|
Post by LymeEnigma on Jun 26, 2008 14:05:56 GMT -8
The highly conserved 60 kDa heat-shock protein (HSP60) has been considered to be another useful phylogenetic marker and sequence comparisons of its gene have been used for species identification and phylogenetic analysis of the genera Staphylococcus (Kwok et al., 1999Go) and Bartonella (Marston et al., 1999Go). In our previous work (Jian et al., 2001Go), it was indicated that the HSP60 gene may also be a useful tool for phylogenetic study of the genus Bifidobacterium. The clustering pattern was not only similar to that of 16S rDNA, but there was also a better correlation with DNA G+C content. Furthermore, HSP60 gene sequence similarities (~84–96 %) between Bifidobacterium species were much lower than those of 16S rDNA. ijs.sgmjournals.org/cgi/content/full/53/5/1619
|
|
|
Post by enochroot on Jul 7, 2008 7:43:30 GMT -8
UNDERSTANDING LYME WESTERN BLOT WILDER Home Quick Facts Charles Ray Jones, M.D. Madison Towers 111 Park St., Suite F New Haven, CT 06511 Tel. 203-772-1123•Fax 203-772-0682
UNDERSTANDING LYME WESTERN BLOT
There are nine known Borrelia burgdorferi genus specie specific KDA Western Blot antibodies (bands): 18 23 30 31 34 37 39 83 and 93.
Only one of these Borrelia burgdorferi genus specie specific bands is needed to confirm that there is serological evidence of exposure to the Borrelia burgdorferi spirochete and can confirm a clinical diagnosis of Lyme disease.
CDC Western Blot IgM surveillance criteria includes only two Borrelia burgdorferi genus specie specific antibodies for IgM 23 and 39 and excludes the other seven Borrelia burgdorferi genus specie specific antibodies.
CDC Western Blot IgG surveillance criteria includes 18 23 30 37 39 and 93 and excludes bands 31 34 and 83.
It does not make sense to exclude any Borrelia burgdorferi genus specie specific antibodies in a Lyme Western Blot IgG and to include only two of these antibodies in IgM because all the antibodies in IgG were once IgM.
IgM converts to IgG in about two months unless there is a persisting infection driving a persisting IgM reaction. This is the case with any infection including the Borrelia burgdorferi induced Lyme disease.
CDC wrongfully includes five non-specific cross-reacting antibodies in its Western Blot surveillance criteria: 28 41 45 58 and 66. This leads to the possibility of false positive Lyme Western Blots. There can be no false positives if only Borrelia burgdorferi genus specie specific antibodies are considered. One can have a CDC surveillance positive IgG Lyme Western Blot with the five non-specific antibodies without having any Borrelia burgdorferi genus specie specific antibodies.
This does not make sense.
CDC recommends that the Lyme Western Blot be performed only if there is a positive or equivocal Lyme ELISA. In my practice of over 7000 children with Lyme disease, 30% with a CDC positive Lyme Western Blot have negative ELISA’s. The Lyme ELISA is a poor screening test. An adequate screening test should have false positives not false negatives.
Rev. 8/23/04
================================================================
just some more fun -
Got a copy of my WB from Dr Cameron - can't make sense of it!!!!
But noticed on my FIRST WB by Quest in last July - neither 41ka IGG "OR" IGM was lit up... HMMMMM
|
|
|
Post by LymeEnigma on Jul 7, 2008 10:48:45 GMT -8
My first WB, done through Quest and tested with the same blood that was used for my positive ELISA, came back with zero bands positive. A repeat Quest WB with new blood came back with only 41 kDa positive.
Feel like posting your WB results here, enoch?
*edited because I'm a spaz and can't type today....
|
|
|
Post by enochroot on Jul 7, 2008 16:34:58 GMT -8
Hmm will try - but it is not laid out too clearly!
|
|
|
Post by enochroot on Jul 10, 2008 15:41:32 GMT -8
Sorry - will be back with the "WB" stuff - much grief...Out of work /feeling lousy, fighting my ins company, waiting to see the spinal surgeon - but seeing an immunologist on Saturday (+) and winning some of the ins battles (+)
|
|
|
Post by LymeEnigma on Jul 10, 2008 15:48:06 GMT -8
Take your time ... you "sound" beat!
|
|
|
Post by enochroot on Jul 12, 2008 14:51:02 GMT -8
Sorry - very bad time now. Feeling WAY "less good"!@ Taking percocets to function. DID figure one thing out off of Enzo Labs WB report from Dr Cameron NOT band 41, but band 23 was active.
