Innova Biosciences has developed a conjugation technology called Lightning-Link™ (Figure 1) that eliminates almost all of the steps employed in a traditional conjugation process. The elimination of column separations from Innova’s process has probably had the greatest impact. Issues that have beset traditional conjugation procedures—losses of material, sample dilution, batch-to-batch variation and difficulties in scaling up—have now been removed.
The Lightning-Link process is summarized in Figure 2. The researcher pipettes the antibody to be labeled into a vial of lyophilized mixture containing the label of interest. Dissolution of the contents activates the chemicals that mediate the conjugation reaction. Despite its apparent simplicity, the Lightning-Link process is sophisticated and generates conjugates with performance characteristics identical with, or better than, those prepared with laborious multistep conjugation procedures.
Moreover, it is possible to use the resulting Lightning-Link conjugates without purification, as the byproducts of the reaction are completely benign. Because the reactive groups are created in situ in a controlled manner the risk of unwanted polymerization is reduced.
The approach is also tolerant of sodium azide, which is commonly employed as an antimicrobial agent in commercially available antibodies. In addition, BSA, another common additive, has only a modest impact on Lightning-Link conjugation reactions.
This simpler approach to conjugation is likely to shift the balance of indirect detection technologies toward those of direct detection. Researchers carrying out immunodetection procedures can eliminate the tedious secondary incubation and wash steps. Intuitively, one can also see how data quality is likely to be improved by a reduction in the number of assay variables.
In flow cytometry, it is quite common to combine three or more directly labeled primary antibodies, each with a different fluorescent label.
The benefits of simple conjugation technology and direct labeling are even greater in the case of multiplex immunoassay technologies, where large panels of antigen-specific reagents can be constructed from the best available antibody tools, without the usual limitations that apply to indirect detection methods.
Simple conjugation technologies also greatly facilitate conjugate optimization and scale-up.
It is now possible with just 100 micrograms of antibody to create 20 trial conjugates, allowing one to reach a level of performance that is limited only by the attributes of the antibody itself. A similar approach can also be used to identify the best antibody, or best antibody pair, from a library of monoclonal antibodies against a common antigen.
The best conjugate can be scaled easily as there are so few variables in the conjugation process. For example, in Figure 3, conjugates prepared at 50 microgram scale and 50 milligram scale show identical ELISA performance; both conjugation reactions required a hands-on time of 30 seconds.
We also expect simple state-of-the-art conjugation methods to be deployed for manufacturing of tomorrow’s immunodiagnostics reagents, because of greater product consistency, higher yields, and reduced costs.
The labels presently available in the one-step Lightning-Link format include enzymes (horseradish peroxidase, glucose oxidase, and alkaline phosphatase), fluorescent proteins (phycoerythrin, allophycocyanin, PerCP), streptavidin, biotin, and fluorescent dyes that cover the entire visible spectrum. Recently, oligonucleotides have also been integrated into the Lightning-Link format allowing the development of reagents for use in ultrasensitive immuno-PCR diagnostics tests.
The availability of an increasing number of labels will facilitate the exploration of the human proteome. Researchers might also expect the percentage of commercially available antibody tools available in labeled form to increase, and for there to be a broader range of labels for each antibody.
Finally, in view of the simplification of bioconjugation technology one can imagine in the near future a vast library of exciting research tools, comprising millions of antibodies and several hundred labels, which can be combined in any way to create an almost infinite number of virtual conjugates, any of one which can be turned into reality on demand.