Western Blot Bands Explanations

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http://www.geocities.com/HotSprings/Oasis/6455/western-blot.txtAnd The Bands

Played On - Western blot serological test for Lyme disease

http://www.geocities.com/HotSprings/Oasis/6455/western-blot.txt

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as of 1 March 2001

Table of Contents

Terminology.

Borrelia burgdorferi bands mentioned in medical literature (MEDLINE).

Named bands - the gene names.

Bands specific for Borrelia burgdorferi.

Cross-reacting bands.

Bands found, or tested for, in IgM analysis in USA or Mexico.

Bands found, or tested for, in IgG analysis in USA or Mexico.

Other bands found in IgM/IgG in other countries.

The " CDC recommended " bands for Western blot testing - CDC/MMWR 1995.

For more information about the Western blot test and Lyme disease.

-----

Terminology:

Bb Borrelia burgdorferi (the Lyme disease bacteria)

Bdr Borrelia direct repeat

Bmp Bacterial membrane protein

Dbp decorin binding protein

Fla flagellin

Fn-B fibronectin-binding protein

GroEL a chaperonin 60 heat-shock protein isolated from Escherichia coli

Hsp heat shock protein

HSP Heat Stress Protein

kDa kilodaltons (a measurement of size)

Mab monoclonal antibodies

MgtE magnesium transporter protein

Oms outer membrane-spanning

Osp outer surface protein

P protein

p protein

PBP penicillin-binding protein

PMID PubMed ID (identification system for citations)

Tbp transferrin-binding protein

Other terms might be found at:

The Jounrnal of Clinical Investigation - Abbreviations

http://www.jci.org/misc/abbreviations.shtml

-----

Borrelia burgdorferi bands mentioned in medical literature (MEDLINE):

Note: Bands preceded with an asterisk are the 11 Western blot bands for

the ASTPHLD, CDC, FDA, NIH, CSTE, NCCLS 1994 conference recommendation

( " CDC recommendation " ) for the serologic diagnosis of Lyme disease -

see 1995 CDC MMWR link below.

5-kDa

7.5-kDa

11-kDa

13-kDa surface protein - sensu stricto, afzelii

14-kDa internal flagellin fragment [specific for Bb]

15 kDa polypeptide [also for syphilis]

16-kDa

17-kDa Osp 17 [b. afzelii]

*18-kDa p18 flagellin fragment

19-kDa immunogenic integral membrane lipoproteins

cross-reactive with other spirochetes/bacteria

Characterization of antigenic determinants of Bb shared by other

bacteria.

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1372635 & form=6 & db=m & Dopt\

=b

19-kDa decorin-binding protein

20-kDa decorin-binding protein

20.5-kDa

20.7-kDa

*21-kDa OspC [specific for Bb]

22-kDa [specific for Bb or cross-reactive depending on what one reads]

immunogenic integral membrane lipoproteins

[cross-reactive with other spirochetes/bacteria]

Characterization of antigenic determinants of Bb shared by other

bacteria.

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1372635 & form=6 & db=m & Dopt\

=b

22-kDa OspC [specific for Bb]

22-25kDa OspC

23-kDa OspC

*24-kDa OspC

25-kDa OspC [specific for Bb]

26-kDa

27-kDa Osp, Hsp

(Europe burgdorferi strain B29, but not American strain B31)

*28-kDa OspD, Oms28 [specific for Bb]

29-kDa OspA?

