PBXL-1: a new fluorochrome applied to detection of proteins on membranes.

An easy, sensitive and direct fluorescent immunodetection method for proteins is described using the new fluorochrome PBXL-1 imaged with the FMBIO II Laser Scanning Imaging System. PBXL-1 is derived from a protein supra-molecular complex that contains a large number of chromophores. This complex, the phycobilisome, is extracted from a red alga then chemically stabilized to allow its use in specific binding assays. PBXL-1 was cross-linked to goat anti-rabbit IgG or streptavidin with heterobifunctional cross-linkers. The detection limit of PBXL-1 was determined by applying it on nitrocellulose membranes then imaging the membrane using an ytterbium aluminum garnet (YAG) laser. Evaluation of PBXL-1 sensitivity in a specific binding assay was tested on streptavidin/biotin and an antibody system. PBXL-1 provides high sensitivity in direct fluorescent applications due to a physical amplification of signal (i.e., a large number of fluorophores per binding event). PBXL-1 provides a linear response over two orders of magnitude while providing sub-amol sensitivity, indicating broad applicability for detection of a variety of targets. To our knowledge, this is the most sensitive direct fluorescent detection method available.

Use of MagniSep chromium dioxide particles in an immunoassay system.

The use of chromium dioxide particles as a solid support for very sensitive and rapid immunoassays, is the result of the combination of large surface area (40 m2) and high protein uptake capacity (40 mg/g) allowing rapid capture kinetics and high binding capacity. Magnetic and physical properties of these particles give a rapid separation, a complete resuspension and a rapid high-efficiency washing, highly desirable characteristics for efficient automation of immunoassays. Good precision and accuracy, exemplified by excellent recovery, parallelism and correlation were demonstrated. Test results prove that the technology is highly flexible and applicable to a variety of assay formats.

Problems in the serodiagnosis of canine brucellosis: dog responses to cell wall and internal antigens of Brucella canis.

Three procedures are commonly used for the serodiagnosis of B. canis infection: 1) The rapid slide agglutination test (RSAT), 2) the tube agglutination test (TAT), and 3) the modified 2-mercaptoethanol tube agglutination test (2ME-TAT). Hemocultures are always essential for diagnosis. The RSAT was developed to provide a presumptive diagnosis rapidly. It has been found accurate in identifying non-infected dogs; however false positive reactions are common due to shared determinants between the surface antigens of B. canis and certain other gram-negative bacteria. The RSAT has recently been modified to include brief reaction of test sera with 2ME (0.2M) prior to adding test antigen. The modification has improved specificity, but it has not eliminated false positive reactions. The TAT and 2ME-TAT are widely used. Although there is good agreement between tests, both suffer from lack of specificity but are valuable in kennels where B. canis has been identified by blood cultures. Agarose gel immunodiffusion (AGID) tests using extracted (SDC or hot PBS) cell wall antigenic complexes reveal precipitins in the sera of infected dogs usually 8 to 12 weeks postinfection (PI) that may persist 5 years. The antigenic complexes consist of at least 3 antigens; one (antigen '2R') appears to possess high B. canis specificity. Sera from noninfected dogs also may react nonspecifically in AGID tests that employ crude SDC or PBS antigenic extracts leading to 'false positive' interpretations. Cytoplasmic antigens gave up to four precipitin lines with sera from B. canis infected dogs. The antigens (protein or glycoprotein) were present in both S and R Brucella cells, but not in other gram-negative organisms examined. AGID tests that employed cytoplasmic antigens revealed precipitins against one or more (usually 2-3) antigens from PI months 4 through 64. In some dogs, precipitins were present 12 months after the bacteremia had ceased, a time when other tests were diagnostically insignificant, or equivocal. No 'false positive' field sera reached with the cytoplasmic antigens.

Biological properties and dog response to a variant (M-) strain of Brucella canis.

Wild type (virulent) B. canis organisms are always rough and growth becomes extremely mucoid (M+) after several days of incubation, especially on media pH 7.2. The mucoid growth of virulent (M+) B. canis and the marked tendency for M+ bacteria, and extracted cell wall antigens, to aggregate at acid pH (pH less than 6.5) are markedly diminished in a less mucoid (M-) variant. The M+ and M- Brucella, and their extracted cell wall antigens, also differ in hydrophobic properties; the M+ organisms (and antigens) being the more hydrophobic. Immunoelectrophoresis and immunodiffusion analysis also revealed differences between the two strains. Prolonged survival (greater than 1 year) of B. canis in host tissues, with the production of abortions or epididymitis/orchitis is a principal expression of B. canis virulence. Pathogenicity studies in Beagles have indicated that the M- variant is reduced in virulence, since dogs given high doses of the M- strain by the subcutaneous or conjunctival/oral routes remained asymptomatic. Abortions did not occur in experimentally infected pregnant bitches and there was no epididymitis/orchitis in inoculated males. Nevertheless, M- bacteria persisted in the spleen, prostate tissue of some males, and in the lymph nodes for 3 to 9 postinoculation months. Serological responses of dogs to the M-organisms were generally weaker and less enduring than in dogs infected with the M+ Brucella. Sera from dogs infected with the M- strain generally reacted weakly in agglutination tests, especially when the antigen was B. ovis. The minimum infectious dose of M + B. canis was at least five times less than that of the M - Brucella (conjunctival or subcutaneous routes).(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of cell wall antigens of virulent Brucella canis and a less mucoid variant of reduced pathogenicity.

Properties of cell wall antigenic extracts of 2 Brucella canis strains (wild type, M+; and a less mucoid variant, M-) were compared. Cell wall antigens of each strain were extracted by 2 different methods and compared with respect to their serologic, chromatographic, and surface properties. In addition, selected enzymes were used to compare antigen sensitivity to degradation. Analysis of extracted antigens was done by gel immunodiffusion and immunoelectrophoresis. Two cell wall antigens (2-R and 3-R) were present in extracts of wild type (M+) B canis that had been treated with hot (65 C) phosphate-buffered saline solution (PBSS). Three cell wall antigens (2-S, 3A-S, 3B-S) were revealed in saline extracts of the M- variant. Hot (100 C) 1% sodium deoxycholate extracted an additional antigen (1-R or 1-S) from cells (M+ or M-, respectively) which had previously been treated with hot PBSS. Certain properties of the B canis antigen extracts were examined by ion exchange chromatography, agarose gel chromatography, affinity chromatography, and enzyme degradation. Hydrophobic properties of the cell wall antigens were examined by their behavior in divalent cation, polyethylene glycol, and acid solutions. The antigens of B canis (M+) cells extracted with hot PBSS were smaller in size, had a lower net negative charge, and were more hydrophobic than those of the M- cells.

Properties of Brucella canis surface antigens associated with colonial mucoidiness.

Rough Brucella strains B. abortus (45/20), B. ovis (1182), wild type B. canis (M+) and a less mucoid (M-) variant possessed cell wall antigens that were extracted by both sodium desoxycholate (SDC) and hot phosphate buffered saline (PBS). The acid precipitability of cell wall surface antigens extracted in SDC from all four strains suggests that these antigenic complexes may be responsible for the bacterial aggregation in broth media at acid pH and for the relatively high colonial viscosity on normal agar media (pH 6.8). The antigens of B. canis (M+) extracted in PBS were serologically related to those from the other 3 strains, but they differed in acid precipitation and hydrophobic characteristics. Differences between the properties of B. canis surface antigens and those of the other rough Brucella may explain the highly mucoid nature of the canine organism when grown on conventional (pH 6.8) media.