The phagocytosis of uniform fluorescent latex particles by pulmonary macrophages in the rat was analyzed by flow cytometric methods. The percentage of phagocytic macrophages and the number of particles per cell were determined from cell-size and fluorescence histograms. A comparison of in vivo and in vitro phagocytosis data showed that the percentage of phagocytic lavaged macrophages reflected the availability of instilled particles. With sodium azide used to model phagocytosis inhibition, it was shown that the percentage of phagocytic cells and the number of particles per cell can be determined simultaneously.

Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in greater than 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90 degrees light scatter, and blue and green autofluorescence signals. In Fc gamma receptor-mediated phagocytic studies, approximately 90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometric analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and beta-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody ED1, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical.(ABSTRACT TRUNCATED AT 400 WORDS)

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1to 3-h test time)4Of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.

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An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody "sandwich"-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.

The rapid, indirect enzyme-labeled antibody (ELA) microplate test has been developed as a diagnostic and surveillance tool to aid in the control of animal disease. The test has been applied to viral (hog cholera), parasitic (trichinosis), and bacterial (brucellosis) diseases of animals. A correlation of greater than 95% was observed between the hog cholera ELA test and the serum neutralization test for hog cholera in greater than 2,000 field samples obtained during the 1976 epizootic in New Jersey. Serum samples from all of 56 swine naturally infected with Trichinella spiralis at a level considered dangerous to humans were ELA-positive, whereas only one of 360 packinghouse sera negative for T. spiralis was ELA-positive. Preliminary experiments with bovine brucellosis (Brucella abortus) indicate that the ELA test is more sensitive than other test methods currently in use. ELA procedures should soon become tests of choice for the detection of antibodies to animal disease agents.

A gelatin sponge model system for tumor cell inoculation and retrieval of tumor-associated leukocytes is described. Gelatin sponges pre-implanted in nude mice harboring tumorigenic Chinese hamster ovary cells (line CHO) were examined at 2 and 11 days after injection of tumor cells for tumor cell content and leukocyte accumulation after digesting the sponge matrix in collagenase solution. The data indicate a progressive influx of host cells consisting primarily of macrophages, neutrophils and lymphocytes. The total number of viable tumor cells as well as the fraction of surviving tumor cells with clonogenic potential also increased with tumor age. Blank sponges not harboring tumor cells elicited an inflammatory response in the animals which did not change appreciably with length of sponge residence. However, when the sponges were harboring tumor cells, the accumulation of host leukocytes far exceeded that which occurred in blank sponges. This observation suggests a host response directed toward the tumor which is absent in animals bearing blank sponges. Apart from providing anchorage for injected cells, the gelatin sponge, by virtue of its digestibility in collagenase, makes possible the easy retrieval and precise quantitation of tumor-associated host cells.

We describe a gentamicin assay in which a peroxidase-gentamicin conjugate competes with gentamicin for binding to a gentamicin antibody adsorbed to a polystyrene solid phase. The assay can be completed in 30 min and requires 50 microliter of diluted serum. The precision and accuracy are equivalent to that of a radioimmunoassay technique and the reagents are stable for several months.