30-32-kDa OspA

*30-kDa OspA substrate binding protein

31-kDa OspA [specific for Bb]

32-kDa OspA

33-kDa outer membrane

34-kDa OspB [specific for Bb]

34-36-kDa OspB

35-kDa OspB [specific for Bb]

35.5-kDa

36-37-kDa

37-kDa P37, FlaA gene product, [specific for Bb]

38.0-kDa FlaA

*39-kDa BmpA [specific for Bb]

40-kDa

*41-kDa FlaB

42-kDa

43-kDa

44-kDa

*45-kDa [appeared in IgM in control group in 1998 study done in Poland]

MEDLINE - 9972057 - " ...whereas in control group only antibodies

against 45 kDa and 58 kDa were present. "

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9972057 & form=6 & db=m & Dopt\

=b

[appears for HGE]

MEDLINE - 9620365 - " ...confirmed the importance of the 42- to

45-kDa antigens as early, persistent, and specific markers of HGE

infection. "

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9620365 & form=6 & db=m & Dopt\

=b

46-kDa

47-kDa P47 fibronectin-binding protein [specific for Bb]

48-kDa

49-kDa

50-kDa [specific for Bb]

51 kDa MgtE

52-kDa Fn-BA

54-kDa [other Borrelia]

55-kDa

56-kDa

57-kDa PBP

*58-kDa (not GroEL)

59-kDa [a genetically engineeried fragment of the 83-kDa protein]

60-kDa Hsp [all Borrelia]

62-kDa Hsp60

63.7-kDa

64-kDa (P64) [cross-reacts to human axonal proteins]

65-kDa

*66-kDa P66 Oms66 Hsp outer/integral membrane protein

67-kDa

68-kDa

70-kDa Hsp

71-kDa

72-kDa Hsp [cross-reactive with other spirochetes]

[cross-reactive with other spirochetes/bacteria]

Characterization of antigenic determinants of Bb shared by other

bacteria.

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1372635 & form=6 & db=m & Dopt\

=b

73-kDa

75-kDa

77-kDa a genetically engineered recombinant hybred

Use of a hybrid protein consisting of the variable region of the

Borrelia burgdorferi flagellin and part of the 83-kDa protein as

antigen for serodiagnosis of Lyme disease.

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8027303 & form=6 & db=m & Dopt\

=b

78-kDa OspA

79.8-kDa

80-kDa

83-kDa p83 high molecular mass protein [specific for Bb]

84-kDa [b. garinii]

88-kDa

92-kDa

*93-kDa an immunodominant protoplasmic cylinder antigen, associated with

the flagellum [specific for Bb]

94-kDa PBP [specific for Bb]

95-kDa

97-kDa associated with flagella

100-kDa P100

110-kDa

200-kDa a fusion protein, a hybrid protein

-----

Named bands - the gene names:

BmpA, BmpB, BmpC " the 39 kDa Bmp protein family " , PMID: 8978084

BmpD, PMID: 8606088

FlaA, an outer sheath protein of the periplasmic flagella

37-kDa, PMID: 9986810

38-kDa, PMID: 9573194, PMID: 8990312

FlaB, 41-kDa PMID: 9573194

FlgE, protein 40-kDa?, PMID: 8548542

ospAB,28-34-kDa, PMID: 9596714

---

OspA = 29-33.5 kDa

Regarding OspA AND Lyme disease the following molecular weights are

associated with OspA in the MEDLINE database:

Note: To see the abstract for a particular PMID number,

simply insert the number, via copy and paste, in the following URL

after the " & list_uids= " term:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=PubMed & list_uids= & \

dopt=Abstract

Example, using the first number below:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve & db=PubMed & list_uids=1\

0638032 & dopt=Abstract

29-31 kDa - PMID: 10638032, 8429231

29.6 kDa - PMID: 1406458

30 kDa - PMID: 7500914

30-32 kDa - PMID: 2461135

31-kDa - PMID: 10030131, PMID: 8005219, PMID: 8406878, PMID: 1520966

PMID: 1554741, PMID: 10699329, PMID: 8825913

32-kDa - PMID: 8005219, PMID: 1520966

32-kDa/rOspA dog vaccine - PMID: 8567917

32.5 kDa - PMID: 9144917, PMID: 8004045

33 kDa - PMID: 1520966, PMID: 8005219

33.5 kDa - PMID: 8004045, PMID: 1520966

---

OspB 34-kDa PMID: 10030131

OspC 21-25-kDa, 22-25-kDa - PMID: 8825913,

OspD 28-kDa, PMID: 8825913

OspE 19.2-kDa [calculated], PMID: 8262642

OspF 26.1-kDa [calculated], PMID: 8262642

-----

Bands specific for Borrelia burgdorferi:

14-kDa ?

