Bernadette M. Manning

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%.

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.

Applications and recent developments in the use of antibodies for analysis

ABSTRACT There are many compounds that require analysis ranging from pesticide levels in corn to disease markers in human patients. There are copious challenges to be met when measuring analytes such as the matrix in which they are to be determined, the amounts present, the cost and the rapidity of the result required. Enzyme immunoassays, immunoaffinity chromatography, immunomagnetic polymerase chain reaction, flow cytometry and immunobiological biosensors have all characteristics that can enhance analytical techniques. Antibody-based methods have found applications in a large number of diverse areas such as food and water analysis, clinical diagnosis and therapeutics The structure and modes of production of antibodies and antibody-based derivatives is described and their applications in analysis critically examined.

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 of ELISA and Sensor-based Assays for the Detection of Ethynyl Estradiol in Bile

A polyclonal antibody, EEAb1, was produced against the synthetic steroid hormone, ethynyl estradiol (EE). EEAb1 demonstrated low levels of cross-reactivity with related compounds. Competitive and inhibitive ELISA assays were developed along with a biosensor-based assay using a BIAcore® 3000 for the detection of EE. For EEAb1 the detection ranges were 6-100 000 ng ml−1 in the competitive ELISA, 3-50 000 ng ml−1 in the inhibitive ELISA and 12-100 000 ng ml−1 in the biosensor-based assay. The competitive ELISA data corresponded more closely to the 4-parameter curve model used and also required less time to perform than the inhibitive ELISA. The assays were optimized for use with bile. The bile was diluted 1/2 with buffer (phosphate-buffered salin or Hepes-buffered saline) prior to analysis. The assay formats described in this paper offer quick and simple methods for the detection of ethynyl estradiol in bile samples from cattle.

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.

Novel generic approach to reliable rapid analytical and bioanalytical measurements

There is a clear need for a portable analytical system with high sample throughput, which yields rapid and clear results at a field level for color-based assays. ELISA is a widely used technique for environmental and clinical analysis. However it is time consuming, non-portable, requires expensive plate reading equipment and skilled analysts. An alternative system has been developed in our laboratories using a digital camcorder as a detection method. Preliminary results generated in our laboratories using this approach to screen antibody-antigen reactions have been very promising.