Saw an immunologist today , going back Monday for tests.
|
|
|
Post by LymeEnigma on Jul 12, 2008 15:19:16 GMT -8
Sorry - very bad time now. Feeling WAY "less good"!@ Taking percocets to function. DID figure one thing out off of Enzo Labs WB report from Dr Cameron NOT band 41, but band 23 was active. Saw an immunologist today , going back Monday for tests. From the list I've compiled, 23 kDa can cross-react with Borrelia hermsi, and nearby 25 kDa will light up in patients with leptospirosis. What tests are you having done? Keep us updated.
|
|
|
Post by LymeEnigma on Jul 15, 2008 11:10:36 GMT -8
From Diagnosis of Lyme BorreliosisMaria E. Aguero-Rosenfeld,1,4* Guiqing Wang,2 Ira Schwartz,2 and Gary P. Wormser3,4 Departments of Pathology,1 Microbiology and Immunology,2 Medicine, Division of Infectious Diseases, New York Medical College,3 Westchester Medical Center, Valhalla, New York4 cmr.asm.org/cgi/content/full/18/3/484#LABORATORY_DIAGNOSIS(iii) Western IB. The use of antigens separated by molecular size in IB assays has contributed to the determination of which antigens of B. burgdorferi sensu lato are immunodominant at different stages of LB. Academic centers in the United States have evaluated in-house IB assays using B. burgdorferi sensu stricto strains other than B31 (84, 89). The few commercial IB assays that are currently available in the United States use B. burgdorferi sensu stricto strains; two manufacturers use B31 (6). Various B. burgdorferi sensu lato species and strains isolated from different geographic locations have been studied in Europe (117, 118, 280). Based on recognized inter- and intraspecies differences among the immunodominant antigens of B. burgdorferi sensu lato, it is not surprising that the source of antigens in IB affects the detection of antibodies in European patients with LB (280). Whether B. burgdorferi sensu lato antigens elicit IgM versus IgG antibodies depends on the duration of infection and the manifestation of LB. Detection of reactivity is also affected by the quality of the antigen used in the immunoassays (including the type and source of antigen). In early LB, IgM antibodies are directed to OspC and the flagellar antigens, FlaB (41 kDa) and FlaA (37 kDa) (7) (Fig. 1, left). Variable rates of IgM response to BmpA (39 kDa) have been observed in sera of patients with early LB, which appears to be related in part to the source of antigen used and/or the duration of disease prior to testing for antibodies (7, 84, 89, 181). The highest rates for IgM reactivity to the 39-kDa protein were reported by Engstrom et al. (89), using B. burgdorferi sensu stricto strain 297 (84%), and by Ma et al. (181), using B31 (50%). In contrast, Dressler et al. (84), using B. burgdorferi sensu stricto strain G39/40, reported IgM reactivity to the 39-kDa protein in only 4 and 8% of acute- and convalescent-phase sera of patients with EM, respectively. The infrequency of reactivity to BmpA antigen reported by Dressler et al. could be attributed to the low expression of this antigen by the B. burgdorferi sensu stricto G39/40 strain used in their study. We have observed IgM reactivity to this antigen in only 3% of acute-phase sera of patients with EM of a duration of less than 1 week and in 35% of acute-phase sera of untreated patients with EM of a duration of more than 7 days, using a commercial IB kit prepared with the B. burgdorferi B31 strain; convalescent-phase sera from the same groups showed IgM reactivity to the 39-kDa protein in 37% and 31%, respectively (7). *snip* The number of antigens recognized in IgM IB and the intensity of the immune response as determined by band intensity are greater in sera of symptomatic patients with EM, those with multiple EMs, or those with EM of a duration of more than 2 weeks at presentation, compared to asymptomatic patients with a solitary EM of a duration of less than 2 weeks (7) (Fig. 1, left). An expanded immunologic response is also found in patients with early neuroborreliosis (84). In early LB, IgG antibodies are directed to OspC and flagellin (41 kDa). IgG reactivity to BmpA (39 kDa) was reported by Engstrom et al. to occur in 85% of acute-phase sera of patients with EM (89). In our experience, antibody to this antigen is detected in 35% of acute-phase sera of untreated patients with EM of a duration of more than 7 days and in 33% and 64% of convalescent-phase sera of treated patients with EM of durations of less than 7 days and more than 7 days, respectively (7). Other antigens that elicit IgG immunoreactivity detectable by IB prepared with B. burgdorferi