Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens

Abstract

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured.

Bead-based immunoassays using a micro-chip flow cytometer

“…Polymeric micro-particles have been used as solid supports in a range of surface binding assays for many years, and examples of bead-based assays include: immunoassays, 1 DNA hybridisation, 2 protein-protein binding 3 and enzyme assays. 4 There are many practical reasons for preferring bead-platforms for assays over solution-based methods or indeed other types of solid supports (e.g. protein microarrays): they provide an increased surface to volume ratio, reducing the reagent volumes required; beads move freely in solution so that the reaction kinetics are faster; immobilisation of biomolecules on the particle surface localises the chemistry to a defined point facilitating rapid analysis; a wide range of surface functionalised beads are commercially available and large numbers of beads can be rapidly analysed by flow cytometry.…”

Επιδημιολογικη Διερευνησηεπιτηρηση Και Προληψη Της Βρουκελλωσης Σε Γεωργοκτηνοτροφικη Περιοχη Της Ελλαδας Με Την Βοηθεια Μηχανογραφημενης Χαρτογραφησης

“…Επιπλέον παρουσιάζει συγκριτικά πλεονεκτήματα από τις συμβατικές μεθόδους (ανίχνευση περισσοτέρων θετικών ορών, υψηλότερων τίτλων και ανίχνευση ανοσοσφαιρινών IgG, IgM και IgA). Η μέθοδος παρουσιάζει ακόμα διαφορετικά αποτελέσματα ανάλογα με το αντιγόνο που χρησιμοποιείται, τους αντιορούς και την ποιότητα της στερεάς φάσης που χρησιμοποιείται (Perera V.Y. et al, 1983;Krambovitis E. et al, 1992;Bardbuddhe S.B.,1994 C.N.…”