The aim of my work was to develop high-throughput chemiluminescent immunoassays, working on the automated LIAISON® system. Two semi-quantitative immunoassays were created to diagnose bacterial infections caused by: 1- Clostridium difficile (detection of Toxin A&B in human stool specimens); 2- Chlamydia trachomatis (detection of anti C.trachomatis human IgG in human serum specimens). Clostridium difficile is a gram-positive, spore-forming anaerobic bacillus. It is a major cause of antibiotic-associated diarrhea and colitis. Antibiotic-associated colitis is an infection of the colon commonly found in individuals who have been using antibiotics. It is the most common acquired infection in hospitalized or extended care patients. Clostridium difficile manifests itself by the production of Toxins A&B. The LIAISON® Clostridium difficile Toxin A&B kit could be used as a screening assay in combination with other more specific assay. For the development of this assay 2 MoMAbs anti Toxin A and Toxin B were produced. These were used as solid phase (PMPs) and conjugate, in a 2 steps “sandwich” chemiluminescent immunoassay, in order to detect the 2 C.difficile Toxins. Some parameters of this new assay were analyzed in this work with the following result: high specificity (99.1%), sensitivity (97.7%) and Limit of Detection (200pg/ml). These values show the high performance of the kit, the Limit of Detection in particular is better than the reference kit of Meridian (PTAB). Nine different strains of toxigenic C. difficile (A-/B+ and A+/B+) were tested by spiking a negative stool with the bacteria. All these microorganisms react in the LIAISON® Clostridium difficile Toxins A&B assay, reflecting a positive result. The ability of stool components to cause interference in general was evaluated. Both the 39 organisms (spiked in negative and low positive stools) and 17 compounds normally present in the stool does not exhibited cross reactivity or interference problems. Finally the short time to results (30 minutes), and the high throughput (> 90 tests / h), together with all parameters see above met the necessary criteria for the marketing of this LIAISON® Clostridium difficile Toxin A&B kit. Chlamydia trachomatis is the most prevalent sexually transmitted bacteria worldwide. It is an obligate intracellular pathogen that can cause numerous disease states in both men and women. Both sexes can display urethritis, proctitis, trachoma, and infertility. The bacterium can cause prostatitis and epididymitis in men. In women, cervicitis, pelvic inflammatory disease (PID), ectopic pregnancy, and acute or chronic pelvic pain are frequent complications. C. trachomatis is also an important neonatal pathogen, where it can lead to infections of the eye (trachoma) and pulmonary complications. The human-pathogenic strains of C. trachomatis have been subdivided into 15 serovars. The 4 peptides mixture (Ct A, Ct B, Ct C and Ct D), used in this thesis, derived from the C.trachomatis major outer membrane protein (MOMP) and are currently used by the C. trachomatis ELISA Savyion Kit. These peptides together are capable to specifically reacting with antibodies specific to any one of the 15 serovars. Several attempts have been tried in the aim to develop this assay: 4 coating concentrations of peptides on solid phase were evaluated with 2 different sample volumes, then 2 modified peptides (with cysteines alkylated) were used in order to improve the recognition by the antibodies (present in the C.trachomatis positive samples). The strategy that allowed the correct functioning of the C. trachomatis LIAISON® kit, has involved the use of biotinylated peptides. These were made to react simultaneously with the solid phase (streptavidin PMPs) and human anti C. trachomatis antibodies in the samples (sera). With the following step the IgG anti human IgG are added as a conjugate. The specificity calculated with this assay is 97.7% and the sensitivity is 99.1%; both these parameters largely satisfy the criteria that were indicated necessary for the marketing of this kit, so the goals were successfully achieved.

(2012). Development of high - throughput chemiluminescent immunoassay for the detection of clostridium difficile and chlamydia trachomatis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).

