the versatile chemical
Immunochemical sorting techniques developed T.M
Now reaching beyond historic roles in building and agriculture, lime to day has become just about the No. 1 chemical in both diversity and ton nage of uses. With remarkable effec tiveness and cost-benefit ratios, among other things modern lime can:
ABSORB ALKALIZE BENEFICIAI CAUSTICIZE CLARIFY COAGULATE DEHYDRATE DISINFECT FLUX NEUTRALIZE PLASTICIZE PURIFY PRECIPITATE
Two similar immunochemical techniques for sorting out molecules and cell types from mixtures are emerging from two re search groups at Harvard Biological Laboratories in Cambridge, Mass. The researchers say the techniques are simple, inexpensive, and highly sensitive. The techniques are based on an obser vation by Australian scientists 11 years ago that antibody molecules stick to plastic. In that work, Kevin Catt and Geoffrey W. Treagar at Monash Univer sity, Melbourne, found that by binding antibody proteins to certain plastics, they could assay for radioactive antigen mol ecules. The two Harvard groups have refined and redirected that approach, applying it to different problems. One such problem is how to sort one cell type from a complex mixture. Graduate student Lawrence J. Wysocki and Dr. Vicki L. Sato say that their new method readily sorts several types of lymphocytes from mouse spleens, apparently without damaging the bio logical activity of the cells. The other group, consisting of Dr. Walter Gilbert and his collaborator, Stephanie Broome, uses a similar tech nique to pluck out one type of molecule from a mix. They note that the technique might prove useful in recombinant DNA experiments in which a tiny amount of a nonbacterial protein that's fused chemi cally to some bacterial protein must be detected. Both techniques rely on the sensitive and highly specific binding that takes place between antibody molecules and antigens. Antigens, which may be pro teins, glycoproteins, or other molecules carrying "recognition sites" for anti bodies, are the ultimate objects of the quest. But to look for them requires a starter supply of purified antigen with which to procure an antibody supply. Antibodies are elicited by injecting antigen molecules into animals such as rabbits or goats. Their blood then con tains antibodies directed against those antigens. Typically, the antibody proteins in the blood are purified partially before use.
Such antibodies stick to plastics such as polystyrene or polyvinyl chloride. Once stuck, they do not wash off readily, hence they bait the trap for the antigen mole cules against which they're directed. The trap then is turned into a multilayered sandwich with the antigen being caught in the middle between layers of antibody that are, in turn, between layers of two kinds of film. And, because one of those antibody layers contains radioactively labeled molecules and one of the film layers contains x-ray sensitive film, the trap-turned sandwich provides a quick and permanent measure of whether a particular antigen is present. For example, Broome and Gilbert apply the method to detect the enzyme protein β-galactosidase within bacteria cells. First, the researchers coat plastic disks with antibodies to that protein. Then those disks are placed over colonies of bacteria growing on an agar surface. Such colonies usually are several millimeters in diameter. Antigen may be released in one of several ways, including exposing cells to chloroform vapor. Once released, antigen molecules—here β-galactosidase—diffuse and bind to the specific antibodies that are stuck on the plastic disks. Next, more antibody mole cules, but ones that are radioactive (la beled with iodine-125), are added to bind to other sites on the antigen molecules. Finally, the x-ray-sensitive film is applied to show which cell colonies contain the antigen and which do not. The technique can detect "a few picograms of protein antigen, a few molecules from each bac terial cell," according to Broome and Gilbert. The same principles apply to the cellsorting method, except that instead of fishing for free antigens, the search is for antigens that are on the surface of par ticular cells. Very little antibody is need ed, Wysocki and Sato note, but cell se lectivity is high. The disadvantage of ei ther technique is that specific antibodies are needed, and that other proteins can interfere with the binding of antibody to the plastic. Jeffrey L. Fox, C&EN Washington
Immunochemical method can detect picograms of proteins First binding site holds protein to antibody on disk
Radioactive antibody binds at other sites on the protein
Proteins with at least two antigenic binding sites
REFINE STABILIZE all this and more can be done with
LIME
Plastic disk
July 24, 1978 C&EN
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