Serodiagnosis of listeriosis based upon detection of antibodies against recombinant truncated forms of listeriolysin O.
Amino-terminal fragments of listeriolysin O (LLO) of 240 and 411 residues (fragments LLO240 and LLO411, respectively) were expressed in Escherichia coli as fusion polypeptides with maltose-binding protein (MBP) with the aim of producing specific antigens for use in serological tests. In Western blots (immunoblots) with crude bacterial extracts of the fusion polypeptides, the reactivities of MBP-LLO240 and MBP-LLO411 with anti-LLO antibody (ALLO)- and anti-streptolysin O antibody (ASLO)-positive human sera were first compared with that of the entire LLO (LLO530) also fused to MBP (MBP-LLO530). Sixteen of 17 (94.1%) ALLO-positive samples reacting with MBP-LLO530 also reacted with MBP-LLO411, whereas this proportion dropped to 11 of 17 (64.7%) with MBP-LLO240. Alternatively, 18 of 19 (94.7%) ASLO-positive samples giving an interpretable result reacted with MBP-LLO530, whereas 1 of 19 (5.3%) of these samples reacted with MBP-LLO240 or MBP-LLO411. The fusion polypeptide MBP-LLO411 was purified by maltose affinity chromatography and was further evaluated as a diagnostic antigen in a Western blot assay. Twenty-one of 21 (100%) serum samples obtained from patients with listeriosis and found to be positive for ALLO by a reference dot blot test reacted with MBP-LLO411, whereas 1 of 20 (5%) ASLO-positive serum samples and 1 of 100 (1%) serum samples from healthy adults were reactive. Thus, a polypeptide limited to the 411 amino-terminal residues of LLO is a specific and sensitive antigen for the detection of ALLO.
Gholizadeh
Y
Y
Laboratoire de Microbiologie, Institut National de la Santé de la Recherche Médicale U 411, Faculté de Médecine Necker-Enfants Malades, Paris, France.
Poyart
C
C
Juvin
M
M
Beretti
J L
JL
Croizé
J
J
Berche
P
P
Gaillard
J L
JL
D000293:Adolescent; D000328:Adult; D000907:Antibodies, Bacterial / Q000097:blood*; D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods / Q000706:statistics & numerical data; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008089:Listeria monocytogenes / Q000235:genetics / Q000276:immunology*; D008088:Listeriosis / Q000175:diagnosis* / Q000276:immunology; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods* / Q000706:statistics & numerical data
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C038705:hlyA protein, Listeria monocytogenes