A standard method for conjugating a protein label to an antibody is depicted in Figure 2 (upper panel). The method requires the use of two tags, “X” and “Y”, one of which is attached to the antibody and the other to the label. The label (but not the antibody) can be sometimes be purchased with the Y tag already attached. After removal of excess reactants from the antibody using spin columns, the two entities are mixed and X and Y react to form a covalent X–Y bond that connects the antibody and label. There are a number of related two-tag methods and although the chemistry varies they all have the same advantages and limitations. The key advantage is that the formation of homodimers is prevented because X must react with Y. The major disadvantage is that the purified antibody has to be chemically tagged and separated from excess reactants before the label can be attached. These operations lead to: (i) losses of antibody on spin columns; (ii) batch-to-batch variation; (iii) difficulty in scaling up; and (iv) difficulty in scaling down (which is highly desirable when making trial conjugates with expensive antibody reagents).
To overcome the limitations of the traditional approaches to conjugation, Innova Biosciences has developed Lightning-Link® technology, which has the same control as the two-tag methods but with none of the tags or process complexity (Figure 2, lower panel). Lightning-Link technology allows direct one-step attachment of the antibody to specially modified labels. No knowledge of chemistry is required; the primary antibody is simply pipetted into a vial of lyophilized Lightning-Link mixture. The necessary reactive groups are created in situ while unwanted side reactions are suppressed. Because there are no spin column separation steps, the concentrations and ratios of reactants are tightly controlled by the operator and very easy to replicate. As little as 10 micrograms of antibody can be conjugated, and the hands-on time is always less than 30 seconds, irrespective of the scale. Presently over fifty labels are available in the Lightning-Link one-step format including fluorescein, other organic dyes (e.g., DyLight®, Cy®, and Atto), fluorescent proteins, tandem dyes, enzymes, biotin, and streptavidin.
There are hundreds of suppliers of antibodies and some have over 100,000 primary antibody reagents, the vast majority of course with no labels. The need to apply different chemistries with different types of labels, the sheer number of labels and the complexity of the two-tag systems deter suppliers from investing in the production of thousands of conjugates. It is easier and less risky for suppliers to maintain collections of antibodies in their unlabeled form. There are ~500,000 commercial antibodies (and perhaps a similar number around the world generated by individual researchers) and all of them are compatible in their native forms with Lightning-Link. This opens up some exciting possibilities and Lightning-Link has become the driving force behind the virtual library concept—a vast collection of some 50 million research tools (1 million primary antibodies x 50 Lightning-Link labels), any one of which can become real with under 30 seconds of effort. This is a fabulous position from an assay developer’s perspective and also attractive to antibody companies that can effectively expand their portfolios with zero risk.
Finally, while antibody conjugation was once practiced only by those with specialist knowledge of chemistry, today everyone is a competent conjugation scientist and making antibody conjugates has never been easier.