Yi-Her Jou

[21] Methods for the attachment of haptens and proteins to erythrocytes

Publisher Summary This chapter discusses the methods for the attachment of haptens and proteins to erythrocytes. Erythrocytes to which antigens or antibodies are adsorbed or conjugated have been used extensively as target cells for the detection of antibodies and antigenic substances. The reaction of antigen-bearing erythrocytes with the complementary antibody results in agglutination or initiates a complement-dependent hemolysis. The antigen-bearing erythrocytes have been used in the quantitation of antigen by the inhibition of passive immune hemolysis. Such indicator erythrocytes have also been used to quantitate antigen-binding cells by rosette assay and antibody-forming cells by plaque-forming cell assays. Moreover, these indicator cells can be used for enumerating antigen-bearing cells by a reverse rosette assay and for counting antigen-secreting cells by a reverse plaque-forming cell assay. Assays using antigen-bearing erythrocytes to detect hybridomas secreting the desired antibody can be found elsewhere. In addition, the new coupling methods are discussed in this chapter.

Monoclonal Antibodies and a Heterobifunctional Reagent: A Novel Approach to the Vectorial Labeling of Selected Membrane Proteins

Many applications exist and others are envisioned for the chemical coupling of macromolecules to membrane proteins on the surface of mammalian cells. The ability to use antibody as a means to label and subsequently to follow the distribution of cell surface proteins is reported here. A new procedure is outlined for covalently coupling monoclonal antibodies to thiol-containing membrane proteins. The key reagent in the coupling reaction is the commercially available heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The coupling proceeds in a simple two-step reaction in aqueous medium under very mild conditions. This results in a very efficient and stable attachment of anti-hapten antibodies to a selected set of cell surface proteins without any loss in cell viability and without denaturing the antibody molecule. The hapten-binding activity of the antibody is exploited to monitor the re-distribution of the antibody-labeled cell surface proteins periodically after the coupling reaction. The hapten binding activity can also be utilized to isolate membrane macromolecules via affinity chromatography.

Succinylation of hapten-protein conjugates facilitates coupling to erythrocytes by water soluble carbodiimide: Preparation of stable and sensitive target cells for use in hemolytic assays☆

A facile method is described for the preparation of haptenated sheep red blood cells (SRBC) for use as targets in hemolytic spot and plaque assays for the detection of anti-hapten antibody. The method involves the use of the water soluble 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDCI) as a reagent to couple hapten-succinyl-rabbit serum albumin conjugates to SRBC. The presence of the succinyl groups on such conjugates is shown to increase the efficacy of the resulting target cells, presumably by acting as a substrate for the EDCI and thus increasing the extent of coupling to SRBC.

A filtration double antibody radioimmunoassay that simplifies and semi-automates the isolation of immune precipitates

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.

Chemical nature of mouse antibodies homologous to the 3-pyridylazo group: The fine specificities of hybridoma and serum antibodies

Rabbit antibodies which bind aromatic annular nitrogen-containing haptens exhibit a specificity wherein such nitrogens are distinguished from the closely related aromatic CH group. The mouse hybridoma system was used to extend this work producing hybridoma antibodies homologous to the 3-pyridylazo group. Fine specificity mapping by double antibody radioimmunoassay revealed differences among the individual hybridomas, as well as a greater resemblance of mouse serum antibodies to rabbit serum antibodies than to hybridoma antibodies. Quantitative structure-activity relationships applying the parameters of hapten molar refractivity had hydrophobicity were used to help elucidate the types of intermolecular forces involved in the interaction of pyridine derivatives with the antibodies. The results are consistent with the interpretation that pyridine binding to antibody does not involve desolvation.