Contract Immunoassay Development
Kalon Biological Ltd have many years of experience in the commercial development of qualitative and quantitative EIA kits as well as rapid assays in both latex agglutination and lateral flow formats. Immunoassays have been successfully developed for pharmaceutical, biotechnology and diagnostic companies.
Kalon Biological offers a custom service in immunoassay development and
start- up manufacturing. We also offer related custom services including: antibody development and purification; conjugation chemistry; design, cloning expression and purification of recombinant proteins.
Enzyme Linked Immunosorbant Assay (ELISA) Kits
Overwhelming the most popular format for EIA. We can fully develop a convenient, accurate and stable ELISA to meet the customer's specifications. Diagnostic kit components are optimised and adjusted to produce an assay which is suited to the customerís requirements and giving maximum signal-to-noise ratio. The tracer can be labeled with peroxidase (HRP), alkaline phosphatase (AP), etc. and the parameters optimised for coated microtitre plates, standards and controls as detailed below. The developed assay in kit form, together with protocol, is dispatched to the customer and full advice is provided on the running and application of the kit in the laboratory. Kalon Biological can also manufacture the kit to order.
We understand the characteristics of each component which contribute to the performance of the final kit and can provide any of the kit components including coated microtitre plates, labeled tracer, calibrated standards or kit controls.
ELISA Kit Components
Coated plates - Production of optimally coated microtitre plates for an ELISA depends on many factors including the binding properties of the plastic and the geometry of the wells as well as the coating process and the possible requirement for a blocking or preservative wash. We are able to optimise these factors in order to minimise the background colour and maximise the signal-to-noise ratio.
Tracer - Different immunoassays require antibodies or antigens to be linked to enzymes or reporter molecules. We routinely link the antigen or antibody for HRP, Alkaline Phosphatase, biotin or avidin by using heterobifunctional reagents. This technique has the advantage of allowing the reaction to be controlled and frequently producing stable intermediates which can be stored.
Standards and Controls - Quantitative ELISA requires a set of standards and at least one control. These, together with the validation data, can be supplied either separately or with the ELISA kit. Qualitative or semi-quantitative assays such as serology tests require control sera which are positive or negative for antibodies directed against the infectious agent together with a cut-off control. Kalon sources these sera, titrates the cut-off control and provides them, together with the validation data, either separately or together with the new ELISA.
Latex Agglutination Kits
These rapid, antigen detection tests employ antibody-coated latex particles. Whilst being extremely simple to run and giving a result in two to three minutes these assays can be highly sensitive; detecting nanograms or less of the antigen. The factors which we examine when optimising these tests are: type and purity of the antibody preparation; the use of intact antibodies or their fragments; latex particle size; immobilisation by hydrophobic absorption or covalent linkage. We can develop both the rapid two minute slide tests and those for latex enhanced laser nephelometry.
Lateral Flow Tests
These are generally antigen detection tests requiring no equipment and taking only a few minutes to run. We coat gold particles with antibodies or other proteins to yield the most sensitive and stable product. We also optimise the antibody or other proteins immobilised on the nitrocellulose membrane as the capture line. Other aspects of the test which are optimised are the assay format and the strip architecture.
Custom Reagent Development
We can supply polyclonal antibodies produced in sheep, goats or other species. Antibody development and characterisation usually takes 4-6 months, with available, purified antigen.
Antibody and Protein Antigen Purification
Proteins such as antibodies or those to be used as immunogens or the analyte can be purified by size exclusion, ion exchange, immunoaffinity chromatography or one of the various protein A, protein G media.
Recombinant Protein Antigen cloning, expression and purification
We prefer to produce recombinant proteins in a baculovirus/spodoptera system which, because of post translational modifications, more closely resemble mammalian proteins than those produced in E. coli. The resultant recombinant proteins are usually tagged with six histidines or glutathione reductase, which allows one step purification by affinity chromatography.