Neospora caninum ELISA
Immunoenzymatic assay for the detection of antibodies against Neospora caninum in bovine serum.
Neospora caninum (N. caninum) is an obligate intracellular protozoan parasite that has been confused previously with Toxoplasma gondii. The dog is the definitive host. Infection can be acquired by ingesting food and water contaminated with oocysts excreted in feces of dogs, by ingesting infected tissues, or transplacentally. Vertical transmission is a major route of transmission in cattle and dogs. In dairy cattle, N. caninum is a major cause of abortion in many countries. Calves may be aborted; stillborn; born underweight, weak, or paralyzed; or they may become paralyzed within 4 weeks of birth. Nonsuppurative encephalitis is the main lesion in aborted fetal tissues. Abortion can occur throughout gestation, and some cows may abort again. Diagnosis of N. caninum abortion consists in identifying the organism in tissue sections of aborted foetus by immunohistochemistry or PCR. A number of serological assays, such as ELISA, have been developed to detect N. caninum antibodies. They are useful to identify chronically infected cows which represent an important reservoir of the organism.
To detect the presence of antibodies against N. caninum in bovine serum, controls and prepared serum samples are incubated in wells coated with N. caninum antigen (Ag). The antibodies (Ab) specific to N. caninum present in positive serum samples will bind to the Ag in the wells. After several washes to eliminate unbound substances, a conjugate (an Ab coupled to an enzyme) targeted at bovine Ab is added. After incubation, the excess of this conjugate is eliminated by a second wash and its attachment is revealed with a chromogenous substrate. The conjugate, if present, reacts with the substrate and a green color develops. The reaction is then stopped and the optical densities are read. The intensity of the color allows the determination of the status of sample tested. A negative sample will show a weak reaction (pale green) whereas a strong positive will show a strong reaction (dark green). All shades of green between dark and pale represent various degrees of positivity.
Materials Provided
- 2 x12 strips of 8 wells coated with N. caninum Ag
- 1 x 2,5 ml Ready-to-use positive control
- 1 x 2,5 ml Ready-to-use negative control
- 1 x 125 μl Concentrated conjugate
- 2 x 100 ml Concentrated wash solution (10X) (Ready-to-use stop solution)
- 1 x 30 ml 1 x 30 ml Ready-to-use substrate
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Materials Required but not Provided:
- Purified water
- Adjustable single- and multi-channel pipettes
- Single-use pipette tips
- Test tubes for sample dilution
- ELISA 96-well microplate reader equipped with 405 nm filter
- Containers for preparation of solutions
