Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor

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Abstract

We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)′2 fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.

Introduction

Interdisciplinary studies in the fields of chemistry, electronics and biology have led to the development of many novel sensor surfaces and detection systems [1], [2], [3], [4]. This has resulted in the commercial availability of a number of multifunction instrumental biosensors, which are rapidly becoming a major tool in the field of biomedical research. These systems are now widely used to study biomolecular interactions between proteins and peptides, DNA, lipids, carbohydrates, drugs and more recently cells, and are suitable for a range of research applications including structure/function studies, epitope mapping, kinetic and equilibrium binding analysis, ligand searching and specific monitoring of chromatographic fractions [5].

The most widely used instrumental biosensors to date have been the BIAcore (BIAcore X, 1000, 2000 and 3000) range (Biosensor, Uppsala, Sweden, http://www.biacore.com) and IAsys (IAsys manual system, IAsys Plus and IAsys Auto+) systems (Affinity Sensors, Cambridge, UK, http://www.affinity-sensors.com). The BIAcore biosensors are flow-based instruments, which use the detection principle of surface plasmon resonance [6], [7]. The sensor surface consists of a glass slide coated with a thin (50 nm) gold film to which is attached, by an inert (alkanethiol) linker layer, a chemical matrix onto which one of the binding partners can be immobilized using well defined chemistries [8]. The flow cells are formed by interfacing the sensor chip with a thermostatically controlled integrated fluidic cartridge (IFC). Four parallel channels (60 nl volume, 1.5 mm2) are formed on the sensor surface and the microfluidics are used to deliver the reagents and samples over the sensor surface.

By comparison, the IAsys sensors are cuvette-based systems, which use a waveguide technique called a prism coupler or resonant mirror for detection [9], [10]. The IAsys sensor surface (4 or 16 mm2) is the bottom of an independent two-well format micro-cuvette, which has a choice of derivatised surfaces for ligand immobilisation. Samples (1 to 80 μl) can be added to, or removed from, the cuvette using a robotic dispenser.

The specific operating principles and design features of these instrumental biosensors suggest that they will be individually suited to particular applications (e.g., kinetic analysis, high throughput screening, ligand fishing). Biosensor analysis can be used in conjunction with other analytical techniques {e.g., micropreparative high-performance liquid chromatography (HPLC) [5], [11], [12], mass spectrometry (MS) [13]}, providing complementary information on the nature of the sample. The sensitivity and sample recovery volumes characteristic of micropreparative HPLC are compatible with biosensor requirements, allowing the two systems to be used in a complimentary manner that we have termed “bidirectional synergy” [12]. In one direction micropreparative HPLC generates well characterised homogeneous reagents for use on the biosensor, whilst in the other mode, the sensor provides specific information to aid the chromatographic process (e.g., development of affinity chromatography systems, vis-a-vis choice of antibody, optimisation of elution conditions [12], [14], or specific immunodetection for chromatographic fractions [14], [15]). Indeed, the biosensor can be considered as a micropreparative affinity surface with on-line detection comparable to a chromatographic immunoaffinity system. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis has recently been interfaced with biosensor analysis (BIA/MS) [13] and has been used to monitor affinity interactions occurring on the sensor surface (e.g., nature of the binding components, identification of unknown components, determination of specific and non-specific binding). The probe element surface of a laser desorption/ionisation TOF instrument has also been chemically and physically modified and used in both the binding process and the subsequent mass spectrometry analysis in a technique call affinity mass spectrometry or SELDI (surface enhanced laser desorption/ionisation) [16].

The interface between biosensor analysis, micropreparative HPLC and MS appears to be particularly attractive for the identification, purification and characterisation of ligands for orphan receptors identified by polymerase chain reaction (PCR) approaches or data base searching strategies [14]. For example, biosensor analysis has been used to identify sources of ligands for orphan proteins [17], [18], [19], [20], [21], [22] and used as a specific affinity detector for the chromatographic fractionation [15], [17], [19], [21], [22]. The potential of BIA/MS has been demonstrated by using the BIAcore sensor chip [23] and BIAcore PROBE [24] directly as MALDI targets. Alternatively, the specifically bound ligand can be eluted from the sensor chip prior to MALDI analysis [25].

