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.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Design and Use of a General Capturing Surface in Optical Biosensor Analysis of Sulphamethazine in Milk

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 microg kg -1 and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cow's milk. The advantages of the new assay format include analyte-independent immobilisation and regeneration. Furthermore, the assay enables measurements with covalent interactions between analyte and detecting molecule. The main disadvantage is the requirement of a conjugate between analyte and the hapten H1. Moreover, it is likely that the antibody surface will have a shorter life span than a surface with the antimicrobial immobilised.     

A new development of measurement of 19-Nortestosterone by combining immunochromatographic strip assay and ImageJ software

A rapid immunochromatographic assay was developed to detect 17β-19-Nortestosterone (19-NT). The 19-NT-OVA conjugate was immobilised to nitrocellulose membrane as a test line and goat anti-rabbit IgG as a control line. Anti-19-NT polyclonal antibody labelled with colloidal gold particles acted as detector reagent. We tested the sensitivity of the strip using spiked swine urine, and analysed the intensity of test line using ImageJ software. The sensitivity by naked eye measurement was determined to be 200 ng/ml and lower limit using ImageJ was distinguished as 5 ng/ml. The total assay time was less than 20 min, suitable for screening and quantifying the residue of 19-NT in swine urine.     

Generation of an anti-NAGase single chain antibody and its application in a biosensor-based assay for the detection of NAGase in milk

Bovine mastitis, an inflammation of the mammary gland in cows, is a major challenge for the dairy industry worldwide as it lowers milk yield, reduces milk quality and increases overall production costs. Early diagnosis is of the utmost importance. N-acetyl-β-D-glucosaminidase (NAGase) is an enzyme released into milk during inflammation and acts as an early indicator of mastitis. This paper describes the selection of anti-NAGase single chain fragment variable antibodies (scFv) from na?ve human antibody libraries and their incorporation into an automated optical biosensor-based immunoassay to detect NAGase in milk. The scFv with the highest affinity for NAGase was first characterized by inhibition ELISA, followed by further evaluation using a surface plasmon resonance platform. Purified NAGase was immobilized on the surface of a CM5 chip and spiked NAGase milk samples were analyzed. The limit of detection for the assay for the assay was determined as 1μg/ml.     

Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system.

An automated biosensor system for measuring molecular interactions has been used to study the kinetics of monoclonal antibody-antigen reactions. The system combines a microfluidic unit in contact with a sensor surface for surface plasmon resonance detection. The specificity of the surface is determined by the operator. Antibody or antigen is immobilised in a dextran matrix attached to the sensor surface. The interaction of matrix bound antibody or antigen with the corresponding partner in solution is monitored in real time. None of the interacting molecules needs to be labelled and it is not necessary to determine the concentration of the the matrix bound component in advance. Two systems were studied: matrix bound monoclonal antibodies (MAbs) interacting with HIV-1 core protein p24 and immobilised aminotheophylline reacting with MAbs. Control of the amount of immobilised ligand and reusable sensor surfaces permits the comparison of different MAbs reacting with antigen under almost identical conditions. Differences in affinity and reaction rates are immediately apparent. The calculated association rate constants for p24 MAbs ranged from 3 x 10(4) - 7.4 x 10(5) M-1 s-1 and for theophylline MAbs association rate constants as high as 1 x 10(6) M-1 s-1 were encountered. The calculated dissociation rate constants were in the region 2 x 10(-4) s-1 to 2 x 10(-2) s-1.     

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg^-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.     

Development of a One Step Strip Test for the Detection of (Dihydro)streptomycin Residues in Raw Milk

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified monoclonal anti(dihydro)streptomycin antibodies. The capture reagent in the assay is a streptomycin- bovine serum albumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, three drops of raw milk are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the streptomycin immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of (dihydro)streptomycin in the sample will thus prevent the binding of the detector reagent to the streptomycin immobilised on the membrane, resulting in disappearance of the test line in the read out zone. Using raw milk samples, spiked with either streptomycin or dihydrostreptomycin, complete disappearance of the test line was obtained with 160 ng ml^-1 and 190 ng ml^-1, respectively. This demonstrates that the test is applicable for screening raw milk samples for the presence of (dihydro)streptomycin residues at MRL level (EU: 0.2 mg (dihydro)streptomycin kg^-1 milk). The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.     

Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B₀ and IC₅₀ determination. The stability of the assay was assessed by the consistency of B₀ in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B₀ and IC₅₀ was evaluated to assess the degree of “matrix” effect that would be present in an assay. Based on these criteria the most promising antibody (2E10, a monoclonal antibody) was selected for further evaluation. This antibody had good sensitivity with IC₅₀=4.47±0.41 ng/ml (n=11) in buffer). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2 ng/ml and 8 ng/ml. A comparison of the biosensor method with a previously developed ELISA demonstrated that both methods give equivalent results (slope of the correlation plot = 1.02) with a high correlation (r²=0.91) between them.     

Enzyme linked immunosorbent assay for milk progesterone

A direct antigen heterologous enzyme linked immunosorbent assay (ELISA) for milk progesterone has been developed using progesterone-3-O-carboxymethyloxime-bovine serum albumin (P-3-O-CMO-BSA) antiserum and 17-α-hydroxy-progesterone-3-O-carboxymethyloxime-horseradish peroxidase (17-α-OH-P-3-O-CMO-HRP) enzyme conjugate. The data of the present study reveal that the homologous assay, which employed P-3-O-CMO-HRP as the label, showed no displacement. On the contrary, replacement of P-3-O-CMO-HRP with 17-α-OH-P-3-O-CMO-HRP as the label showed significant displacement and led to the development of a sensitive and specific assay. The recovery of the exogenously spiked progesterone from milk pools was in the range of 94.3-97.88% for toned milk and 97.6-101% for full-cream milk. The intra-assay and interassay coefficients of variation (CVs) ranged from 4.1-7.8% and 4.4-7.0%, respectively. A high ionic strength buffer was used to obtain released progesterone from binding protein/fat. The progesterone values measured in toned and full-cream milk ranged from 1.198-9.745 ng/mL and 6.949-14.923 ng/mL, respectively. The milk progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.95 (n = 65).     

Detection of human adenovirus hexon antigen using carbon nanotube sensors

Human adenoviruses (HAdVs) have been implicated in a wide range of diseases affecting primarily the respiratory, ocular and gastrointestinal systems. A rapid and efficient method for the detection of HAdV hexon antigen is described using carbon nanotube (CNT) sensors. Anti-HAdV antibody was immobilised on the reverse surface of a CNT sensor. As a control, non-specific mouse IgG was immobilised on another CNT sensor. I-V_gate curves were measured after incubation of various concentrations of recombinant HAdVs hexon antigen with anti-HAdVs antibody-immobilised or non-specific mouse IgG-immobilised sensors. The curves showed a positive shift that was dependent on the hexon antigen concentration in the anti-HAdV antibody-immobilised sensor, whereas no such shift was observed in the non-specific mouse IgG-immobilised sensor. The sensitivity of the CNT sensor method was greater than that of enzyme-linked immunosorbent assay. Hence, this method offers a new tool for HAdV detection by analysing antigen-antibody interactions.     

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a mycotoxin produced by certain fungi that infest cereal grains. A hybridoma cell line producing a monoclonal antibody (Mab) was used as the starting point in the development of a recombinant single chain variable fragment antibody (scFv) recognising DON. The scFv and Mab were characterised using two immunoassay formats: competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and biolayer interferometry (BLI). Using CD-ELISA the IC₅₀s for DON were 36.1 and 13.8 ng/ml for assays based on the scFv and Mab, respectively. The cross-reactivity to DON analogs was very similar for the scFv and the Mab. The real-time binding of the antibodies to an immobilised DON-protein conjugate was also monitored. In competitive BLI assays the IC₅₀s using the scFv and Mab were 68.3 and 15.8 ng/ml, respectively. The results suggest that sensitivity of assays, but not selectivity, was affected by removal of the constant regions of the Mab.     

