Plant glycoprotein biosynthesis. Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root.

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radio-labeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-D-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,

Semi-automatic solid-phase radioimmunoassay for carcinoembryonic antigen.

A solid phase, double antibody radioimmunoassay has been semi-automated for the quantitation of carcinoembryonic antigens. Sepharose-bound rabbit anti-goat IgG immunoglobulin was used as the second antibody, reactions were carried out in 96-well microtiter plates, and samples processing (filtration and washing) was accomplished with the aid of a 24-sample harvester.

Stability of T- and B-cell numbers in human peripheral blood.

A comparison was made between the percentage of T and B cells present in human blood on the day of collection and those recovered from whole blood specimens stored for one, two, and three days. In the peripheral blood of normal individuals, the percentage of E-rosetting and surface-membrane immunoglobulin-positive cells was unchanged throughout the three day period. Furthermore, whole blood samples from patients with various hematological diseases maintained for three days exhibited T- and B-cell percentages equivalent to those tested on day zero.

Monoclonal antibodies: expanded potential for labeled antibody ligand assays.

The impact of the availability of monoclonal antibodies is just beginning to make itself felt. This paper describes some of the advantages monoclonal antibodies have over polyclonal for clinical laboratory applications. The use of monoclonal antibodies in the two-site immunoradiometric assay is discussed in detail.

Soluble HL-A antigens in serum. I. Isolation and purification.

A new method was developed which utilizes fractional salting out, gel filtration, ion exchange chromatography and polyacrylamide gel electrophoresis for the isolation and purification of soluble HL-A antigens from serum. The method produced purified antigens possessing HL-A 5,9 specificities with yield up to 60% of that detected in the original serum. Up to 150-fold increase in specific HL-A antigenic activity above that in the starting material could be achieved as measured by the ability of the purified antigens to specifically inhibit the cytotoxic activity of HL-A alloantisera against selected target cells. Electrophoretically purified HL-A antigens with different specificities appear to have a similar molecular size, i.e. 33 000, but differ in their electric charge properties, beta2-microglobulin (beta2m) seems to be noncovalently associated with HL-A antigens present in serum, but during purification beta2m in progressively lost until at the final purification by polyacrylamide gel electrophoresis, the material is completely devoid of beta2m.

Monoclonal antibodies in clinical immunology.

Advances in clinical immunodiagnostics resulting from the application of hybridoma technology are starting to appear. Monoclonal antibodies are beginning to displace their antiquated progenitors, polyclonal antisera, in many facets of immunology. Their homogeneity, specificity, and availability make hybridoma-derived antibodies the immunological reagents of the future in immunoassays, immuno-affinity chromatography, immunotherapy, and areas yet to be defined.

The hybridoma-an immunochemical laser.

The advent of hybridoma technology has provided the immunotechnologist a finely tunable instrument that should permit a marked advance in the immunologic sciences. The ability to choose the precise antibody required and the virtually unlimited availability of easily purified antibodies have already resulted in the simultaneous immunometric assay and potential reagents for immunoscintigraphy and immunotherapy. Using an antibody with a specific affinity and recognition for a specific antigenc determinant can greatly influence the shape and range of a radioimmunometric calibration curve. With monoclonal antibodies, assay systems based on immune complexes (e.g., turbidimetric assays and counterimmunoelectrophoresis) can be made more precise, thus permitting study of the basic physicochemical principles underlying the antigen-antibody reaction and the development of greatly improved quantitative assays. On the other hand, the ability to select an antibody exhibiting specific characteristics implies the necessity to select. One no longer has the luxury of using a mixture of antibodies, hoping to take advantage of the fact that some will have desirable secondary characteristics such as electrophoretic mobility or the ability to absorb to plastics.