Scientific Validity

1. Scientific Validity • Section 3 of the LymeCryme/ActionLyme Powerpoint Series What is meant by “scientifically valid?”What are chromatography (Western Blotting), detection, specificity and quantification? What are the FDA criteria to validate an analytical method?Were these methods applied in “Lyme Disease” testing?

2. Let’s answer the last question first: • No.

3. I am going to torture you but it’s not going to be that bad: Chromatography or “Separations Science” • Chromatography literally means “color writing” and referred to the likes of the separation of plant pigments on a paper. The techniques were about simply separating out what was in a mix. Later came methods or attempts to quantify the junk in the mix.

4. Light/Prisms • Everybody knows about this kind of separation so I won’t go into it:

5. Let’s steal a graphic on plant pigments separations… • http://www.regentsprep.org/Regents/biology/units/laboratory/instrumentation.cfm

6. In the real world of science, we have newer ways to separate and quantify say like the plant pigments. • We use, usually, something like HPLC or High-Pressure Liquid Chromatography or High-Performance LC, or we gave Gas Chromatography, etc, but it’s all about, really, creating a molecular sieve, or a very, very small sieve that acts on the compounds in question at a molecular level. All of this relies on the properties of the compounds, and when they come out of the sieve, they’re usually hit with a light beam, which the compound absorbs in a way dependent upon the compound’s atomic/electron array.

7. Example of electron cloud/array • It may be hard to envision what all these big words mean, but let’s start anyway with a complex example: Don C. Wiley’s HLA molecules (white, clam-shell shape):

8. Shoot the crystallized compound with X-rays and compare to a database of previous shoots.

9. Molecules or compounds are what they do. (So what do they look like? is what you, the chemist, want to know.)

10. (Slide 10) X-Ray Diffraction Scatter PatternThis is what the sumbich looks like, but the requirement is that the compound is a crystal.

11. Problem with X-Ray diffraction to determine OspA’s structure (Refer to Section 2 of this series for the OspA/Pam3Cys explainer) • You can’t re-crystallize a lipid. Like, you can’t put butter in the freezer and then try to crack it along cleavage lines. It won’t freeze, and if it won’t freeze, you can’t get a true X-ray scatter pattern from the crystal. • That’s why there is no structure for OspA or Pam3Cys. As shown in Section 2 of this series on Pam3Cys, the best these scientists (Radolf, Weis) have been able to do is add on radio-labelled (H³) palmitate and then remove it, intact.

12. (Slide 12) Kapisch? You follow? • Don Wiley was able to extract and then recrystallize the HLA molecules to determine the kinetics (what are the electron interactions between 2 molecules) of a bound antigen. That’s how you were able to see the cloud pattern. The HLA crystal was shot with X-rays to get a scatter pattern, which was then compared to a library of knowns. But this can’t be done with OspA or mycobacterial lipopeptides because there are “lipo” or fatty acid or oily or lipid attachments (palmitoyl). So, no one really knows what the structure of OspA is. But worse, the Lyme crooks don’t even ask or try to explain it to us.

13. OspA

14. Separate stuff, make sure it’s only one compound, recrystallize it, blow it up, shoot it with X-rays, RNA/DNA sequence… • You get the idea. We are GONNA find out what stuff is, so we can tell what it’s going to do.

15. I’m starting at the end. Usually you start with the periodic table, but the goal is lost unless you see the big picture, first. Now, electrons are funny things. Unlike human Lyme activists, they like to do stuff in bunches or groups. And the groups have to be a particular size before they say, “This group is full. Move on.” • You’ve heard of orbitals and shells and blah, blah, blah, but the down and dirty is because of charges and atomic forces, the number of protons (positively charged “particles”) tends to dictate where the electrons go, and how many of them there are, and how tightly they like to hang out around the nucleus or the proton bunches.

16. Borings, Gayons, and Floozons • Now, there is no perfect atom. A perfect atom would be like the Noble Gases, but they actually are so perfect (electron shells are “satisfied” or don’t want to either add or get rid of extras), they don’t really do much. The rest of them tend to, well mate. Sodium goes with Chlorine (salt), etc. Carbon likes everybody but especially Hydrogen, on Earth, at least. Carbon’s Atomic Number is 6, which means it has 6 protons, and 2 electrons in the first “shell,” and 4 in the next. It will tend to share the 2 outer pairs of electrons. This makes it, well, the most Gay atom. Hydrogen is flighty and tends to go with anyone and everyone, so it’s kinda, you know, floozy.

17. You the chemist are going to detect the arrangements of the Gayons and Floozons. • There are some bonding tendencies - a wordier person would call them “properties” – and chemists like to categorize the general, naturally occurring tendencies (and also our human interactions with them) of the elements. In short, when you see a lot of “C, H, O, N, S, P,” it’s probably going to either smell bad or be a problem for humans or both. • A lot of times chemists like to stick them together in natural or arrangements similar to stuff we see in nature… and then run to the US Patent Office.

