Plastic is the ultimate unnatural material. Its persistence in the environment as a pollutant stems from the fact that the natural world has not had time to evolve mechanisms to degrade it in a timely fashion. Wood, bone and leather are foodstuffs for various microorganisms, but they have all existed for a very, very long time. Many of the plastics we deal with daily are formed by injection molding. In this process, raw pellets of polypropylene or polystyrene are melted into a hot liquid that is then forced into metal molds. Cooling takes place in the mold so that the plastic becomes solid. The solid piece, which is still quite hot, is then ejected from the mold into oxygen-rich air. Oxidation ensues and produces organic acids, aldehydes, ketones and epoxides[1] on the exposed surface.
Antibodies and other proteins have evolved over hundreds of millions of years. It is a lucky accident that proteins and injection-molded plastics were found to interact in such a useful way[1]. Not only do antibodies spontaneously bind tightly to injection-molded plastic surfaces without the aid of other chemicals or treatments, but these antibodies also retain their ability to bind to their target antigens. This is analogous to making a delicate design in clay and then throwing the clay object at a wall and expecting the design to stay intact. But amazingly enough, it does!
One of my first assignments as an industrial chemist was to get antibodies off of previously coated polypropylene test tubes. There was an oil crisis at the time, and plastic tubes were in tight supply. It was thought that if we could remove antibodies from antibody-coated tubes that had failed to pass quality control, they might be reusable. Many reagents such as chromic acid, alcoholic potassium hydroxide and permanganate destroyed the binding ability of previously coated antibodies, but they failed to produce a surface that could be coated with another antibody. The only treatment that could make the surface suitable for recoating was boiling water for several minutes. In effect, the protein had to be cooked off.
To better understand how antibodies coat onto plastic, we also tried various pretreatments. Detergents, lipids and organic solvents had little or no effect on the ability of antibodies to coat the plastic surface. The only reagent that seemed to make the surface uncoatable was elemental iodine in a sodium hydroxide solution. This is the classic iodoform reagent, which is a test reagent for the presence of ketones and aldehydes[2]. It also destroys the ketones and aldehydes in the process.
A quality problem provided further evidence of the interaction of hot plastic in an oxygen-rich environment. A company was coating 12 mm by 75 mm polypropylene test tubes with antibodies and had been successfully marketing them for several years. A problem occurred with tube-to-tube reproducibility. About 3 percent of the tubes tested showed a higher level of binding than the rest of the tubes, thereby causing the batch of coated tubes to fail. Weeks of hard work with variations in coating procedures and blocking reagents failed to correct the problem. An observant quality control technician noted that all the tubes that showed the enhanced binding had a number 8 embossed on the outside bottom of the tube. No one at the company knew the significance of the numbers on the bottoms of the tubes. A call to the molder revealed that the number identified a particular cavity in a 36-cavity mold. When the molder examined the mold, they found that the cooling line for cavity number 8 had been blocked. That meant that a tube ejected from cavity number 8 was hotter than tubes molded in the other cavities. This higher temperature likely led to more oxidation products forming on the tube’s inner surface and higher amounts of antibody being bound. Clearing the cooling line on cavity number 8 solved the reproducibility problem. The so-called edge effect seen in microtiter plates may have a similar origin. Wells on the periphery of a microtiter plate would be expected to cool quicker than wells in the plate’s interior.
The observations above are evidence that coating antibodies onto injection-molded plastic is not a passive process. It is likely a chemical process that involves the reaction of amines and possibly other functional groups with reactive oxygen species on the surface of the plastic.
How much antibody can be coated on a plastic surface is a question that frequently occurs in assay development. Studies conducted decades ago[3] suggested that it was possible to coat antibodies at 1 ug/cm2. This agrees well with closest-packing calculations that take into account the known dimensions of antibodies. Experiments with rabbit polyclonal to digoxin and cortisol showed that if polypropylene tubes were first coated with digoxin antibodies and subsequently with cortisol antibodies, no cortisol antibodies could be co-coated until the digoxin antibody coating concentration fell below 0.5 ug/mL. Yet countless experiments have shown that coating concentrations higher than 0.5 ug/mL do produce higher amounts of active bound antibody, up to a limit of 3-4 ug/mL. How can these two seemingly contradictory observations be reconciled?
Enhanced coating strategies are known and have been employed to improve assay performance and conserve costly antibodies. One approach is to coat an anti-species antibody such as a goat anti-rabbit antibody and then let it capture the rabbit antibody of interest. Another approach used successfully is to chemically label the primary with a biotin or fluorescein thiocyanate (FITC) molecule and then coat the corresponding secondary reagent streptavidin or anti-FITC. All of these techniques require less of the primary antibody to be used in the assay. In my hands, an anti-FITC system used 20 times fewer primary antibodies than when that same primary antibody was directly coated. This observation may help explain the apparent paradox about coating concentration cited above. Secondary coating strategies may deliver more useable antibodies than direct coatings. This may also explain why some antibodies pretreated with a cross-linking agent such as glutaraldehyde have enhanced coating[4]. The glutaraldehyde may “shrinkwrap” antibodies and prevent them from being deactivated on coating.
While we have learned much over the years about coating antibodies on plastic surfaces, there are still many questions that remain. Polyclonal antibody coating solutions with coating concentrations above 1 ug/mL can be recovered and used to coat other tubes or wells. In my hands, monoclonal antibody solutions are not reusable even at coating antibody concentrations of 3 ug/mL. Methods using labeled goat anti-mouse antibodies inhibited by measured amounts of unlabeled goat anti-mouse antibodies can be used to estimate the amount of mouse antibody coated on microtiter wells. In several systems where 100 uL of mouse antibody at a coating concentration of 3 ug/mL was coated, it resulted in only 10 ng of mouse antibody being coated on the well out of 300 ng of antibody being offered. The remaining 290 ng that was recovered was uncoatable on a fresh surface. The mechanism of this inability to coat is currently unknown.
Coating antibodies on plastic surfaces has been practiced on an industrial scale for decades and is central to products that affect human health and other important areas. It is still amazing that it works at all, and as outlined above, there is still much to be learned about the process.
Learn more about DCNovations products, including antibodies, here.
[1] K. J. Catt and G. W. Tregear, Science 158, 1570 (1967)
[2] https://www.chemguide.co.uk/organicprops/carbonyls/iodoform.html
[3] A. J. Pesce, D. J. Ford, M. Gaizutis, and V. E. Polak, Biochim. Biophys. Acta 492, 399 (1977)
[4] U.S. Patent 4,069,352