21-kDa

22-kDa OspC

25-kDa OspC

28-kDa OspD

31-kDa OspA

34-kDa OspB

35-kDa

37-kDa

39-kDa

47-kDa

50-kDa

83-kDa

93-kDa

94-kDa

Notes:

14-kDa ? PMID: 9920119, PMID: 1556546

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9920119 & form=6 & db=m & Dopt\

=b

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1556546 & form=6 & db=m & Dopt\

=b

B. burgdorferi: 22 kDa, 31 kDa, 34 kDa, 39 kDa, 83 kDa - PMID: 9440203

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9440203 & form=6 & db=m & Dopt\

=b

p35 and p37 are Borrelia burgdorferi genes - PMID: 9175831

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9175831 & form=6 & db=m & Dopt\

=b

at least one is an apparently specific band (25, 28, 39, 47, 50, or

93 kDa). - PMID: 7791177

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=7791177 & form=6 & db=m & Dopt\

=b

antibodies against the 94 kDa, 31 kDa and 21 kDa proteins are largely

species-specific. - PMID: 8223404

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8223404 & form=6 & db=m & Dopt\

=b

-----

Cross-reacting bands:

19-kDa

22-kDa ??

20-kDa

30-kDa

33-kDa

34-kDa ??

36-kDa

40-kDa

41-kDa

60-kDa

66-kDa

72-kDa

Notes:

Search terms:

cross-react*

cross-antigenicity

crossreact

Immunoblot analysis indicated the presence of cross-reacting antibodies

directed to B. burgdorferi antigens with apparent molecular weights of

60, 41, 40, 36, 30 and 20 kDa. - PMID: 1385332

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid= & form=6 & db=m & Dopt=b

P66, P60, P41 which are dominant immunogens of all types of borrelias

PMID: 9162453

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9162453 & form=6 & db=m & Dopt\

=b

19, 22[??], 72 - PMID: 1372635

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1372635 & form=6 & db=m & Dopt\

=b

60-75 kDa range, p40, p33 and two proteins in the range of 20 kDa. -

PMID: 1597198

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1597198 & form=6 & db=m & Dopt\

=b

Whereas the 60 kDa, 41 kDa, and 34 kDa[??] constituents reveal a

marked cross-antigenicity with other spirochetes and even more distantly

related bacteria,...PMID: 8223404

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8223404 & form=6 & db=m & Dopt\

=b

-----

Bands found, or tested for, in IgM analysis in USA or Mexico.

18-kDa

21-kDa

23-kDa

24-kDa

25-kDa

28-kDa

37-kDa

39-kDa

41-kDa

45-kDa

55-kDa

58-kDa

60-kDa

66-kDa

93-kDa

Notes:

37 - PMID: 9986810

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9986810 & form=6 & db=m & Dopt\

=b

41 kDa and 58 kDa - PMID: 9972057

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9972057 & form=6 & db=m & Dopt\

=b

24 kDa (OspC), 41 kDa, and 37 kDa - PMID: 8748261

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8748261 & form=6 & db=m & Dopt\

=b

39, 58, 60, 66, or 93 kDa - PMID: 8748261

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8748261 & form=6 & db=m & Dopt\

=b

55-kDa - PMID: 7642278

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=7642278 & form=6 & db=m & Dopt\

=b

Recommended criteria for the immunoglobulin M (IgM) immunoblot are the

recognition of two of three proteins (24, 39, and 41 kDa). PMID: 7714202

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=7714202 & form=6 & db=m & Dopt\

=b

25-kDa antigens - PMID: 8308100

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8308100 & form=6 & db=m & Dopt\

=b

23-kDa - PMID: 8225587

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8225587 & form=6 & db=m & Dopt\

=b

at least 2 of the 8 most common IgM bands in early disease

(18, 21, 28, 37, 41, 45, 58, and 93 kDa) - PMID: 8380611

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8380611 & form=6 & db=m & Dopt\

=b

-----

Bands found, or tested for, in IgG analysis in USA or Mexico.

