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.     

Development of a Biosensor-based Immunoassay for Screening of Chloramphenicol Residues in Milk

Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 μg l^-1) and the decreased consumption of antibody (four times less than on the CAP surface). This assay is rapid (3 min/run), sensitive, and specific for CAP and CAP glucuronide. It could be integrated in a multi-residue screening test and applied to other matrices (bile, urine, meat).     

Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab1). In this format the primary Ab1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 +/- 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.     

Screening the interactions between HIV-1 neutralizing antibodies and model lipid surfaces

Our work is motivated by the observation that rare, broadly neutralizing antibodies (NAbs), 4E10 and 2F5, associate with HIV-1 lipids as part of a required first step in neutralization before binding to membrane-proximal antigens. Subsequently, induction of these types of NAbs may be limited by immunologic tolerance due to autoreactivity with host cell membranes. Despite the significance of this lipid reactivity there is little experimental evidence detailing NAb-membrane interactions. Simple and efficient screening assays are needed to select antibodies that have similar lipid reactivity as known NAbs. To this end we have developed a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol self-assembled monolayers (SAMs) that replicate salient lipid surface chemistries and NAb binding to lipid surfaces. Specifically, we probed the relative importance of charge and hydrophobicity on antibody-surface interactions. We found that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies (mAbs). Furthermore, we confirmed the importance of charge-mediated antibody surface interactions, originally suggested by results from mAb interactions with conventional lipid vesicle/bilayer surfaces. Our approach, using self-assembled thiol monolayers that replicate the binding behavior of NAbs on lipid surfaces, thus provides an efficient and useful tool to screen interactions of mAbs and lipid-reactive NAbs.     

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.     

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.     

High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100

Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.     

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.     

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.     

Localized surface plasmon resonance biosensor integrated with microfluidic chip

A sensitive and low-cost microfluidic integrated biosensor is developed based on the localized surface plasmon resonance (LSPR) properties of gold nanoparticles, which allows label-free monitoring of biomolecular interactions in real-time. A novel quadrant detection scheme is introduced which continuously measures the change of the light transmitted through the nanoparticle-coated sensor surface. Using a green light emitting diode (LED) as a light source in combination with the quadrant detection scheme, a resolution of 10⁻⁴ in refractive index units (RIU) is determined. This performance is comparable to conventional LSPR-based biosensors. The biological sensing is demonstrated using an antigen/antibody (biotin/anti-biotin) system with an optimized gold nanoparticle film. The immobilization of biotin on a thiol-based self-assembled monolayer (SAM) and the subsequent affinity binding of anti-biotin are quantitatively detected by the microfluidic integrated biosensor and a detection limit of 270 ng/mL of anti-biotin was achieved. The microfluidic chip is capable of transporting a precise amount of biological samples to the detection areas to achieve highly sensitive and specific biosensing with decreased reaction time and less reagent consumption. The obtained results are compared with those measured by a surface plasmon resonance (SPR)-based Biacore system for the same binding event. This study demonstrates the feasibility of the integration of LSPR-based biosensing with microfluidic technologies, resulting in a low-cost and portable biosensor candidate compared to the larger and more expensive commercial instruments.     

CD4-binding compounds: An assay to detect new classes of immunopharmacological agents

The interaction of antibodies with protein antigens is accepted as a paradigm of protein — protein interactions. In searching for a new generation of immunomodulatory compounds based on the interaction of the T-cell surface glycoprotein CD4 with MHC class II antigens, a model assay has been developed in which MHC molecules have been substituted by a monoclonal antibody (anti-Leu3a) to the CD4 amino-terminal domain-specific epitope, Leu3a. This assay can detect diverse classes of molecules including proteins such as HIV envelope glycoprotein gp120 and low molecular weight compounds such as aurin tricarboxylic acid, dextran sulphate and Evans blue. The interaction of these molecules with CD4 in the assay appears to be identical to their interaction with native CD4 on intact cells. Other protein—antibody pairs could be substituted for CD4—anti-Leu3a enabling this assay format to be used for the detection of proteins or small organic compounds which interfere with a wide range of therapeutically-relevant macromolecular interactions.     

