Please revise the washing procedure. The PBS solution should contain no detergents, as these will loosen the slide coating and cause the antigen to be washed off.

As part of our production process of Rickettsia and Bartonella, we completely remove all traces of the Vero cells where they are grown. The reason we remove the rest of the Vero cells is that a lot of times the cells react with the patient's serum, creating a fluorescence that can be misinterpreted as positives. By removing the Vero cells, we are eliminating the possibility of false positives, and this is actually an advantage of our slides over the other of commercial brands.

There are several reasons which can cause background on the IFA slides. All of them are in relation to the way of performing the technique, which should always follow the instructions for use given in the package insert. 1.- Prepare and store the wash solution according to the above mentioned instructions. 2.- Check the washing protocol. It is important to respect the washing times specified. Washing must be performed in agitation to ensure that all of the wells are washed uniformly. The slides must be rinsed in distilled water after each wash, because traces of salts from the wash solution can be a cause for background. 3.- Check incubation times (incubation for longer than specified can cause background also). 4.- Check that the wells have not dried during incubation. Especially if background affects only a few wells, it can be due to the fact that these wells dried up during incubation. It is advisable to use a humid chamber during this step.

Such a problem could indicate desiccation of the globulin. Ensure that the incubation of the 20ul of sample and globulin takes place in a humid chamber. Please check that the washing step is being performed correctly. Some samples can present prozone, however these are very rare cases and only happen in samples with very high titers.

Please consider the prevalence of the disease in the area. If you are finding many positive results at titers of 1/64 it may be possible that these titers correspond to past infection, and it may be necessary to perform titration for an accurate diagnosis.

The evaluaton of the immunological response to Chlamydia, such as measured in the MIF technique, must be carried out over the three Chlamydia species simultaneously. This is a way of controlling cross reactivity; the differential responses to the three Chlamydia species will render the final diagnosis. Therefore the kit may be used to detect responses to any of the species. The positive control is positive for the 3 Chlamydias.

Unfortunately, neither ELISA nor IFA are suitable tests to measure responses to vaccination. Both tests use whole antigens, and will not be able to discriminate responses to the vaccine components (hemagglutinin and neuaminidase) from antibodies against other proteins of the virus. Haemagglutination inhibition (HAI) and even better, neutralization test (NT) are the only assays suitable to measure responses to vaccination.


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