The success of ligand fishing experiments depends critically on the ability to conveniently recover in high yield the bound ligand for subsequent characterisation. Therefore, a recent area of biosensor development has been the automated elution and recovery at high concentration in small volumes (≥1 μl) of specifically bound material from the sensor surface in a form suitable for sensitive and specific downstream analysis [e.g., MS, N-terminal sequence analysis, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE)]. The latest generation of BIAcore (BIAcore 3000, Biosensor) has been designed to minimise the dilution effect during recovery following desorption by reversing the flow through the flow cell to return the sample back to the auto-sampler. However, the surface area of the flow cells (1.6 mm2) in this instrument limits the overall capacity and hence the amount of material that can be recovered at each cycle. The use of the BIAcore probe will overcome this limitation (surface area 12–20 mm2) although this instrument is not automated. The IAsys cuvette based system appears to be particularly suited for ligand fishing due to the potential for long binding contact times and the confined environment which facilitates recovery post-desorption in small (μl) eluent volumes. The availability of a high-capacity carboxymethyldextran sensor surface (CMD-SELECT cuvette, 16 mm2 surface) will also increase the level of recovered ligand, further facilitating post-elution analysis.

We present herein the use of a cuvette based biosensor (IAsys Auto+) for preparative ligand fishing using the A33 antibody–antigen interaction [15] as a model system. A33 antibody (IgG) or antibody fragment [F(ab)′2] were immobilised onto a preparative CMD-SELECT cuvette and used to purify the A33 epithelial antigen from Mono Q fractions generated from transfected Sf9 cell lysates or detergent extracts of LIM1215 colonic cells lines [26]. The recovered ligand was subsequently analysed and characterised using sodium dodecyl sulphate (SDS)–PAGE, Western blot analysis and micropreparative HPLC. Using automated repetitive injection and recovery, microgram quantities of essentially homogeneous ligand could be recovered in high yield using the F(ab)′2 surface.

Section snippets

Cell culture techniques

The LIM1215 colonic carcinoma cell line was grown in RPMI medium (Irvine Scientific, Santa Ana, CA, USA) containing 10% foetal calf serum. Confluent cells (2.2·105/cm2) were passaged using Trypsin-Versene solution (Life Technologies GIBCO BRL, Gaithersburg, MD, USA). Cells were seeded 1/10 into tissue culture dishes (150×20 mm, Nunclon, Roskilde, Denmark) containing 25 ml RPMI 1640 supplemented with 10% foetal calf serum, 1 μg/ml hydrocortisone, 0.025 U/ml insulin and 10.82 μg/ml

Results and discussion

The monoclonal antibody A33 (mAbA33) detects a tissue specific cell surface antigen expressed by both normal and transformed intestinal epithelium [31]. The A33 antigen has recently been identified as a novel member of the immunoglobulin superfamily [32]. The A33 antigenic system is the focus of several clinical studies on patients with colon cancer. Phase I/II clinical studies have shown that the monoclonal A33 antibody (i) localises with high specificity to colon cancer, (ii) is retained for

Conclusions

The cuvette-based IAsys biosensor, which uses the resonant mirror optical detection principle, was used to perform micropreparative ligand fishing. The A33 antigenic system was used to validate the methodology, using both the recombinant A33 expressing Sf9 cells as well as the LIM1215 colonic carcinoma cell line, from which the A33 antigen was originally identified and purified to homogeneity [15], [30], as sources of antigen.

Using a high capacity dextran biosensor surface (surface area 16 mm2)

Acknowledgements

We are especially thankful to Dr. Ivan Gout, Ludwig Institute for Cancer Research, University College Branch, London, for providing the A33 antigen baculovirus construct and Dr. Detlef Geleick, Ludwig Institute for Cancer Research, Melbourne Branch, for the bioreactor production of the A33 monoclonal antibody.

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