Ref.CDVB018
96 tests/kit
E. COLI K99
Bovine
General Information
Diarrhea is a major cause of mortality in young cattle under six
months. Bovine neonatal gastroenteritis is a multiffactorial disease. It
can be caused by viri : coronavirus or rotavirus, by bacteria : Salmonella
or by protozoan microorganisms such as Cryptosporidium parvum. In calves
youger than 3 days, K99 positive enterotoxigenic E. Coli are frequently
isolated, particularly in colustrum-deprived calves or in calves which
received colustrum free of anti - K99 specific antibodies.
The diagnosis of the etiologic agent of diarrhea can only be performed in the laboratory because clinical signs do not allow to differentiate between the different microorganisms; It is possible to identify these agents by means of different culture and floating techniques including staining. However these techniques are labor intensive and unpracticable. These classic techniques have rapidly been replaced by the Elisa technology because of its simplicity and the limited requirements in laboratory equipment. The sensitivity and specificity of the Elisa technique for the detection of these pathogens is at least as good as that of the more classical techniques : results are very similar. The Elisa technique is rapid, reliable and is particularly suited to the analysis of important numbers of samples.
Principle of the test
The Cypress E Coli K99 test uses 96- well Microtitration plates sensitized by a monoclonal antibody specific for an antigenic determinant of E. Coli K99. This antibody allows specifically the capture of the corresponding pathogen which is present into the faces samples. Some rows have been sensitized with these antibodies and others are containing non specific biological material. These non - sensitised rows allow the differentiation between specific immunological reaction and non specific bindings. A large number of false positives are eliminated.
Faces are diluted in dilution buffer and incubated on the microplate for 1 hour at room temperature. After this first incubation step, the plate is washed, then the conjugate - peroxidase labeled anti-K99 specific monoclonal antibodies - are added to the wells. The plate is incubated for 1 hour at room temperature.
After a second incubation step of 1 hour at room temperature, the plate is washed again and the enzyme substrate (hydrogen peroxide) and the chromogen (tetramethyl benzidine TMB) are added. This chromogen has the advantage of being more sensitive than other peroxidase chromogens and not being carcinogenic.
If K99 positive E. Coli is present in the tested faeces, the conjugate remains bound to the corresponding microwells and the enzyme catalyses the transformation of the colorless chromogen into a pigmented compound. The intensity of the resulting blue color is proportional to the titer of K99 in the sample. Enzymatic reaction can be stopped by acidification and resulting optical density at 450 nm can be recorded using a photometer. The signals recorded for the negative control microwells are subtracted from the corresponding positive microwells.
The positive antigen supplied with the kit gives the reference value. From this value and a specific coefficient it is possible to determine a limit of positivity for K99.
Rev.09.95
