George L. Mayers

A new method for studying cell cycle characteristics in ANLL using double labeling with BrdU and 3HTdr

Ten patients with acute nonlymphocytic leukemia (ANLL) received bromodeoxyuridine (BrdU) at 100 mg/M2 intravenously over 1 h. BrdU is incorporated into the DNA by S-phase cells and was detected by using a monoclonal anti-BrdU antibody in the bone marrow aspirate (BM) and biopsy specimens obtained at the end of the infusion. Additionally, BM was incubated in vitro with tritiated thymidine (3HTdr) and processed by our previously described double-label method. This allowed us to measure the duration of S-phase (Ts) and total cell cycle time (Tc) of myeloblasts. Data revealed a higher number of S-phase cells from biopsies (21%) than BM (5%). The Ts ranged from 9 to 35 h and Tc ranged between 36 and 152 h in different patients. Using this method, data are available within 48 h and if shown to be clinically relevant, may be useful for prospective planning of therapy in individual patients.

[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.

Arginine and lysine in binding sites of anti-4-azophthalate antibodies

Abstract Antibodies from individual rabbits, directed against 5-azoisophthalate have been examined for the presence of lysine in their combining sites by complete maleylation of amino groups in the absence and presence of hapten, and for arginine by glyoxalation of guanidinium groups in the absence and presence of hapten. By each procedure, antibody sites were lost when hapten was absent and this loss could be partially prevented when hapten was present during the chemical modification procedure. The results indicate that a large proportion of the sites of anti-5-azoisophthalate antibodies studied contain arginine and that a small proportion contain lysine.

Clonogenic potential of myeloid leukaemia cells in vitro is restricted to leukaemia cells expressing the CD34 antigen

Cells from patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) were separated into CD34-enriched and CD34-depleted subpopulations. The clonogenic capacities of these two subpopulations were then compared to each other and to the original unseparated cell population. In every study, the CD34-enriched subpopulation demonstrated a substantial increase in clonogenicity in vitro in comparison with the original cell population, while the reverse was the case for the CD34-depleted subpopulations. For reasons not clear at present, the enrichment for clonogenic cells far exceeded the enrichment for cells expressing the CD34 antigen. Additionally, the clonogenic potential was found to be unrelated to the level of myc expression in the various cell populations.

The use of protein A in solid-phase binding assays: a comparison of four radioiodination techniques☆

Preparations of protein A radioiodinated by 4 different methods have been compared in indirect radioimmunoassays. The oxidative methods (chloramine-T and iodogen) for direct iodination of tyrosyl and histidyl residues were applied with high efficiency and gave a suitable product, provided the substitution ratio was kept low (1 iodine atom/molecule of protein A). Higher levels of modification tended to perturb the Fc-binding characteristics of the protein, especially with the use of iodogen. Introduction of the isotope via substitution of lysyl residues (Bolton-Hunter and Wood reagents) was also examined. The Bolton-Hunter modification of protein A gave an unsuitably low labeling efficiency; in contrast, the Wood reagent gave efficiencies approaching 50%. Protein A could be extensively substituted with the latter reagent (greater than 5 diiodinated benzimidate molecules per protein molecule). Thus, the use of the Wood-labeled protein A could raise the sensitivity of the binding assay at least an order of magnitude compared to using protein A iodinated by the oxidative methods. The effects on the biological activity of protein A exerted by the different labeling procedures are rationalized on the basis of the amino acid composition and tertiary structure of the protein.

Comparison of methyl-p-hydroxybenzimidate and 1,3,5-trichlorotriazine in producing sensitive target cells for use in the hemolytic spot assay☆

Two bifunctional reagents were shown to be useful in the coupling of immunogens to the surface of either nucleated or non-nucleated cells. The heterobifunctional reagent methyl- para -hydroxybenzimidate was used to couple aromatic amino-haptens to the surface of SRBC which resulted in a stable and sensitive target cell capable of detecting as little as 20 pg of purified anti-hapten antibody in the hemolytic spot assay. The multifunctional reagent 1, 3, 5-trichlorotriazine yielded similar results when coupling amino-haptens to the surface of SRBC for use in the hemolytic spot assay. This reagent was also used to couple protein to the surface of SRBC which were able to detect as low as 1 ng of purified anti-protein antibody in the hemolytic spot assay. The sensitized SRBC produced using either of these coupling reagents were shown to remain stable for several months giving reproducible results from one test to another. Lastly, 1, 3, 5-trichlorotriazine was used to couple the hapten 4-aminophthalate to the surface of nucleated cells with retention of greater than 90% cell viability with continued growth and cellular division in culture.

Immune Response of BALB/c Mice to Phthalate: Characterization of a New and Useful Model for Studying Immune Regulation

The humoral immune response to a unique haptenic determinant has been characterized at the single cell level. We report here that the hapten, phthalate, when conjugated to keyhole limpet hemocyanin (4-azophthalate-KLH) is highly immunogenic in adult BALB/c mice, and that the immunogenicity of the hapten is largely restricted to the two charged carboxylate groups on phthalate. Based upon plaque-forming cell (PFC) inhibition with increasing concentrations of free phthalate, relative affinity distribution profiles of antibody-forming cells have been established for individual mice. The antibody-forming cells in the spleens of these mice consist of five or six individual relative affinity sets. PFC inhibition studies with hybridomas secreting phthalate specific monoclonal antibodies reveal, as expected, a more restricted inhibition profile. The majority of PFC produced by the hybridomas could be assigned to one of the relative affinity sets. PFC inhibition studies employing a panel of cross-reactive phthalate...

Detection of single-stranded DNA damage using monoclonal anti-thymidine antibody

A method to detect single-stranded DNA damage from individual cells has been developed using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 micrograms/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 micrograms/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion.