Madison Area Lyme Support Group

INTRODUCTION

Antibody staining can detect specific proteins when a sample contains matching antigens. This experiment used polyclonal antibodies to borrelia burgdorferi targeting at least 5 different proteins. The antibodies used are proprietary off-the-shelf products conjugated with the popular FITC fluorescent molecule and sold by KPL and Abcam. When FITC is excited with blue light it fluoresces and emits green light. With the correct filtering, it is possible to observe only subjects which are fluorescing at specific wavelengths including agents to which the antibodies have bound.

SAMPLE DONORS

All seven UK adult blood sample donors were chronically and long-term ill and six had previously been diagnosed with Myalgic Encephalomyelitis (M.E.). All had symptoms which correspond with Lyme disease.

METHOD

Donors provided 4mls of venous blood in lavender-top vacutainer tubes with EDTA. The blood collection tubes were kept upright for several hours to allow the cells to settle and achieve a degree of cell layering. Gentle centrifuging for 20 minutes at ~20 to 40g’s further compacted and layered the cells. Most of the plasma was drawn-off with a pipette without disturbing the cell layers. 1.5 to 2mls of BSK was gently added to the tubes, minimising disturbance of the cell layers. The tubes were kept at room temperature or incubated at 30°C. Incubation appears to speed the development of a biofilm. After 3 to 7 days all samples developed a bound cluster at the top of the cell layers. This was associated with a cohesive fluid more viscous than the culture medium and which appeared clear to the naked eye. Under the microscope it was slightly milky. The cluster and its associated slime were obtained by removing most of the BSK with a pipette. The cluster could then be ‘grasped’ with a 3ml disposable pipette and with the tube tilted to ~45° or more – was carefully dragged up the side of the tube and deposited on a slide. Sometimes it was easier to grasp the cluster with tweezers, but these have to be very narrow to reach into the tube. Each sample was usually sufficient to prepare 3 – 4 slides. To each slide a drop of FITC conjugated anti-borrelia burgdorferi conjugated antibody, pre-diluted at 10 or 20 to 1, was added and gently mixed.

RESULTS

The results are presented as pairs of micrographs. The top photo in each matched pair, show a normal darkfield image with only the fluorescence emission filter in place. The bottom photo of the pair shows the same field of view with the fluorescence excitation filter installed. In some images refocusing may have slightly offset some subjects. The images can be seen here:  https://www.flickr.com/photos/76898309@N08/albums/72157713385691712

DISCUSSION

The experiment requires replication which includes healthy controls which I will not conduct.

It is notable that in experiments with freshly drawn or stored whole blood, minimal signs of staining with anti-B.b antibodies occurs. After simple culture with BSK, some white blood cells appear to contain granules that react and aside from these a few tiny agents are seen to have stained. It is only after culture with BSK using the method described above that spectacular staining occurred. The culture method layers white blood cells and probably a good number of platelets at the top of the sample. This is where the slime substance is found and where the clumped blood cells contain and/or are surrounded by antigenic material. This suggests the possibility that some white blood cells may be reactive. However, further experiments showed that with lymphocytes, eosinophils and basophils, only a small proportion contained granules within the cytoplasm that bound the antibodies.

Replication of the experiment with monoclonal antibodies could help to determine whether a cross-reaction is occurring, and potentially improve efficiency by identifying which particular B.b. proteins are being expressed, if any.

All donors in the current experiment had been ill and symptomatic for many years, mostly for over a decade. If replication as suggested proved the method for identifying infection with B.b., further experiments with shorter term infections would be required to identify within what time range a 4ml blood sample would be reactive.

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For more:  https://madisonarealymesupportgroup.com/2018/09/27/spirochete-culture-microscopy-videos-see-whats-inside-you/

https://madisonarealymesupportgroup.com/2020/03/02/spirochetes-in-sputum-of-chronic-lyme-patient/

https://madisonarealymesupportgroup.com/2018/09/29/microscopy-of-spirochaete-biofilm/

https://madisonarealymesupportgroup.com/2019/02/27/advanced-imaging-found-bartonella-around-pic-line/