B31 are the 93 (also referred as P83/100)-, 66-kDa, 45-, 35-, 30-, and 18-kDa antigens. IgG antibodies reacting with a large number of antigens are typically seen in sera of patients with neuroborreliosis or late LB (84, 280) (Fig. 1, right). In an attempt to standardize serologic diagnosis of LB, criteria for IB interpretation have been established in the United States (53). These guidelines were derived from the systematic evaluation of IB in LB by two academic centers. The IgM criteria adopted were those of Engstrom et al. (89) based on a study of patients with EM using B burgdorferi sensu stricto strain 297. According to these criteria, a positive IgM blot is defined by the presence of two of three particular immunoreactive bands (OspC, 41 or 39 kDa). The IgG criteria are based on a study by Dressler et al. (84), who used B. burgdorferi sensu stricto isolate G39/40 as the source of antigen for evaluation of sera from patients with various manifestations of LB. These criteria require the presence of at least 5 of 10 particular bands (93, 66, 58, 45, 41, 39, 30, 28, 21 [OspC], or 18 kDa). The guidelines for IB interpretation further state that IgM or IgG criteria can be used during the first 4 weeks of illness, but only IgG criteria can be used after 4 weeks after onset of disease.Guidelines for IB interpretation in Europe have recently been published, but consensus on criteria has not been reached (118). Criteria applicable to each species causing LB may be needed in Europe (280). IB studies using members of each of the three different B. burgdorferi sensu lato species causing LB in Europe as a source of antigen to test sera from German patients with various manifestations of LB indicated that the overall highest sensitivity was achieved with B. afzelii pKo (118). Levels of immunoreactivity are also a function of the specific manifestation of LB in Europe. For example, OspC of B. garinii strains has better immunoreactivity than OspC of other B. burgdorferi sensu lato species in detecting IgM antibodies in patients with neuroborreliosis. This is most likely explained by the fact that B. garinii is the species that most frequently causes neuroborreliosis in Europe (197). In general, IB and ELISA have similar sensitivities except in the detection of acute-phase antibodies in early LB (7, 8). In a study of well-characterized sera from 46 culture-confirmed U.S. patients with EM, IgM IB was positive in 43% of acute-phase sera, compared with 33% by whole-cell ELISA (7). IB and ELISA have similar sensitivities when sera of patients with extracutaneous manifestations or late stages are tested (Table 4). The specificity of IB is greater than that of ELISA, as interpretation of IB relies on the presence of specific immunoreactive bands; nevertheless, it is important to emphasize that the specificity of IB is not 100%. Sera of individuals who received the recombinant OspA vaccination may show several bands on IB, depending on the source of antigen used in IB. Most frequently there is IgG reactivity to the 31-kDa antigen (OspA) and to other fragments of this antigen migrating below OspC (Fig. 1, right). These latter bands might be confused with the 18-kDa antigen included in the IgG criteria for IB interpretation (9, 205). Major limitations of IB include the visual scoring and subjective interpretation of band intensity that may lead to false-positive readings, the cost, and the variability of antibody responses in patients with the same clinical manifestation of LB. False-positive readings are particularly seen in IgM IB due to the presence of low-level reactivities to the 41-kDa and OspC antigens in sera from individuals presenting with other infectious and noninfectious illnesses (84, 89). False-positive IgM IB results have been reported in 6% of patients with illnesses such as rheumatoid arthritis, infectious mononucleosis, and systemic lupus erythematosus (89). Furthermore, IgM reactivity may persist for prolonged periods of time after treatment of early Lyme borreliosis (7). In addition, there is lack of standardization of the antigen source and preparations used in IB. Important methodological considerations include the use of appropriate positive control sera. Alternatively, the use of monoclonal antibodies raised to immunodominant antigens may assist in band location and interpretation. In the absence of an objective means of determining band intensities (densitometry), the use of intensity cutoff materials (weak control sera) is recommended. ______________________________________________________ LE note: all bold mine.
