In this course about Lyme disease. Let us now focus on the diagnosis. The clinical diagnosis of Lyme disease usually starts with the clinical history, a history of vacations, outdoor activities use of tense exposure to wounded, bushy and grassy areas. It also has to do with clinical signs such as rash, arthritis, Bell's palsy and clinical symptoms, such as each pain and numbness. Now, the laboratory diagnosis of Lyme disease has to do with spiracle antigens and the antibodies created against the spiral key and its antigens. According to the CDC guidelines, the laboratory diagnosis of Lyme disease is made by a two step process.

The first step has to do with Eliza or an IFA test. Eliza stands for ends linked immunosorbent assay an IFA stands for indirect immunofluorescence assay. If any one of these is negative, that result is final that means it is not Lyme disease, or it could be and that an alternative diagnosis is to be looked at such as other illness not related to Lyme disease. The final possibility is that you would have to wait for the convalescence sera that has to do with timing because it may take a few weeks for that to come up. convalescence, Sarah Sara is a server that has been obtained from a patient that has recovered already from the infectious disease and is considered special reaching antibodies against that infection stage is not the result of a license or if it is positive or even if it is a cubicle that is doubtful.

One needs to do a second step that is Western blot also known as even a blocked Western blot is an immuno block used to detect and analyze proteins. Shown here is a representation by CDC of what I had just explained about the results being positive or negative. And it has details in terms of the timing of the symptoms and in terms of the types of immunoglobulins that is it M and RG to be detected through the western blot Eliza shown below is an Eliza test result in a 98 Well, acid plate for Lyme disease. That test is based on the principle that if a sparrow fetal antigen is quoted at the bottom of And therefore, immobilize the antigen can then be conjugated with the patient's own antibody. And then a substrate can be linked to the antibody and this link creates a color change and the signal can be detected and quantified in a machine for the presence of the patient's antibodies.

Now, a blank are a negative control with no antibodies, but just Lyme disease antigens and positive control with manufacture antibodies are known antibodies already present and therefore, at negative control that is no substrate can be used as reference points. So he is shown are several tests, some being positive and others being negative. In the next set of slides I'll explain in detail in each of these tests. If he in an IFA, our indirect immunofluorescence the fluorescent dye, shown here in green is used to detect the spiral cattle antibodies. It that means it has a principle that is similar to an Eliza and I affair can be done in life and multilevel therapies. The Western blot technique This procedure is used to detect borrelia proteins from patients blood samples shown on yellow What is the procedure for Western blot?

Western blot starts with an SDS page agarose gel in which proteins are loaded and then separated as it moves from the negative electrode to the positive electrode. The proteins in the gel are then transferred on to a pvdf membrane, shown here in blue. The membrane is then blocked with neutral proteins such as BSA or Milkha Singh. The membrane is then incubated with antibodies against the specific target in this case, spiral keto protein Next, the membrane is incubated with antibodies against the primary antibody that are labeled with h r p enzyme. Finally, the membrane is incubated with a common luminescent HRP substrate that is then exposed to an extra film in order for us to detect the signal as shown here and here was a marker showing the different sizes of the proteins in Killa Dalton's Dr. Wright is an example of the Western blot test results showing several borrelia proteins that can be detected through this technique including of course, our Pay us B, C and D. All those are surface.

How to surface lipoproteins. Of course our main focus throughout this course was asked B and C because of their importance in the transmission between the tick and the host of the borrelia during transmission for Lyme disease. The numbers shown here represent the sizes of the proteins in kilo daltons. This concludes the laboratory diagnostic aspect of Lyme disease.