A rapid,sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots

A rapid, sensitive method has been developed to detect antibody-antigen complexes on “Western blots.” The methods of H. Towbin, T. Staehlin, and J. Gordon were used to separate and blot the antigens onto nitrocellulose. The remaining sites of attachment were blocked and the nitrocellulose was washed with polyoxyethylenesorbitan monolaurate (Tween 20). The blot was then reacted with the antiserum or hybridoma supernate to be tested. After the antigen-antibody reaction was completed, the blot was washed and treated with anti-antibody which has been conjugated to alkaline phosphatase. The alkaline phosphatase was detected by the reduction of the tetrazolium salt to diformazan by the hydrogen ions released in the formation of indigo by the reaction of the phosphatase on the indoxyl phosphate. The advantages of this method over previously described techniques are (1) use of Tween 20 allows the blot to be stained with Coomassie blue, (2) the substrates of the alkaline phosphatase reaction are stable for long periods of time, (3) the reaction products form an intense blue color which does not fade, (4) the resolution is extremely good with little to no band broadening, (5) the reaction is sensitive to picogram quantities of antigen, and (6) the reaction is quantitative.     

Immunoregulatory T-cell defects in B-cell chronic lymphocytic leukemia: cause or consequence of the disease? The contributory role of decreased availability of interleukin 2 (IL-2)

Several phenotypic and functional defects have been described within the residual T-lymphocyte population of patients with B-cell chronic lymphocytic leukemia (B-CLL), particularly in those in the more advanced stages of the disease. In this study, we review these abnormalities and discuss their possible effects on the course of the illness. Particular emphasis is devoted to the role of interleukin 2 (IL-2) in B-CLL. Evidence is provided that the IL-2 released by B-CLL T-lymphocytes may be utilized by the neoplastic B-cell clone that expresses the IL-2 receptor and that decreased availability of IL-2 may play a contributory role in some of the T-cell defects encountered in B-CLL.     

The isolation of spirochaetes from the rat caecum

Spirochaetes indigenous to the conventional rat caecum were cultivated on agar medium using selective isolation techniques. The 27 axenic isolates represented four morphological types of spirochaetes differing in cell size, cell coiling pattern and number of protoplasmic flagella.     

Plasma catecholamines and erythrocyte swelling following capture stress in a marine teleost fish

Plasma concentrations of adrenaline and noradrenaline were measured at rest from cannulated fish and following net capture. Adrenaline and noradrenaline concentrations in capture-stressed fish averaged 36,740 pmol l-1 and 38,860 pmol l-1 respectively, whereas resting values were less than 200 pmol l-1 for both amines. Erythrocyte swelling and raised blood lactate were evident in stressed fish. In vitro effects of 5 mmol l-1 adrenaline on erythrocyte suspensions suggested that the catecholamine had a direct effect on erythrocyte volume. The significance of these results is discussed in relation to the oxygen transport properties of the blood.     

The imprecision of the ratio of two percentages observed in differential white blood cell counts: a warning

Ratios of two percentages observed in differential counts of leukocytes or lymphocytes are very imprecise if they are based on relatively small numbers of cells. This is shown by the length of the 95% confidence intervals for such ratios. A formula is given for computing the limits of such intervals. They should accompany each observed value for a ratio.     

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Assembly of a functional F1 of the proton-translocating ATPase of Escherichia coli

Assembly of the F1 portion of the proton-translocating ATPase of Escherichia coli was examined in vivo. Analysis of strains lacking genes which specify the Fo polypeptides a, b, and c showed that the F1 subunits were able to assemble into a complex in the absence of the Fo subunits. In addition we have investigated the effects of mutations in the individual genes which specify the F1 polypeptides on the assembly process. Mutations of the uncA(alpha), uncG(gamma), or uncD(beta) genes result in a defective assembly of the F1 complex. In contrast, mutations in the uncH(delta) or uncC(epsilon) genes did not prevent assembly of the core alpha beta gamma complex. In these cases, however, the partial F1 complexes were incapable of restoring energy-linked functions to F1-depleted membranes.