18. Corporate Floozons • And then the chemists who ran to the USPTO try to think up a disease for which they own the unique arrangement of the Cs, Os, Hs, Ns, etc. • But try to think of all the COHNPS products you know that are simply a mimic of nature, a direct copy of nature (“Pharming”), or a genuine aximadent: • Aspirin (Birch Bark, prostaglandin or inflammation inhibitor), Mold (penicillin), Insulin, Dyes or Insecticides (psychotropics are all mimics of microbial paralytic agents or dyes, like Methylene Blue), Peach Pit (alkaloids as chemotherapeutics), … (So, you can be unimpressed.)

19. Armageddon • So: You got the plain old regular Floozons and the Corporate Floozons and then the plain old regular chemists (PORCs) in the background who do the grunt chromatography work of determining whether the USPTO product actually is what it is – and is PURE – or free from contaminants before it is sold. • There is no unethical messy stuff. You, the chemist, are chugging along performing the separations science according to the FDA’s rules of determining certainty of the CONCENTRATION of the PURE product (dosage, milligrams, etc)… And then Yale comes along with this mystery compound called OspA.

20. (Slide 20) Ka-chug, ka-chug… Chromatograms http://www.statisticaldesigns.com/images/bd_02.gif

21. Western Blot Chromatographyhttp://alpha1.mpk.med.uni-muenchen.de/bak/nrz-borrelia/miq-lyme/Frame-MiQ-microbiological53.html

22. Turn it on it’s side

23. The peaks are the bands are the compounds separated out like the plant pigments…

24. You follow now? • In the real world of BigPharma, there is real chromatography and analytical chemistry, and there are rules to this work - rules produced by the FDA as “guidelines” (I know, now we all hate that word) - for how to VALIDATE a METHOD like a chromatography method for one purpose only: To prove your product is pure and that other scientists can repeat the method in their labs. • None of the rules were applied in Yale Lyme Psychosis Science.

25. So, what are the FDA rules? • Number One Rule:Have you shown that you are only detecting the analyte in question, and that two or more species don’t co-elute at the same time (this is proven with Diode Array or scanning over multiple wavelengths) . The “retention time” or the “approximate kilodalton molecular weight” (we call them, like, “band 41” in LymeCryme speak) tends to be the unique identifier of the peak or band in question. For example (next slide):

26. SPECIFIC identifiers for the SPECIFIC analyte in question

27. FDA’s definition of SPECIFICITY • FDA requires that your METHOD to detect something, only detects the SPECIFIC analyte in question, like decanoic acid or OspA antibodies and does not detect ANYTHING ELSE!!! at the same retention time. • Secondarily, and this is very important, an ANTIBODY itself has SPECIFICITY.

28. Disregard how crappily I drew this spirochete; Look at the SPECIFIC antigens. • They’re specific to the spirochete (to some degree), and you will make specific antibodies against them: • These dots are like OspA, B, C, …

29. The dots are the Osps, but they vary or change or mutate, so antibodies do no good. And vaccines do no good. What changes is the protein ends of the Pam3Cys OspA.

30. (Slide 30) SPECIFIC Borrelia Flagella • http://www.nature.com/nrmicro/journal/v3/n2/fig_tab/nrmicro1086_F1.html • Wavelength, number of flagellar filaments, amplitude, are characteristics of a certain spirochete’s internal flagella and that’s how we tell spirochetes apart from other spirochete or how they are ordered taxonomically.

31. Taxonomy database- look at how Borrelia are organized – (Part I)

32. Taxonomy database- look at how Borrelia are organized – (Part II) • Fukunaga M et al. (1996b) • Fukunaga, M., Okada, K., Nakao, M., Konishi, T., and Sato, Y. "Phylogenetic analysis of Borrelia species based on flagellin gene sequences and its application for molecular typing of Lyme disease borreliae." Int. J. Syst. Bacteriol. (1996) 46:898-905. • Ras NM et al. (1996) • Ras, N.M., Lascola, B., Postic, D., Cutler, S.J., Rodhain, F., Baranton, G., Raoult, D. "Phylogenesis of relapsing fever Borrelia spp." Int. J. Syst. Bacteriol. (1996) 46:859-865.

33. All Borrelia are Relapsing Fever Spirochetes and they’re known apart by their differences in Flagellin. • All Borrelia are Relapsing Fever Spirochetes and they’re known apart by their differences in Flagellin. • So, if they’re all known by their SPECIFIC DIFFERENCES in flagellin wavelength and number of filaments, then why don’t we look for SPECIFIC Borrelia antibodies, since almost everyone who has Lyme has band 41 or antibodies against flagellin?

34. “Lyme Disease” is a strawman • It turns out, Borrelia burgdorferi SPECIFIC flagellin antibodies are and have been identified, such that most cases of not only “Lyme” Relapsing Fever [defined by the presence of OspA or Pam3Cys (?)], but any cases of Borreliosis or Relapsing Fever are identifiable by the SPECIFIC flagellin method.

