StaphGold™ ELISA Test
Sampling and Testing
How should samples be stored?
Fresh, preserved or frozen samples can be used. Clotted samples (due to extended storage at ambient temperature) may be used but clots should be removed before samples are diluted, as clots may cause pipetting errors. Over time, sample exposure to proteolytic conditions will degrade the antibodies, and therefore samples should be frozen for long-term storage.
Can diluted samples be stored?
Yes, dilutions can be stored frozen. Make sure to thoroughly mix the sample by repeated pipetting before re-testing. A slight decline in antibodies may be occasionally observed.
Can milk samples from sheep and goats be tested?
The test has not yet been evaluated for goat and sheep milk samples, so at this stage, the use for goat and sheep milk samples is not recommended. There is, however, early indications that the test will work on milk from these other species.
How can transmission be reduced when a milking robot is used?
A system that enables sanitation of cups after each cow is milked can be used instead of a milking order. Establishing a ‘smart’ milking order or a ‘smart’ sanitation cycle where extra cleaning actions are put in place after milking of S. aureus-positive cows might also be possible.
How does the StaphGold™ ELISA handle sample carryover during routine milk recording or herd testing?
There are typically two sources of sample carryover (from one cow to the next) in a routine milk recording or herd testing operation:
- Milk meter carryover on-farm
- Sample needle carryover at the milk testing laboratory
Both carryover sources present the possibility of pathogens in the first sample being transferred (or ‘carried over’) into the sample of the next animal. For direct pathogen detection methods such as microbial culture or PCR (where specific bacterial DNA are amplified and detected), this presents an opportunity for false-positive results.
The StaphGold™ ELISA test procedure involves a ten-fold dilution step during sample preparation. Should milk from a cow with high antibody levels be carried over, the subsequent sample will first be greatly diluted (approximately 100-fold) by the next milk sample, then diluted ten-fold during test sample preparation. Any remaining antibodies will therefore come in well under the limit of detection and thus generate a ‘Negative’ ELISA result for S. aureus. If the second animal has a somatic cell count below 250,000/mL, it would not be a candidate for StaphGold™ ELISA testing, in any case.
NB: Standard practice in a lab is to change pipette tips between samples.
Does the test work with the different S. aureus genotypes?
Yes, very likely, although Koru has not looked specifically at antibody responses to certain genotypes. But we know that we can detect the antibody response to different S. aureus phenotypes that occur around New Zealand.
For example, the StaphGold™ ELISA detects antibodies to typical coagulase-positive, haemolytic S. aureus strains, but it also detects antibodies to coagulase-positive or coagulase-negative non-haemolytic S. aureus strains.
As we can detect antibodies to a range of different phenotypes, we believe that it is likely that we can detect antibodies to different S. aureus genotypes as well.
How long do the antibodies remain after the infection has cured?
This depends on the stage of the lactation when the cure occurs. If the infection cures during the dry period, specific antibodies will not be detected, or detection will be below the test cut-off during the next lactation, except for the very early stage of the lactation. Similarly, if the infection clears during the lactation and inflammation has subsided (as measured by SCC returning to normal levels), S. aureus-specific antibodies are not normally detected during the next herd test in four weeks.
What should I do with suspect positive cases?
Suspect positive cases should be retested during the next herd test. If the re-test is negative, the animal can be considered negative. Conversely, a positive result indicates an infected animal.
In the meantime, suspect positive cows should be milked after the negative cows but before the confirmed ELISA-positive cows, to reduce the spread of infection.
Can I select animals for testing based on previous SCC data?
Yes, because in many cases, an infection with S. aureus is already chronic, or is in the process of becoming chronic. In these animals, antibodies can be detected in milk, even though the elevation of the SCC was determined weeks earlier. In the case that the infection has cured in the meantime and SCC have returned to normal, the test will return a negative result in any case.
Laboratory Operations and Test Validation
Is any special equipment required to perform the assay?
No, only standard ELISA equipment is required.
An automated plate reader with a generic software that reads at A450 and A650 wavelengths is required.
Data analysis can easily be performed in Excel using the template provided on the Downloads page.
Plates can be washed manually (using bath or pipette techniques) or with an automated plate washer, for which the program should be carefully optimised.
The use of electronic or manual multi-channel pipettes is highly recommended.
For more information, please see the Materials and Equipment document on the Downloads page.
How should the plates be washed?
Hand washing procedures generally outperform automated plate washers. Therefore, plate washer settings should be carefully optimised. Overall, gentle plate washing settings may cause less variation.
Can different samples produce a different intra- and inter-plate variation (CVs)?
Yes, an important part of the overall variation is caused by the interaction of the assay with the sample. Different samples may produce slightly different CVs, depending on the nature of the sample.
How can subsampling logistics work in high throughput milk recording labs where samples are currently discarded after compositional analysis has been performed?
For especially high throughput labs, high-SCC cows can be pre-identified from the previous month, then subsampled (0.2 – 1.8mL) for testing before the milk recording samples are tested as normal for the current month. These subsamples would be chilled overnight before testing using the StaphGold™ ELISA the next day and reporting to farmers and their vets shortly afterwards. Note that given the chronicity of S. aureus infections, the urgency of S. aureus reporting is not as great, so a one-day delay in reporting is fine.
Are there any published case studies?
Not yet, however, a farm trial involving five farms and approximately 5000 cows in total is underway and publications are expected in the first half of 2022.
Are there any science publications?
Koru Diagnostics has not yet published any scientific articles, except for an abstract at the 2019 IDF Mastitis Conference in Copenhagen, Denmark. A number of publications are expected in 2022.