|
|
|
Post by LymeEnigma on Jul 15, 2008 11:17:00 GMT -8
From Recombinant Chimeric Borrelia Proteins for Diagnosis of Lyme DiseaseMaria J. C. Gomes-Solecki,1 John J. Dunn,2 Benjamin J. Luft,3 Jonathan Castillo,3,dagger Daniel E. Dykhuizen,4 Xiaohua Yang,3 John D. Glass,1 and Raymond J. Dattwyler3,* Brook Biotechnologies, Inc., Long Island High Technology Incubator, Stony Brook, New York 117901; Biology Department, Brookhaven National Laboratory, Upton, New York 117932; and Department of Medicine3 and Department of Ecology and Evolution,4 State University of New York at Stony Brook, Stony Brook, New York 11794 Whole B. burgdorferi assay mixtures contain many proteins with epitopes that cross-react with antibodies against other common infectious agents. Bruckbauer et al. demonstrated extensive cross-reactivity between B. burgdorferi and the bacterial pathogens Borrelia hermsii, Treponema pallidum, Treponema phagedenis, Leptospira interrogans, Neisseria meningitidis, Haemophilus influenzae, Yersinia enterocolitica, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, E. coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and Legionella micdadei[/b] (1). Many healthy adults with no history of B. burgdorferi infection have detectable levels of anti-41-kDa IgG, and many individuals also have antibodies directed against other spirochetal antigen. The Borrelia proteins in the 60- to 75-kDa range, p33 and two proteins with a mass of about 20 kDa, are among the most highly cross-reactive (1, 9, 13). The immunodominant 41-kDa flagellin antigen is not cross-reactive with nonflagellated bacteria but it is highly cross-reactive with similar proteins from other spirochetes. In addition, the accuracy of indirect assays for B. burgdorferi infection can be compromised by infectious and immunopathological conditions such as viral infections, subacute bacterial endocarditis, rheumatoid arthritis, and systemic lupus erythematosus.
jcm.asm.org/cgi/content/full/38/7/2530
|
|
ginger
Welcome Newbie!
Posts: 1
|
Post by ginger on Jul 15, 2008 21:23:34 GMT -8
I am wondering if there is another lab besides Igenex which does bands 31 and 34 and the 31 band confirmation epitope (sp?) so that there is a way we can double check our positive results from Igenex.
|
|
|
Post by LymeEnigma on Jul 15, 2008 21:42:00 GMT -8
I have yet to find one, at least here in the States, but we are definitely on the same page in that line of thinking. I turn to my Igenex tests as proof that I was, indeed, infected with Lyme and babs, and yet in the back of my mind I have to question even those results....
|
|
|
Post by LymeEnigma on Jul 27, 2008 9:41:53 GMT -8
|
|
|
Post by enochroot on Jul 27, 2008 18:48:47 GMT -8
Hey! Been an UGLY UGLY past few weeks... really relapsed(out of work). But I got my boatload of tests from the immunologist.
He is sending me to a reumatologist as I came back with autoimmune positives including schleraderma(?) He also said I could come back to him for glutathione injections if the autoimmune direction is a dead end.
My IGG western blot for Lyme came out "no bands lit"
|
|
|
Post by LymeEnigma on Jul 28, 2008 8:49:42 GMT -8
Damn, enoch! Talk about a growing puzzle.... The only autoimmune positives I ever came back with were ANA and decreased C3 and C4 complements, which can indicate lupus. I truly believe, at this point, that there is a pathogenic basis behind many autoimmune diseases.
Might just be time for me to write that paper I've been sitting on; I have some novel views on molecular mimicry.
I'm so sorry that you have taken such a nose-dive. I really hope that your doctors are able to get to the bottom of it sooner than later. How did your IgM look?
|
|