Determination of human transferrin concentrations in mouse models of neisserial infection.
Transferrin constitutes the major protein involved in the transport of iron from the sites of absorption to the sites of storage and utilization. Despite the high affinity of transferrin for iron, most bacterial pathogens, such as the human restricted Neisseria meningitidis, have developed iron acquisition mechanisms. Several animal models of bacterial infection that include the exogenous supply of human transferrin have been implemented, and tests using transgenic mouse models are underway. Here we describe an ELISA sandwich procedure based on two monoclonal antibodies with negligible cross-reactivity to murine transferrin, to estimate human transferrin concentrations in mouse sera. The assay can detect as little as 10 ng/ml of human transferrin with coefficients of variation ranging from 1.6% to 4.4% (intra-assay) and 3.8% to 5% (inter-assay). The recovery values range from 90% to 110% in the assay working range (25-400 ng/ml). Human transferrin concentrations estimated in sera from 41 human transferrin transgenic mice ranged from 2 to 14 microg/ml. Further estimations of human transferrin levels in mouse sera of a previously described mouse model of N. meningitidis were also carried out. The intraperitoneal injection of 8 mg of human transferrin achieved a sustained value of human transferrin in mouse sera in the range of 1-2mg/ml over the first 24h, indicating that bacteria reaching the blood stream during this time would be exposed to levels of hTf found in normal human serum.
Perera
Yasser
Y
Center for Genetic Engineering and Biotechnology, Havana, Cuba. yasser.perera@cigb.edu.cu
Cobas
Karen
K
Garrido
Yainelis
Y
Nazabal
Consuelo
C
Brown
Enma
E
Pajon
Rolando
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D015153:Blotting, Western; D003429:Cross Reactions / Q000276:immunology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D008585:Meningitis, Meningococcal / Q000097:blood*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009345:Neisseria meningitidis; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D014168:Transferrin / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014168:Transferrin