18-kDa

20-kDa

21-kDa

22-kDa

23-kDa

24-kDa

28-kDa

29-kDa

30-kDa

31-kDa

34-kDa

35-kDa

39-kDa

41-kDa

45-kDa

55-kDa

58-kDa

88-kDa

62-kDa

66-kDa

93-kDa

Notes:

A serum sample was considered positive by WB [igG] if at least three of

the following protein bands were recognized: 18, 24, 28, 29, 31, 34, 39,

41, 45, 58, 62, 66, and 93 kDa. PMID: 10071428

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=10071428 & form=6 & db=m & Dop\

t=b

93, 39, 34 or 23 kDa IgG bands - PMID: 9580180

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=9580180 & form=6 & db=m & Dopt\

=b

55-kDa - PMID: 7642278

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=7642278 & form=6 & db=m & Dopt\

=b

The recommended criteria for a positive IgG immunoblot are the

recognition of two of five proteins (20, 24 [> 19 intensity units], 35,

39, and 88 kDa). Alternatively, if band intensity cannot be measured,

the 22-kDa protein can be substituted for the 24-kDa protein with only

a small decrease in sensitivity. PMID: 7714202

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=7714202 & form=6 & db=m & Dopt\

=b

at least 5 of the 10 most frequent IgG bands after the first weeks of

infection (18, 21, 28, 30, 39, 41, 45, 58, 66, and 93 kDa)-PMID: 8380611

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=8380611 & form=6 & db=m & Dopt\

=b

66 kDa and 31/34 kDa - PMID: 3819479

http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=3819479 & form=6 & db=m & Dopt\

=b

-----

Other bands found in IgM/IgG in other countries:

Europe/Germany - PMID: 9163458

The following interpretation criteria resulting in specificities of

greater than 96% were recommended: for IgG WB, at least one band of

p83/100, p58, p56, OspC, p21, and p17a for PKa2; at least two

bands of p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for

PKo; and at least one band of p83/100, p39, OspC, p21, and p17b

for PBi; for IgM WB, at least one band of p39, OspC, and p17a or

a strong p41 band for PKa2; at least one band of p39, OspC, and

p17 or a strong p41 band for PKo; and at least one band of p39 and

OspC or a strong p41 band for PBi.

IgG titers to P35 and P37 Mice/Northern blot - PMID: 9175831

Italy - PMID: 8809552

The overall reactivity of the

three Borrelia strains in IgG immunoblots indicated that ten

protein bands were significant, with a different prevalence of some

of them in the two groups of patient sera: bands at 60-58, 30-33,

36-37 and 28-27 kDa were markers for neuroborreliosis sera;

proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for

Lyme arthritis. The IgM Immunoblots revealed significant bands

at 100-83, 72-70, 51, 24- 21 and 18-17 kDa only with

neuroborreliosis sera.

Russia - PMID: 7791165

IgG reactivity with > or = 5 spirochetal proteins, particularly the

37, 39, 41, 60, and 93 kDa antigens

Germany - PMID: 8167425

IgG - 95 and 19-17 kDa

France - PMID: 8405312

[igG] Reactions with specific protein bands (94, 73, 30 and 21 KDa)

-----

The " CDC recommended " bands for Western blot testing - CDC/MMWR 1995.

