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From monoplex to multiplex. Bacterial capsular polysaccharide antibody immuno assays

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From monoplex to multiplex. Bacterial capsular polysaccharide antibody immuno assays

Rechten: Alle rechten voorbehouden

Samenvatting

Some species of disease causing bacteria, such as Streptococcus pneumoniae (Pneumococci, Pn) and Heamophilus influenza type b (Hib), have a polysaccharide (PS) capsule to protect their selves against the human immune system. Immunity against these bacteria appeared to depend mainly on anti-capsular PS antibodies. Pneumococci have 90 different types of PS. Immunity needs to be obtained for each type. The worldwide accepted technique to do determine antibody concentrations is the ELISA (Enzyme Linked ImmunoSorbent Assay). This technique uses 96-wells plates coated with PnPS to bind antibodies and analyse the concentration by colouring with a substrate and determine optical density. Although this is an accurate technique, antibodies for each PS type need to be analyzed separately, which makes this technique time consuming and labour intensive.
A new technique to muliplex assays was developed, promising to be able to do 100 tests at once in a single well, instead of one test as in ELISA's. This is realized by conjugating analytes, in this case PnPS, to microsphere beads. The microspheres are internally dyed with a combination of two fluorescent dyes. By incubating these mircrospheres with a serum sample, antibodies from the serum will bind to the microspheres. Antibody concentrations are determined by binding of a phycoeritrin labled antibody and analyze the fluorescence intensities of microspheres and analytes on the Luminex or Bio-Plex Workstation. Hib capsular PS (PRP) and PnPS serotypes 4, 6B, 9V, 14, 18C, 19F, 23F and CWPS (cell wall PS) have been conjugated to microspheres. An anti-PS antibody assay was created and optimized.
Microspheres were tested on specificity, correlation with ELISA and inter and intra assay CV were determined. There was also looked whether the CWPS microspheres are capable of capturing the cross-reactive CWPS antibodies. This was the case. Results of experiments with immunized rabbit sera and inhibition experiments with 100 μg/ml PnPS showed that the microspheres are not completely serotype specific. Serotype 14 had a 17.3% cross-reaction when incubated with rabbit serum immunized with type 23. Serotypes 3, 6B, 9V and 14 had a high inhibition percentage (37-41%) when they were incubated with serum from rabbits immunized with serotype 4.
Correlation of the MIA (Multiplex Immuno Assay) and ELISA techniques is not sufficient yet, shown by a correlation (R2) of 0.567 with a P-value of 0.133 for 11 calibration sera [Goldblatt, 2000]. A number of 78 samples pre and post vaccination gave a slightly lower correlation; 0.482 with significant P-values (ranking from <0.001 to 0.030 per serotype). The mean inter assay CV was 9.8% ranking from 6.2 (type 14) to 19.6 (type 23), indicating reproducibility. The mean intra assay CV of 46.3% show that results are non-reproducible within an assay. Still this multiplexing technique is very promising; smaller sample volumes are required, speed is increased, larger dynamic ranges and equivalent or increased sensitivity (better reaction kinetics) are obtained. The protocol needs to be optimized more and more PnPS need to be conjugated to microspheres. There also needs to be looked more closely to the conjugation method and (intra) reproducibility of the assay.

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OrganisatieHZ University of Applied Sciences
OpleidingChemie
AfdelingAcademie voor Technologie & Innovatie
PartnersWilhelmina Kinder Ziekenhuis Utrecht, Afd. Pedriatische Immunologie, UMC Utrecht
Datum2006-06-29
TypeBachelor
TaalEngels

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