Solid-state capture and real-time analysis of individual T cell activation via self-assembly of binding multimeric proteins on functionalized materials surfaces

Polyfunctional T cell responses are increasingly underpinning new and improved vaccination regimens. Studies of the nature and extent of these T cell responses may be facilitated if specific T cell populations can be assessed from mixed populations by ligand-mediated capture in a solid-state assay format. Accordingly, we report here the development of a novel strategy for the solid-state capture and real-time activation analyses of individual cognate T cells which utilizes a spontaneous self-assembly process for generating multimers of biotinylated class I major histocompatibility-peptide complex (MHCp) directly on the solid-state assay surface while also ensuring stability by covalent interfacial binding. The capture surface was constructed by the fabrication of multilayer coatings onto standard slides. The first layer was a thin polymer coating with surface aldehyde groups, onto which streptavidin was covalently immobilized, followed by the docking of multimers of biotinylated MHCp or biotinylated anti-CD45.1 monoclonal antibody. The high binding strength at each step of this immobilization sequence aims to ensure that artefacts such as (partial) detachment, or displacement by proteins from solution, would not interfere with the intended biological assays. The multilayer coating steps were monitored by X-ray photoelectron spectroscopy; data indicated that the MHCp proteins self-assembled in a multimeric form onto the streptavidin surface. Immobilized multimeric MHCp demonstrated the capacity to bind and retain antigen-specific T cells from mixed populations of cells onto the solid carrier. Furthermore, real-time confocal microscopic detection and quantification of subsequent calcium flux using paired fluorescent ratiometric probes facilitated the analysis of individual T cell response profiles, as well as population analyses using a combination of individual T cell events.     

Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 1. Kinetic characteristics and comparison with other Elisa formats

Solid‐phase two‐site enzyme immunoassays were developed using a number of monoclonal antibodies with differing specificities for wheat grain gluten proteins. While polystyrene microwells performed well in the assay, very high levels of non‐specific binding occured with PVC (Polyvinylchloride) microwells in the absence of ‘capture’ (solid‐phase bound) antibody. This was due to binding of complexes of antigen and labelled antibody, and could only be reduced by the use of rather stringent blocking conditions. Assays involving either the simultaneous or sequential addition of antigen and labelled antibody were developed, the latter providing superior results. A number of successful two‐site assays were constructed using single monoclonal antibodies, suggesting that repeating epitopes, known to occur in gluten proteins, were commonly recognized. Pairs of monoclonal antibodies with dissimilar antigenic specificites did not function in the two‐site assay. A comparison of monoclonal antibody based two‐site and antigencompetition immunoassays for gliadin was made. Several antibodies performed considerably better in one format than the other; thus, both assay formats will be of use in quantification of specific gluten protein subclasses.     

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.     

Microminiaturized immunoassays using quantum dots as fluorescent label by laser confocal scanning fluorescence detection

An immunoassay readout method based on fluorescent imaging analysis with laser confocal scanning is described. The ZnS-coated CdSe quantum dots (ZnS/CdSe QDs) were linked to a detection antibody. Immunoassay was carried out on a glass chip using a sandwich assay approach, where antibody covalently bound to a glass chip was allowed to capture antigen specially. Afterwards, the detection antibody labeled with QD was allowed to bind selectively to the captured antigen. The fluorescent signals of the sandwich conjugate were detected by a laser confocal scanner. A diode laser was used to excite efficiently the fluorescent signals while bovine serum albumin was used to eliminate nonspecific binding sites. The detection limit of this approach was up to 10(-9) M under current experimental conditions. The specificity of the QDs-labeled immunoglobulin (IgG) was tested by an experiment using goat IgG and human IgG samples. The result was consistent with the binding specificity in a sandwich-type assay. The potential of this method to function as a simple and efficient readout strategy for immunoassay in biochip is discussed.     

Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments

Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.     

Proteome Analysis Based on Human Recombinant Antibody Microarrays

Protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform high-throughput global proteome analysis. A chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. We aim to develop protein microarrays based on human recombinant scFv antibody fragments for global proteome analysis. The concept of comparing proteomic maps of healthy versus diseased samples will allow disease-specific proteins to be detected. In fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. However, the complexity of proteomes, containing several thousands of different proteins, is a problem. Here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. To this end, an anticytokine antibody array was developed and human dendritic cells (±activation) was used as model system. The results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. Furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. Due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. In more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. The results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable.     

Development of an assay for the quantification of type I collagen synthesis in the guinea pig

There is a need for a reliable assay for the quantification of collagen type I synthesis in the guinea pig, an important model for many connective tissue diseases. Procollagen type I C-terminal propeptide (PICP) is the established marker of type I collagen synthesis but, to date, no assay has been developed to measure PICP in guinea pig tissue extracts. A monoclonal antibody, known to cross-react with intact guinea pig procollagen type I (anti-PICP), was tested for its ability to bind soluble guinea pig PICP in crude skin extracts using a biosensor. Anti-PICP was immobilised to the surface of a sensor chip and antibody-antigen binding was detected using the phenomenon of surface plasmon resonance (SPR). The binding component in the SPR-immunoassay was identified as PICP by purification and N-terminal sequencing. Guinea pig PICP was purified from skin by gel filtration, ion exchange chromatography and lectin affinity chromatography. Purified PICP was then biotinylated and used with anti-PICP to develop a competition ELISA that was able to selectively and sensitively measure PICP in extracts of guinea pig connective tissue.     

一种表面等离子共振芯片的制备及其应用

目的 研制一种新型的还原态牛血清白蛋白（rBSA）芯片,使其适用于Biacore系列仪器并具有与CM5芯片相同的功能。方法 利用二硫键还原剂将天然态牛血清白蛋白（nBSA）的所有巯基还原出来,形成rBSA,然后通过物理吸附将rBSA固定于裸金芯片表面,经交联和羧基化处理后得到rBSA芯片,接着利用原子力显微镜（AFM）表征其表面修饰效果,并与CM5芯片比较在磺胺甲唾唑（SMX）检测中的应用效果。结果 通过AFM扫描,发现rBSA芯片表面基质均一;其检测SMX的抑制标准曲线与CM5芯片相比较,曲线形状十分相似,各抑制浓度的抗体结合信号均具有很好的稳定性;此外,rBSA芯片还具有再生更容易、分析时间更短的优势。结论 rBSA芯片修饰成功,制备的芯片具有优良的性能,能够满足实验的需要。     

Simple quantitative live cell and anti-idiotypic antibody based ELISA for humanized antibody directed to cell surface protein CD20

Rituxan, a chimeric anti-CD20 antibody, has been used for treating non-Hodgkin's lymphoma and some autoimmune diseases. However, a humanized anti-CD20 antibody is desirable for long-term treatment of autoimmune diseases. CD20 is an integral membrane protein with a small intervening extracellular loop. Lacking a native soluble CD20 protein, we developed a simple cell-based enzyme-linked immunosorbent assay (ELISA) using live WIL2 cells in a 96-well format to measure relative binding affinity to support the humanization process. Although WIL2 cells grow in suspension and require centrifugation during the wash steps, the assay was quantitative and reproducible. We also demonstrated that cloned adherent transfected Chinese hamster ovary (CHO) cells could be used to improve assay throughput. For clinical studies requiring quantification of the humanized antibody in serum, we used an alternate approach and developed a high throughput ELISA using an anti-idiotypic antibody as a surrogate antigen for capture and an anti-idiotypic antibody for detection to overcome serum effects. These assay strategies may be applied for characterization of other antibodies directed to multitransmembrane proteins.