The ASTPHLD, CDC, FDA, NIH, CSTE, NCCLS 1994 conference recommendation

( " CDC recommendation " ) for the serologic diagnosis of Lyme disease -

CDC/MMWR 1995:

ASTPHLD - Association of State and Territorial Public Health Laboratory

Directors

CDC - Centers for Disease Control and Prevention

FDA - Food and Drug Administration

NIH - National Institutes of Health

CSTE - Council of State and Territorial Epidemiologists

NCCLS - National Committee for Clinical Laboratory Standards

According to CDC's Morbidity and Mortality Weekly Report (MMWR) 1995;

44 (31):590-591, an IgM immunoblot is considered positive if two of the

following three bands are present:

24 kDa (OspC)*

39 kDa (BmpA)

41 kDa (Fla)

An IgG immunoblot is considered positive if five of the following 10

bands are present:

18 kDa

21 kDa (OspC)*

28 kDa

30 kDa

39 kDa (BmpA)

41 kDa (Fla)

45 kDa

58 kDa (not GroEL)

66 kDa

93 kDa

* The apparent molecular mass of OspC is dependent on the strain of

B. burgdorferi being tested. The 24 kDa and 21 kDa proteins referred

to are the same.

Recommendations for Test Performance and Interpretation from the Second

National Conference on Serologic Diagnosis of Lyme Disease,

CDC MMWR, August 11, 1995 / 44(31);590-591

http://www.cdc.gov/epo/mmwr/preview/mmwrhtml/00038469.htm

or

http://wonder.cdc.gov/wonder/prevguid/m0038469/entire.htm

-----

For more information about the Western blot test and Lyme disease, see:

The National Lyme Disease Network LymeNet Newsletter

Volume 4 - Number 13 - 9/23/96

SPECIAL ISSUE - Understanding the Western Blot

http://www2.lymenet.org/domino/nl.nsf/UID/4-13

Explanation of the Lyme Disease Western Blot by Carl Brenner

http://www.lymealliance.org/Medical/MedCategory4/Lab4/lab4.html

Laboratory Tests by Tom Grier - Part 2 - Western Blot and ELISA

http://www.lymealliance.org/Medical/MedCategory4/Med12A/med12a.html

IGeneX, Inc. - Lyme Disease Western Blot

http://www.igenex.com/lymeset2.htm

MRL Diagnostics' Lyme Disease B. burgdorferi Genogroup 1

Western Blot IgG test

http://www.mrldiagnostics.com/insert/wb0400g.htm

Immuno-Biological Laboratories (IBL) - Hamburg [Germany] -

Borrelia burgdorferi/Lyme IgG Western Blot

http://www.ibl-hamburg.com/prod/re_97228.htm

The western immunoblot for Lyme disease: determination of sensitivity,

specificity, and interpretive criteria with use of commercially

available performance panels.

Tilton RC, Sand MN, Manak M

BBI Clinical Laboratories, Inc., New Britain, Connecticut 06053, USA.

Clin Infect Dis 1997 Jul;25 Suppl 1:S31-S34

http://www.x-l.net/Lyme/BBI_TEST.htm

SIMULTANEOUS ELISA AND WESTERN BLOT TESTING IN EVALUATION OF PATIENTS

FOR SUSPECTED LYME DISEASE

Janice M. Kochevar, FNP-C, B. Liegner, M.D.

LDF 10th Annual International Scientific Conference on Lyme Borreliosis

& Other Tick-borne Disorders: April 28-30, 1997

http://www.x-l.net/Lyme/elisawb497.htm

MEDLINE -

Western Immunoblot*/blot* [in Title] AND Lyme Disease - 33 on 1 Mar 2001

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=search & db=PubMed & orig_db=PubMe\

d & dispmax=1000 & doptcmdl=DocSum & term=%28western%5BTI%5D+AND+%28immunoblot*%5BTI%5\

D+OR+blot*%5BTI%5D%29+AND+%28lyme+OR+burgdorferi+OR+%28borreli*+AND+ixodes%29+OR\

+%28%28erythema+AND+migrans%29+NOT+glossitis%29+OR+garinii+OR+afzelii+OR+neurobo\

rreli*%29

-----

For more information on Lyme disease, see:

Lots Of Links On Lyme Disease

http://www.geocities.com/HotSprings/Oasis/6455/lyme-links.html

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Prepared by

Art Doherty

Lompoc, California

doherty@...