Fasciola ELISA- Bulk tank milk assay:​

An ELISA to detect Fasciola hepatica (Liver fluke) infection in cattle.

 

Instructions for Use

 

Intended Use:

 

The Fasciola ELISA has been developed for rapid screening for fasciolosis in ruminants.  The assay detects anti-Fasciola antibodies in both sera and milk. 

 

Introduction:

 

Fasciolosis (liver fluke infection) is common in many temperate areas where the environment is suitable for the causative parasite, Fasciola hepatica, and its intermediate host (mud snails of the genus Lymnaea).  Infection can cause acute, sub-acute and chronic infection in sheep, with clinical signs ranging from sudden death through to anaemia, oedema, lethargy, poor wool growth and ill-thrift. 

 

 

 

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In cattle, the disease is more insidious with some cases presenting with anaemia and oedema but in others the lack of obvious clinical signs masks serious production losses.  A specific diagnosis of infection can be made on the basis of detection of F. hepatica eggs in faecal samples.  However, this method is not feasible during the prepatent phase (1-12 weeks post-infection).  In addition, faecal egg counts in cattle are often low or intermittent, thus resulting in failure to make a specific diagnosis. 

 

The Fasciola ELISA detects specific antibodies to F. hepatica in bovine serum or milk.

 

Principle:

 

The Fasciola ELISA is based on a recombinant mutant antigen of F. hepatica (Collins et al., 2004).  The antigen was produced in P. pastoris, the use of the recombinant antigen renders the test highly specific and highly sensitive for Fasciola infection.   On the 96-well ELISA plate, odd numbered columns, marked with a black dot, are coated with antigen, while even numbered columns are uncoated. Results are reported as the ratio of the sample to the positive control.

 

 

Test kit:

 

Each kit contains:

5 individually wrapped ELISA plates

50X wash buffer tablets

Sample dilution buffer (red)

Conjugate dilution buffer (green)

Positive control serum

Negative control serum

Conjugate, anti-ruminant IgG conjugated to horseradish peroxidase

Revelation solution (TMB)

Stop solution (1M H2SO4)

 

 

Storage: 

 

For optimal performance the kit should be stored at 4°C.

 

 

Instructions for use:

 

Serum is diluted on plate at a dilution of 1/20. 

 

For milk samples, centrifugation may be necessary to remove the fat layer.  Alternatively, samples can be stored overnight at 4C to allow fat layer to settle.  Milk can then be applied to the plate “neat” using 200 ml per well.

 

For serum controls:

 

  1. Dispense 190 ml of sample dilution buffer per well.

  2. Add 10 ml of sera to each well.

  3. Agitate plate gently to mix sample.

  4. Add milk samples to wells, 200 ml per well.

  5. Incubate plate at 37°C for 20 min.

  6. Wash plate three times in 1 x wash buffer (PBST), drum plate on tissue paper to remove excess wash solution. 

  7. Dilute antibody conjugate 1/100 (Per plate, add 100 ml of conjugate to 10 ml of conjugate dilution buffer) in conjugate dilution buffer and dispense 100 ml per well.

  8. Incubate plate at 37°C for 20 min.

  9. Wash plate as per step 6 above.

  10. Dispense 100 ml per well of revelation solution (TMB) and incubate at room temperature for 10 min.

  11. After 10 min, stop reaction by adding 100 ml/well of stop solution.

  12. Read at O.D. of 450 nm on plate reader.

 

 Recommended plate layout:

 

 

 

 

 

 

 

 

 

 

All samples should be duplicated in adjacent columns.

 

 

Interpretation of test results:

 

To calculate the corrected O.D. 450 nm values for a sample, subtract the O.D. 450 nm from the uncoated well (even numbered column) from the corresponding coated well (odd numbered column). 

 

The sample to positive ratio can be calculated as:

 

 [O.D. 450 nm sample/O.D. 450 nm positive control]*100. 

 

 

 As with any diagnostic assay, results from the ELISA should be interpreted with consideration of the clinical, epidemiological and other laboratory findings.

 

 

References:

 

Collins P.R. Stack, C.M., O’Neill S.M. Doyle, S., Ryan T., Brennan, G.P., Mousley, A., Stewart, M., Maule, A.G., Dalton, J.P.  and Donnelly, S. (2004).  Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence.  J. Biol. Chem. 279:17038-17046.