripheral blood sample volumes required for isolation of mRNA for polymerase chain reaction (PCR) amplification of human IgE Fd encoding sequences. Cells secreting chimeric human IgE monoclonal antibody specific for the hapten NIP were used to establish the conditions for specific amplification of C epsilon 1 domain and Fd encoding sequences, as determined by Southern hybridisation. Amplification of C epsilon 1 domain sequences could be achieved using as few as ten cultured cells as the source of RNA. Specific IgE+ B cell enrichment using immuno-magnetic particles prior to RNA extraction was, however, required to obtain amplification of IgE C epsilon 1 and Fd fragments from lymphocytes prepared from 40 ml human peripheral blood. IgG1+ B cell enrichment from similar samples was not required for detectable amplification of human C gamma 1 cDNA sequences. However, this procedure improved amplification efficiency. Optimisation of methods to separate specific B cell populations, or specific RNA/cDNA sequences, will facilitate in vitro generation of human IgE Fab fragments from peripheral blood.

B-Lymphocytes↗
PubMed1992

Sequencing of PCR-amplified DNA.

Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the use of the PCR product for other purposes either prior to or subsequent to generation of sequence data. The single-stranded sequencing methods generally require some prior decision regarding sequencing of the product. Assisted by automated workstation development, sequencing of single-stranded DNA PCR products generated either during thermal cycling or following affinity-capture strand separation may have significant future utility, particularly in genome mapping and routine clinical diagnosis. Despite template type and protocol differences, in all situations the purity and concentration of PCR-amplified DNA template used remains the most critical factor determining the efficiency and reliability of nucleotide sequencing methods.

Bacterial Proteins↗
PubMed1992

Fundamentals of the polymerase chain reaction: a future in clinical diagnostics?

The polymerase chain reaction (PCR) is an example of a technique that is having a profound impact on both fundamental and applied clinical science research. The availability of PCR-based diagnostic kits for the detection of polymorphisms within the HLA-DQA1 locus portends a technology that will undoubtedly become part of the clinical laboratory's diagnostic arsenal, and will extend and/or refine laboratory-based diagnosis in many areas. With current research effort directed to increase our knowledge of the overall structure of the human genome, and the identification of disease-associated genes and sequences, we can anticipate correspondingly rapid advances in its applications. This paper briefly reviews the basic facets of the PCR, which suggest it will fulfill such a role in future clinical diagnosis.

Clinical Laboratory Techniques↗