[Detection of LOS-specific antibody-secreting cells by ELISPOT assay].
OBJECTIVE
To detect dynamically the response of specific antibody-secreting cells elicited by a detoxified-lipooligosaccharide-cross-reactive mutant (dLOS-CRM) of diphtheria toxin conjugate vaccine for Moraxella catarrhalis (M.cat).


METHODS
BALB/c mice were intranasally immunized with dLOS-CRM conjugate vaccine. The specific antibody-secreting cells responding to LOS of M. cat in different inductive and effective sites, including nasally associated lymphoid tissues (NALT), spleen, cervical lymph nodes (CLN), nasal passages (NP), lungs and Peyer's patches (PP) were detected by an enzyme-linked immunospot assay (ELISPOT). The levels of LOS-specific antibodies IgA, IgG and IgM in serum, nasal flush fluid, alveolar douche fluid, saliva and fecal extract were also detected by ELISA.


RESULTS
Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent enhancement of immune response. LOS-specific antibody (IgA,IgG or IgM)-secreting cells were found in NALT, spleens, CLN, NP, lungs and PP with most LOS-specific IgA antibody-secreting cells located in nasal passages, and next, NALT and lungs. It was correlated well with the level of LOS-specific IgA, IgG or IgM antibody titers in nasal flush fluid, alveolar douche fluid, saliva, serum and fecal extract.


CONCLUSIONS
dLOS-CRM can induce specific mucosal and systemic humoral immune response through intranasal immunization. ELISPOT assay is quick, sensitive, specific, and would be a very useful tool to analyze dynamically the mechanism of single antibody-secreting cell response.
Jiao
Xin-an
XA
College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China. xajiao@yahoo.com
Hirano
Takashi
T
Gu
Xin-xing
XX
D000281:Administration, Intranasal; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis; D000921:Antibody-Producing Cells / Q000276:immunology*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000276:immunology*; D004167:Diphtheria Toxin / Q000276:immunology*; D005260:Female; D007114:Immunization; D007070:Immunoglobulin A / Q000032:analysis; D008070:Lipopolysaccharides / Q000276:immunology*; D008168:Lung / Q000276:immunology; D008221:Lymphoid Tissue / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D001936:Moraxella catarrhalis / Q000276:immunology*; D009305:Nasopharynx / Q000276:immunology; D010581:Peyer's Patches / Q000276:immunology; D013154:Spleen / Q000276:immunology; D018074:Vaccines, Conjugate / Q000008:administration & dosage / Q000276:immunology
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D004167:Diphtheria Toxin; D007070:Immunoglobulin A; D008070:Lipopolysaccharides; D018074:Vaccines, Conjugate; C023023:lipid-linked oligosaccharides