Simultaneous Identification of Antibodies toBrucella abortus and Staphylococcus aureus in Milk Samples by Flow Cytometry

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureusantibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortusor S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-μm-diameter latex particles. The difference in size between B. abortus and S. aureuspermits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.

Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies.

The indirect immunodetection method is powerful in detecting antigens in situ, but to date mouse monoclonal antibodies (MAbs) could not be used in immunohistochemical studies of murine tissues without severe background staining. We report here a modification of this method in which mouse MAbs are used to detect murine antigens in cryosections. Before application to the section, mouse MAbs and conjugated anti-mouse antiserum were allowed to complex in vitro. After blocking of the unbound secondary antiserum with normal mouse serum, standard immunohistochemistry was performed. Fifty percent of a randomly chosen panel of over 40 mouse MAbs recognized their antigens in our model system. Adaptation of, for example, the fixation protocol can probably even increase this number. An MAb to the intermediate filament protein desmin, staining both smooth and striated muscle, was used to demonstrate this technique in cryosections of 15-day-old mouse embryos. In contrast to standard immunohistochemistry with the same antibodies under the same conditions, background staining was completely absent with this technique. With this modification to the well-established indirect detection method, the usefulness of mouse MAbs is significantly increased.

A simple method for immunofluorescent double staining with primary antisera from the same species.

We have developed a new double immunofluorescence technique by which two neuroactive substances in the same tissue section can be labeled with primary antisera raised in the same species. The optic lobes of the locust Schistocerca gregaria were used as a model system to develop the staining procedure. FMRFamide-immunoreactive neurons were detected by rabbit antisera against FMRFamide and FITC-conjugated secondary antibodies. Antibodies against the second peptide, pigment-dispersing hormone (PDH), also raised in rabbit, were biotinylated and detected via streptavidin-Texas Red. Crossreactivity of the PDH immunoglobulins with the FITC-conjugated secondary antiserum was prevented by pre-incubation with rabbit gamma globulins. The two peptide immunoreactivities could be conveniently observed on the same section with the different fluorescent markers. This double labeling technique with modified antibodies is easily performed and highly useful for co-localization studies with antisera raised in the same species.

Simultaneous characterization of efferent and afferent connectivity, neuroactive substances, and morphology of neurons.

We present a method for establishing in a single experiment four characteristics of individual neurons: the efferent and afferent connectivity, the morphology, and the content of a particular neuroactive substance. The connectivity of the neurons is determined by retrograde fluorescent tracing with Fast Blue and anterograde tracing with the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). After fixation, the brain is cut into 300-micron thick slices. Neurons containing retrogradely transported Fast Blue are intracellularly injected with the fluorescent dye Lucifer Yellow to fill their dendritic trees. The slices are then resectioned at 20-40 microns. One section through the soma of a Lucifer Yellow-filled neuron is selected for the detection of a neuroactive substance contained by this cell [immunofluorescence, secondary antiserum conjugated to tetramethylrhodamine (TRITC)]. Using appropriate filtering, it can be determined in the fluorescence microscope whether a Lucifer Yellow-containing cell body has also been labeled with TRITC, i.e., whether it is immunoreactive for this neuroactive substance. The adjacent sections are subjected to dual peroxidase immunocytochemistry with different chromogens to visualize the PHA-L-labeled afferent fibers (nickel-enhanced diaminobenzidine, blue-black reaction product) and to stabilize the Lucifer Yellow (diaminobenzidine, brown reaction product) in the dendrites of the intracellular injected cells. The other sections are used for electron microscopic visualization of the transported PHA-L. The relationships between the PHA-L-labeled afferent fibers (blue color) and the dendrites of the intracellularly Lucifer Yellow-injected, retrogradely Fast Blue-labeled cells (brown color) are studied by light microscopy. The electron microscope supplies ultrastructural data on the PHA-L-labeled axon terminals.

Three-color immunofluorescence histochemistry allowing triple labeling within a single section.

We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances within the same section and were then reacted with three appropriate species-specific antisera conjugated with fluorescein, rhodamine/Texas red, or biotin. The biotinylated secondary antiserum was subsequently reacted with diethylaminocoumarin (DAMC) conjugated to avidin. This combination resulted in green, red, and blue fluorescent labeling of each antigen, respectively. Each fluorescent marker was viewed and photographed discretely using appropriate excitation and suppression filter combinations. The method is well suited for analyzing instances of multiple coexistence at both the level of the cell soma and within terminal regions. More broadly, the feasibility of three-color immunofluorescence histochemistry extends the range with which antigen localization can be used to investigate the morphological bases of relationships and interactions between immunohistochemically characterized neuronal elements.

Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens.

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.

Immunogold-silver staining: new method of immunostaining with enhanced sensitivity.

A new method for demonstrating antigens in paraffin sections of formol sublimate-fixed tissue is described that utilizes an "indirect" immunohistological technique employing immunoglobulin adsorbed to colloidal gold as the secondary antiserum. The gold particles introduced to antigenic sites are revealed by a silver precipitation reaction. This technique, the immunogold-silver staining method, is of much enhanced sensitivity (up to 200-fold) as compared with standard immunoperoxidase and immunogold staining methods. The results have been confirmed in a study of immunoglobulins in reactive human tonsil. The use of this new method for double immunolabeling is also described.