Rapid three-dimensional biointerfaces for real-time immunoassay using hIL-18BPa as a model antigen

With the goal of designing a rapid and affordable system of real-time immune monitoring for future diagnostic applications in sepsis, we have developed a biointerface composed of polyethyleneimine (PEI) and carboxymethylcellulose (CMC) to provide a means of prompt and facile immunoassay. Biointerface assembly is complete within 30 min, with all preparation performed and monitored within the measurement chamber of a quartz crystal microgravimetry with dissipation (QCM-D) sensor. Optimised biointerface composition, as determined by the mass of antibody immobilised, the level of antigen detection, and the amount of non-specific binding of human serum albumin, was determined to consist of a 4.0 mg/mL CMC hydrogel layer cross-linked to a 0.5 mg/mL PEI sub-layer. Tapping mode atomic force microscopy (AFM) in liquid demonstrates highly uniform and smooth surfaces using these hydrogels. Sensitivity of the biointerface for rhIL-18BPa is 400 ng/mL, with detection of 1 microg/mL achievable following 25 surface regenerations. Performance of the biointerface is verified using surface plasmon resonance (SPR), demonstrating the ability of the biointerface to be applied across platforms.     

Measurement of affinity of viral monoclonal antibodies using Fab'-peroxidase conjugate. Influence of antibody concentration on apparent affinity.

The binding affinity of a monoclonal antibody to tobacco mosaic virus (TMV) was studied using a Fab'-peroxidase conjugate. Measurement of the enzymatic activity allowed the determination of the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. The method was very sensitive and allowed measurements over a 1000-fold range of antibody concns. The calculated affinity constant decreased about 25 fold when the antibody concn used in the binding assay was increased from 30 ng/ml to 35 micrograms/ml.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

Automatic On-Line Analysis Of Milk Constituents (Urea, Ketones, Enzymes And Hormones) Using Biosensors

Traditional methods of monitoring health changes in animals are based entirely on the human senses. However, in modern dairy production systems humans are rarely present, this is particularly the case with the introduction of robotic milking. In these systems all the functions of milking are automated and cows visit at times of their own choosing. Systems of automatic health monitoring are therefore a priority for research to ensure that the health and reproductive status of the animals can be assessed for management purposes. These systems must be automatic, work in field conditions without technical support and cost a few pence per analysis. The first task is to obtain representative biological samples automatically and non-invasively. As milk is flowing into the milking machine from the cow this can be achieved with ease, except that milk is non-homogeneous with a changing lipid fraction during milking. Lipid soluble components such as progesterone and vitamin A are affected by this change and a model has to be established to determine thresholds at different times during milking. Our main interests in dairy cows are in predicting ovulation, detecting metabolic imbalance and detecting preclinical mastitis inflammatory response. Our team is developing a fully automated ovulation prediction system based on the screen-printed carbon electrode biosensor for progesterone demonstrated by Pemberton et al. (1998). In recent experiments the automated system was able to detect concentrations of progesterone between 2 and 30 ng/ml in stored milk samples (r ²= 0.96). The results of field tests are presented showing a good correlation between ELISA and the biosensor (r ²= 0.91) on samples of fresh milk. The results of the recent field tests show the ability of the biosensor to characterise ovulation cycles of cows and to detect pregnancy. We have identified a major lack of other biological models to detect disease with on-line sensors. Our next objective is to create an integrated system for biological research with sensor systems for urea, ketones, lipids and enzymes in milk. This will allow the development of diagnostic models based on analysing numerical sensor-derived data rather than human visual observations for signs of ill health in dairy cows.     

A high throughput electrochemiluminescent cell-binding assay for therapeutic anti-CD20 antibody selection

A cell-based ELISA using suspension WIL2 cells in 96-well format was previously developed for measuring relative binding affinities of humanized anti-CD20 variants. We further developed a new cell-binding assay that uses high binding capacity carbon electrode plates for rapid attachment of suspension WIL2 cells and electrochemiluminescence for detection. Compared to the cell-based ELISA, which requires centrifugation for the manual wash steps, significant improvement in assay throughput was achieved by using a microplate washer. The assay can be performed on both 96- and 384-well plates with a standard curve range of 2.74-2000 ng/ml, which is wider than the range of 15.6-1000 ng/ml for the cell-based ELISA. Using CD20 expressing CHO cell clones, surface expression of >or=33,000 CD20 molecules was sufficient to obtain a dose-response curve in 384-well format. Relative affinities of 15 humanized variants correlated well (r(2)=0.94) between electrochemiluminescent cell-binding assay and cell-based ELISA. A competitive assay format, using mouse anti-CD20 antibody as the tracer, with a dose-response range of 27.4-20,000 ng/ml was also developed. The new cell-binding assay method can be used to efficiently support humanization process for selection of anti-CD20 antibody drug candidates and to characterize antibody binding to other cell surface proteins.     

Detection of nucleoside triphosphate hydrolase as a circulating antigen in sera of mice infected with Toxoplasma gondii.

The nucleoside triphosphate hydrolase, which is present in the tachyzoite form of Toxoplasma gondii, was detected as a circulating antigen in sera of mice infected with a virulent (RH) or an avirulent (Beverly) strain of T. gondii. The enzyme was detected with a monoclonal antibody incorporated into an enzyme-linked immunosorbent assay. The lower limit of sensitivity of the assay was about 0.3 ng/ml, and standard assays provided a linear plot of nucleoside triphosphate hydrolase concentration over a range of 0.3 to 12 ng/ml. In mice inoculated intraperitoneally with tachyzoites of the RH strain, nucleoside triphosphate hydrolase emerged in the serum 1 day after injection, and then the concentration increased and reached a value of 30 micrograms/ml on day 5. In mice inoculated intraperitoneally with cysts of the Beverly strain, nucleoside triphosphate hydrolase was detected at day 3 after injection, and a peak concentration of 89 ng/ml was seen on day 10. The concentration of enzyme decreased thereafter, and the enzyme disappeared from the circulation on day 56.     

Development and evaluation of a new ELISA for the detection and quantification of antierythropoietin antibodies in human sera

Assays for the analysis of antierythropoietin antibodies (anti-EPO Abs) currently suffer from a high degree of nonspecificity or are cumbersome and time consuming to perform. They are therefore not well suited for the analysis of large numbers of human sera samples, a task that has become increasingly important due to an increase in the number of patients developing anti-EPO Abs. The objective of this study was to develop and validate a sensitive and specific ELISA for the determination of anti-EPO Abs that would suit these purposes. In this new double antigen bridging ELISA, anti-EPO Abs bind via one site to recombinant human erythropoietin (rhEPO)-biotin immobilized to streptavidin-coated microtiter plates (MTPs) and by a second site to rhEPO labelled with digoxigenin (DIG). The amount of bound antibody is determined using an anti-DIG antibody coupled to peroxidase. A rabbit polyclonal anti-EPO Ab purified by immunoadsorption is used as reference antibody preparation. The dynamic range of this ELISA was 1-75 ng/ml per assay calibrated with the reference antibody preparation. The assay was specific for anti-EPO Abs and did not react with other immunoglobulins (Ig) present in human serum. The lower limit of detection (LLD) of the assay was 0.5 ng/ml, and the lower limit of quantitation (LLQ) was 1.0 ng/ml. Anti-EPO Abs could be detected in the sera of pure red cell aplasia (PRCA) patients. In contrast to previous reports, no anti-EPO Abs could be detected in the sera of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome (SS), or in the sera of dialysis patients.     

A Biosensor-based Immunoassay for Rapid Screening of Deoxynivalenol Contamination in Wheat

A surface plasmon resonance (SPR) based indirect inhibitive immunoassay was developed for the rapid quantification of concentrations of the trichothecene mycotoxin deoxynivalenol (DON). A DON-biotin conjugate was synthesized and immobilized to a streptavidin coated SPR sensor surface to measure free anti-DON antibody added to a sample. For analysis, ground wheat was extracted with 10% (v/v) methanol in water with 6% (w/v) polyvinyl-pyrrolidone, filtered and cleaned up with MycoSep™ columns. Extracts were mixed with a polyclonal anti-DON antibody and pre-incubated prior to injection into a BIAcore® device. The sensor surface was regenerated with a rinse of 6 M-guanidinechloride in 10 mm-glycine (pH 2.9). The assay had a working range between 0.13 and 10.0 μg ml -1 of DON, a 50% inhibition concentration (IC 50 ) of 0.72 μg ml -1 , and a detection limit of 2.5 pg μl -1 . Recovery of DON in spiked wheat was 104 ±15%. The system enables analysis of a sample to be completed in 15 min including sample preparation (10 min) and quantification (5 min). The assay was applied to the analysis of wheat samples with different levels of DON contamination. Statistical analysis revealed a positive, linear correlation between concentrations of DON measured with the new biosensor and GC/MS or HPLC as reference methods. Coefficients of correlation of R 2 = 0.9464 (GC/MS data) and R 2 = 0.9066 (HPLC data) were found.