Development of high - throughput chemiluminescent immunoassay for the detection of clostridium difficile and chlamydia trachomatis

ROSSINI, CLARA
2012

Abstract

The aim of my work was to develop high-throughput chemiluminescent immunoassays, working on the automated LIAISON® system. Two semi-quantitative immunoassays were created to diagnose bacterial infections caused by: 1- Clostridium difficile (detection of Toxin A&B in human stool specimens); 2- Chlamydia trachomatis (detection of anti C.trachomatis human IgG in human serum specimens). Clostridium difficile is a gram-positive, spore-forming anaerobic bacillus. It is a major cause of antibiotic-associated diarrhea and colitis. Antibiotic-associated colitis is an infection of the colon commonly found in individuals who have been using antibiotics. It is the most common acquired infection in hospitalized or extended care patients. Clostridium difficile manifests itself by the production of Toxins A&B. The LIAISON® Clostridium difficile Toxin A&B kit could be used as a screening assay in combination with other more specific assay. For the development of this assay 2 MoMAbs anti Toxin A and Toxin B were produced. These were used as solid phase (PMPs) and conjugate, in a 2 steps “sandwich” chemiluminescent immunoassay, in order to detect the 2 C.difficile Toxins. Some parameters of this new assay were analyzed in this work with the following result: high specificity (99.1%), sensitivity (97.7%) and Limit of Detection (200pg/ml). These values show the high performance of the kit, the Limit of Detection in particular is better than the reference kit of Meridian (PTAB). Nine different strains of toxigenic C. difficile (A-/B+ and A+/B+) were tested by spiking a negative stool with the bacteria. All these microorganisms react in the LIAISON® Clostridium difficile Toxins A&B assay, reflecting a positive result. The ability of stool components to cause interference in general was evaluated. Both the 39 organisms (spiked in negative and low positive stools) and 17 compounds normally present in the stool does not exhibited cross reactivity or interference problems. Finally the short time to results (30 minutes), and the high throughput (> 90 tests / h), together with all parameters see above met the necessary criteria for the marketing of this LIAISON® Clostridium difficile Toxin A&B kit. Chlamydia trachomatis is the most prevalent sexually transmitted bacteria worldwide. It is an obligate intracellular pathogen that can cause numerous disease states in both men and women. Both sexes can display urethritis, proctitis, trachoma, and infertility. The bacterium can cause prostatitis and epididymitis in men. In women, cervicitis, pelvic inflammatory disease (PID), ectopic pregnancy, and acute or chronic pelvic pain are frequent complications. C. trachomatis is also an important neonatal pathogen, where it can lead to infections of the eye (trachoma) and pulmonary complications. The human-pathogenic strains of C. trachomatis have been subdivided into 15 serovars. The 4 peptides mixture (Ct A, Ct B, Ct C and Ct D), used in this thesis, derived from the C.trachomatis major outer membrane protein (MOMP) and are currently used by the C. trachomatis ELISA Savyion Kit. These peptides together are capable to specifically reacting with antibodies specific to any one of the 15 serovars. Several attempts have been tried in the aim to develop this assay: 4 coating concentrations of peptides on solid phase were evaluated with 2 different sample volumes, then 2 modified peptides (with cysteines alkylated) were used in order to improve the recognition by the antibodies (present in the C.trachomatis positive samples). The strategy that allowed the correct functioning of the C. trachomatis LIAISON® kit, has involved the use of biotinylated peptides. These were made to react simultaneously with the solid phase (streptavidin PMPs) and human anti C. trachomatis antibodies in the samples (sera). With the following step the IgG anti human IgG are added as a conjugate. The specificity calculated with this assay is 97.7% and the sensitivity is 99.1%; both these parameters largely satisfy the criteria that were indicated necessary for the marketing of this kit, so the goals were successfully achieved.
TORTORA, PAOLO
DAL CORSO, ANDREA
chemiluminescent immunoassays, Clostridium difficile, Chlamydia trachomatis
BIO/10 - BIOCHIMICA
English
12-gen-2012
Scuola di dottorato di Scienze
BIOLOGIA - 48R
24
2010/2011
open
(2012). Development of high - throughput chemiluminescent immunoassay for the detection of clostridium difficile and chlamydia trachomatis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/28034
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