The EndoCAb® IgM ELISA has been developed for determination of endotoxin core antibodies in human plasma or serum in patients or healthy individuals. Several studies show a consistent drop in postoperative levels of circulating anti-endotoxin core antibodies compared to the preoperative value. This drop has been interpreted as consumption of antibodies to endotoxin by systemic release of endotoxin. A hypothesis is that if the patients pre-operative endotoxin-core level is low, even moderately low, patients may not be able to cope with the efflux of endotoxin, which may have mild to severe clinical consequences. The assay is of interest for various experimental conditions ranging from in vitro LPS neutralization by plasma components to in vivo studies on kinetics of antibodies to endotoxin in health and diseases.
The EndoCAb® standard median-units IgM (MU) are arbitrary and are based on medians of ranges for 1000 healthy adults in a particular locality. It has not been established whether normal endotoxin-core antibody values vary by region, culture or race. Users should establish appropriate local statistical controls for their studies. It is advised that studies of any patient group should always be controled by studies of appropriately selected contrasting clinical groups and/or healthy individuals recruited locally.
EndoCAb® is a registered trademark. Used under license from the Scottish National Blood Transfusion Service.
The EndoCAb® IgA ELISA kit is to be used for the in vitro quantitative determination of endotoxin-core antibodies in serum, plasma and cell culture supernatant.
The EndoCAb® IgA ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 2½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are incubated in microtiter wells coated with equimolar amounts of endotoxin rough-lipopolysaccharides from four Gram-negative bacterial species, each comprised of a complete inner core, but lacking complete outer core or O-specific polysaccharide chain.
Anti-endotoxin-core antibodies are captured by the solid phase antigen.
Peroxidase conjugated anti-human IgA antibody will bind to captured endotoxin-core antibodies.
The peroxidase conjugated antibody will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the EndoCAb® IgA standards (log).
The endotoxin-core concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.