Borrelia Spirochete Morphology in Peripheral Blood part 5

Supporting Evidence from Fluorescent Antibody Staining Experiments

Further evidence demonstrates that borrelia morphological forms could be detected in 2 of the donors who provided a fingertip blood drop and 1 donor who provided a venous sample (Lavender-top EDTA) for long-term BSK culture. A sample was mixed with KPL's anti-borrelia spp. polyclonal FITC antibody (cat: 02-97-92 Link to product page) and/or Abcam's Anti-Borrelia burgdorferi garinii polyclonal FITC antibody (cat: ab20118 Link to product page).

A brief explanation of antibody staining

The antibodies used here were produced in host animals against dead spirochetes. The antibodies that their immune system generated against the infection were collected and refined. These are then conjugated (combined) with flourescein which glows when it absorbs certain light frequencies (colours). Fluorescein absorbs blue light but it emits green light. With correct filtering it is possible to view ONLY the fluorescence.

When an antibody finds a matching protien in a sample it locks onto it, behaving exactly as an antibody should. This also means that the combined fluorescein is also locked onto the matching target. If enough antibodies lock to a target then with fluorescence filtering the target will glow brightly.

There are 2 other types of fluorescence that need to be considered in fluorescence microscopy:
1. Background-fluorescence, which occurs because of imperfect filtration, thermal effects and the tendency for many substances to have a tiny amount of fluorescence - including blood plasma.
2. Auto-fluorescence, which occurs when cells have naturally occurring fluorescence at higher levels. This could potentially be misinterpreted as successful fluorescent staining. Auto-fluorescence is particularly notable with lymphocytes (white blood cells) and also occurs with platelets. Furthermore, Dr Lida Mattman considered borrelia to have auto-fluorescent properties.

For these reasons the experiments demonstrated below include control-slides. It will be noted that auto-fluorescence was consistently very low but this is probably due in part to mixing with distilled water to match the similar dilution of stains. When whole blood is not diluted the auto-fluorescence of cells can be considerably higher.

Examples of fluorescent antibody staining from one donor showing fluorescence filtered image on the left of each matched pair and normal darkfield image on the right.:

Please see these pages for the complete fluorescenct antibody microscopy results from 3 donors:

Borrelia Garinii FITC stain of culture FITC staining borrelia species
Borrelia Garinii FITC stain of blood2 FITC staining borrelia species
Borrelia Garinii FITC stain of blood3 FITC staining borrelia species
Borrelia Garinii FITC stain of blood4 FITC staining borrelia species
KPL's 'Bactrace' FITC stain of blood1 FITC staining borrelia species
KPL's 'Bactrace' FITC stain of blood2 FITC staining borrelia species
Borrelia burgdorferi and Borrelia garinii FITC combined FITC staining HIGHLY RECOMMENDED

Next: Discussion, Limitations, Conclusion and Acknowledgements

Navigate this article:
Borrelia Spirochete Morphology Introduction
Borrelia Spirochete Morphology Results
Borrelia Spirochete Morphology Extended Results
Borrelia Spirochete Morphology Supporting Evidence
Borrelia Spirochete Morphology Fluorescent Antibody Experiment
Borrelia Spirochete Morphology Discussion, Limitations, Conclusion and Acknowledgments
Borrelia Spirochete Morphology References

Return to Microscopy Navigation