35. Why? • Because somebody grabbed and coded the DNA sequence that codes for all the flagellin that allowed the scientists to perform sequence similarities studies to see how closely related all these spirochetes are and THENCE, … Where do they go in the Taxonomy database?

36. How? • So, has this been done? • Has someone ripped and de-ripped the code and created recombinant flagellin against which to test Lyme/Borreliosis victims to see if their band 41 is SPECIFIC for the burgdorferi (for example, as opposed to syphilis spirochetes)? • Yes, and that was Yale (Fikrig, US Patent # 5,618,533) in 1991.

37. 1991, Yale, PubMed ID # 1894359 / US Patent# 5, 618, 533

38. Yale: Ya with me so far? • Yale has a patent for antibody band 41, and antibody band 41 is SPECIFIC to the SPECIES, Borrelia burgdorferi. • Most people with Lyme or other spirochetes have band 41, and all the rest of the surface antigens (Osps) mutate (undergo antigenic variation). • Yale applied for a patent for this test in 1993, a year before they applied for the patent for LYMErix (OspA). • They don’t really know what OspA is, they just know by now that it’s not a vaccine, but won’t admit it.

39. Graphical Yale Specific Bb Flagellin

40. (Slide 40) REVIEW - I • So, where are we?1) Structure is Function, so once you isolate (separate) a pure compound, you want to know the shape of it. 2) There are rules for validating an analytical method to determine the purity of a compound (say, a pill of “medicine”). We are often talking about chromatography. Chromatography is Separations Science. The method should only detect the analyte in question (is Specific).3) On spirochetes are surface antigens called Osps (Outer Surface Proteins), and these are variable, which is why Relapsing Fever is called Relapsing Fever (antibodies do no good). But Borrelia also have SPECIFIC flagellin and Borreliosis has SPECIFIC flagellar antibodies.

41. Okay, now don’t go away because I haven’t gotten to the real FDA stuff yet. • FDA: • ** Specificity** Sensitivity (Limit of Detection)LinearityRuggedness** AccuracyPrecision, Etc.

42. The important FDA stuff for Lyme testing • Specificity (DOES IT ONLY DETECT, say OspA or Bb-specific flagellin?) • Accuracy (We will take this to mean, Does it detect most cases of Lyme?)Limit of Detection (Does this detect cases of Lyme with VERY LOW antibodies so we don’t miss any cases?)

43. Steere in Europe - 1 • Now, previously we have seen that in 1991, Yale came up with an accurate (17/18), and specific (does not detect the flagellin from other bacteria). In addition to that patent and PubMed report, Yale later went on to say that spiking the ELISA mix with extra flagellin would make the test even better…

44. Steere in Europe – 2; Spiking the ELISA with extra flagellin [PMID 1280650]

45. Steere in Europe – 3; Stuff ya can’t make up • In the previous slide you just saw Yale increasing the FDA - Sensitivity of the Lyme test, by adding extra FDA - Specific flagellin (from their patent 5,618,533), and that it detected cases that were “seronegative” without adding this extra flagellin. That was 1992…

46. Steere in Europe – 4; 1992… • So, by 1992, Yale had an FDA approvable, sensitive, specific test, that detected 17/18 or 94.4% of all cases. • But the same year Allen Steere went to Germany alone will illegal recombinant strains from the USA (B31, that has little or no OspC in it) and strain FRG (Federal Republic of Germany), that were “high passage” (dropped plasmids, like the OspA-B plasmid), with recombinant-OspA-and B-with-no- lipids-attached to come up with the current (Dearborn antibody panel), which is a scientific fraud crime.

47. Steere in Europe – 5; using recombinant OspA-B (no lipids attached) and “high-passage” strains. Stuff ya can’t make up.

48. Steere in Europe – 6; text of the previous clip out from the 1992 Antigens in Europe report. • Steere used “97 German patients,” recombinant OspA-B (the protein or amino acid sequence only and not the lipid end which is most likely to produce antibodies or is immunogenic) from USA strain B31 (has little or no OspC or the brain invasion antigen) with no lipids attached. • And Steere is clearly aware of the 16S RNA method which is SPECIFIC RNA to these spirochetes (meaning he is aware that OspA gene detection in Lyme victims is fraudulent science, since not all people are infected with burgdorferi among the all the Relapsing Fever strains or species).

49. Steere in Europe – 7; the Dressler/Steere/Dearborn “report,” screwing up the FDA validation criteria

50. (Slide 50) Steere in Europe – 8; “Receiver Operating Characteristic” or the ROC • As you have seen in the previous slides, FDA criteria are Sensitivity (or how low can you go in analyte concentration, and your test still detects, reliably, some concentration of the analyte in question) and the likes of Selectivity or Specificity (means only detects one analyte or species). And Yale met these criteria with their patented Flagellin method. But Steere went to Europe anyway with “high passages strains,” recombinant OspA-B with no lipids (which was meant to come up with a standard where OspA and B are missing because of the intended vaccine trials), an d came up with this insane idea that “the more antibodies, the more valid the test.” Everyone better hope Allen Steere never works for the Department of Homelame Stupidity.