Paul P. Dillon

Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor.

Polyclonal antibodies were produced for the development of competitive ELISAs and surface plasmon resonance (SPR)-based BIAcore inhibition assays for the detection of morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine). A conjugate consisting of M3G and ovalbumin was produced and used for the generation of antibodies, for the coating of immunoplates and for immobilisation onto BIAcore chips. Competition ELISAs were developed in PBS and urine to characterise the antibodies ability to recognise free M3G. SPR-based inhibition immunoassays on BIAcore were developed. The regeneration of the surface of a chip immobilised with conjugate following antibody binding, essential for the development of inhibition assays was investigated. Regeneration of the conjugate-coated surface was optimised for both polyclonal antibodies resulting in binding-regeneration capacities of approximately 60 cycles for one antibody and 50 cycles for the second antibody. The inhibition assays developed in urine had ranges of detection of 762-24,400 (antibody 1) and 976-62,500 pg ml(-1) (antibody 2). The inter-day coefficients of variation for the assays ranged from 1.48 to 11.24%.

The Development of Novel Miniaturized Immuno-sensing Devices: A Review of a Small Technology with a Large Future

Abstract Miniaturized screening devices (MSDs) are an emerging technology with a variety of possible applications e.g., detection of diseases, analysis of antibiotics or illicit drugs. MSDs include a variety of different working principles and components. They are generally referred to in the literature as biochips, nanochips, micro-arrays, µTas systems and high throughput screening devices. They consist of a miniaturized device or ‘chip’ with different probes immobilized on the surface that may carry out multiplex analysis (i.e., detection of genes, proteins, illicit or novel drugs etc.,). Such a device is envisaged to be fully autonomous, incorporating micro-fluidics and appropriate means of signal transduction. The vast majority of MSDs that have been developed, to date, are based on DNA probes. DNA-based arrays have been generated to analyse genomes, detect diseases e.g., cancer, and cellular responses to various stimuli e.g., carcinogens, at the genetic level. MSDs may provide a relatively inexpensive, rapid and user-friendly method of diagnosis. However, there is an increasing shift in research toward proteomic analysis, yielding MSDs that incorporate protein probes e.g., antibodies, for the detection of a much wider range of compounds. The specificity and relative ease of production of antibodies facilitate their use as ideal probes for the development of the ‘lab on a chip’ or µTas system. In this review, particular emphasis will be on optical based MSDs with antibody probes and their future application in the developing fields of point-of-care testing (POCT), µTas systems and high throughput screening (HTS). There are further sub-divisions of this nomenclature depending on the biological components or probes they may contain and the method of signal transduction that they utilize.

Production of a recombinant anti-morphine-3-glucuronide single-chain variable fragment (scFv) antibody for the development of a "real-time" biosensor-based immunoassay.

A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was produced using genetic material obtained from the spleen cells of mice immunised with a morphine-3-glucuronide-bovine serum albumin (M3G-BSA) conjugate. Immunoglobulin light (V(L)) and heavy (V(H)) chain genes were amplified and cloned into pAK vectors for generation of recombinant antibody fragments in Escherichia coli. A competition ELISA assay was developed in PBS to characterise the ability of the antibody fragments to recognise free drug and the detection limits were found to be as low as 3 ng ml(-1). Surface plasmon resonance-based inhibition immunoassays were developed. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in solution. Regeneration conditions for antibody binding to the surface were optimised resulting in a binding-regeneration capacity of at least 30 cycles. The inhibition assay for M3G was tested with assay ranges between 3 and 195 ng ml(-1) and 3 and 97 ng ml(-1) in PBS and urine, respectively.

Application of an immunosensor for the detection of the β-lactam antibiotic, cephalexin

Public concern surrounding antibiotic contamination in food and food products has made it imperative to develop analytical methods for their detection. Polyclonal antibodies were used in the development of a surface plasmon resonance (SPR)-based inhibition immunoassay for cephalexin. A conjugate consisting of cephalexin-bovine serum albumin (BSA) was immobilized on the dextran gel surface of the sensor chip. Binding/regeneration studies of antibody to immobilized cephalexin were studied and dissociation of the antibody from the immobilized cephalexin was easily achieved with 10 mmol l−1 NaOH. Forty surface regeneration cycles were carried out and found to be reproducible with only a 7.4% decrease in binding over this number of regenerations. Model inhibition immunoassays for cephalexin were developed in PBS and spiked milk samples with detection ranges of 4.88 to 2,500 ng ml−1 and 244 to 3,906 pg ml−1, respectively.

Development and Use of Antibodies in Surface Plasmon Resonance-Based Immunosensors for Environmental Monitoring

The interaction between antibody and antigen is characterised by relatively high affinity and specificity, making this type of reaction a prime candidate for use as an analytical tool. The interaction may be combined with biosensors in the production of immunosensors for environmental monitoring. Polyclonal and monoclonal antibodies have had a significant impact in analytical detection systems over the past few decades with antibody fragments becoming important in recent years. Production of antibodies to small haptens requires the initial conjugation of hapten to a larger carrier molecule. Once hapten-carrier conjugates have been produced, polyclonal, monoclonal and various antibody fragments may be produced by differing protocols. A critical step in the production of antibody fragments is the development of efficient screening procedures to identify suitable antibody-producing clones and this has been reviewed in this article. Various antibody types may then be used in the generation of immunosensors for the monitoring of environmental pollutants. The selection of the appropriate sensor technology applicable for the determination of an antibody-antigen interaction is of prime importance for immunosensor development. One example of such an application is surface plasmon resonance-based biosensors, as they provide real-time analysis of interactions between the antibody and antigen of interest.

PRODUCTION AND ANALYTICAL APPLICATIONS OF scFv ANTIBODY FRAGMENTS

Polyclonal and monoclonal antibody molecules have had a significant impact in analytical detection systems throughout the last few decades. Recently, the emergence of recombinant antibody technology is also playing a part in diagnostic applications. Recombinant antibody display technology is being exploited to produce antigen binding fragments (Fab) of an antibody and single chain Fv (scFv) antibodies. These recombinant molecules can be as sensitive and specific as their parent molecules, and can often be produced relatively easy and cheaply. This manuscript will focus on antibody phage display technology, and other novel expression systems for the production of recombinant antibodies. It will also critically examine their applications in different assay formats, residue analysis, and, more recently, their role in proteomics.

Development of surface-plasmon-resonance-based immunoassay for cephalexin

The public concern surrounding antibiotic contamination in food and food products has made it imperative to develop analytical methods for their detection. Polyclonal antibodies and protein-hapten conjugates to cephalexin were used in the development of a surface plasmon resonance (SPR)-based inhibition immunoassay to cephalexin. A conjugate consisting of cephalexin-bovine serum albumin (BSA) was immobilised on the dextran gel surface. Dissociation between the antibody and antigen was easily achieved with 10 mmol l-1 NaOH and was very reproducible. Standards of free hapten were prepared and premixed with antibody and, after a suitable incubation time, passed over the surface of the chip with the protein-hapten conjugate immobilised. The hapten in solution inhibited the binding of antibody to the surface resulting in higher response units of antibody bound at lower concentrations of free drug. Model inhibition immunoassays to cephalexin were developed in PBS and spiked milk samples. These assays had detection ranges between 4.88 to 2,500 ng ml-1 and 244 to 3,900 ng ml-1, respectively, with reproducible results.