ELISA-BASED PEPTIDE ASSAY FOR SIMULTANEOUS MEASUREMENT OF ANTIBODIES AGAINST B. SENSU STRICTO, B. GARINII, B. AFZELII, AND DETECTION OF CROSS-REACTIVE

ELISA-BASED PEPTIDE ASSAY FOR

SIMULTANEOUS MEASUREMENT OF

ANTIBODIES AGAINST B. SENSU STRICTO, B.

GARINII, B. AFZELII, AND DETECTION OF

CROSS-REACTIVE ANTIBODIES WITH

BABESIA AND EHRLICHIA

Aristo Vojdani, Ph.D., M.T.; Bernard Raxlen, M.D.; Shirley Scott, M.D.

Elisa-Based Peptide Assay* We applied the commercially available ELISA, Western Blot, and the newly-developed peptide-based ELISA to more than 100 clinical specimens from patients with possible Borreliosis.

* The chronic nature of Borreliosis and antigenic diversity of the spirochetes suggest that antigenic variation plays an important role in immuneinvasion, therefore:

* Peptides from different components of Borrelia during different cycles were selected* Outer surface protein* Leukocyte function* Associated antigens* Immunodominant antigens* Variable major proteins* Decorin-binding proteins from Borrelial subspecies (B. sensu

stricto., B. afzelii, B. garinii)

* In a similar manner, antibodies against specific peptides from Babesia, Bartonella, and Ehrlichia were measured to exclude cross-reactivity.

Methods:•Applied these assays to 24 specimens which were classified as positive (12 specimens) or negative (12 specimens) for Lyme disease by the College of American Pathologists and the New York Department of Health surveys.

Findings:*The 12 specimens with a positive IgG and / or IgM did not correlate at all with the number of bands in the Western Blot Assay and with the multi-peptide ELISA assay.

i.e. – a sample with an ELISA IgG level of 133 resulted in more bands in the Western Blot Assay than a sample with an ELISA IgG level of 995.

*Out of 12 specimens with low IgG and IgM ELISA, three specimens were found to be positive for IgM Blot Assay with 23, 39, 41, 45, and 58 kDa. The same three specimens were highly positive for IgM against immunodominant protein of invariable region, variable major protein, and decorin-binding protein of B. sensu stricto.

Conclusions:

*The traditional ELISA-based Borrelia lysate antigens may suffer from BOTH false negative and false positive results

*A comparison of the new multi-peptide based ELISA assay to the Western Blot might result in a more sensitive, more specific assay for the early detection of Lyme disease.

_______________________________________________________________

This comparison of the new multi-peptide based ELISA assay to the Western Blot was applied to 64 different specimens from patients with Lyme disease symptoms from two different clinics, one for the east coast and the other from the west coast.

West Coast Specimens• In 19 / 24 specimens the Western Blot assay correlated very well with the ELISA peptide assay.

• The new peptide-based ELISA assay was not only quantitative, but 5 specimens classified negative or weak positive by the Western Blot were clearly positive by the peptide ELISA.

• 3 samples were positive with peptides of B. garinii or B. afzelii

• 1 sample has elevation of antibodies against Ehrlichia when using the peptide ELISA

• 4 samples had elevation of antibodies against Babesia when using the peptide ELISA

East Coast Specimens• 33 / 40 specimens the Western Blot assay correlated very well with the

ELISA peptide assay.

• 2 specimens were positive by IgG Western Blot but negative by the peptide Elisa

• 4 samples were positive by peptide ELISA but negative by Western Blot IgG and IgM

• 7 / 33 specimens positive by the peptide ELISA were highly positive with B. garinii

• 11 / 33 specimens had simultaneous elevation in antibodies against Borrelia and Babesia

• 1 / 33 specimens was positive for antibodies against Borrelia, Babesia, and Ehlrichial peptides

Final ConclusionsCorrelation between the Western Blot and the peptide ELISA is good, however, the peptide ELISA has the following advantages over the Western Blot:

•Peptide ELISA is quantitative

•Peptide ELISA is species and sub-species specific

•Peptide ELISA may detect antibodies against unrelated peptides

•Peptide ELISA may detect cross-reactive antibodies against Babesia peptides

•Peptide ELISA may detect cross-reactive antibodies against Ehrlichia peptides

PCR-based methods are insensitive for routinelab testing due to the absence or low number

of bacteria in clinical samples.

45

Lyme Western Blot Assay

The Western Blot Assay has been widely used to detect the presence of antibodies to various infectious disease agents in human serum and plasma. In this procedure, component proteins of purified, inactivated bacteria are electrophoretically separated by SDS-polyacrylamide electrophoresis followed by electrotransfer to nitrocellulose sheets. Each strip serves as the solid-phase antigen for an ELISA test. The Western Blot Assay is more reliable since the cross-reactive antibodies are excluded and peptide specific antibodies are observed. However, results are yes or no and cannot be quantitated.

IgG and IgM antibody pattern with different peptides of Borrelia burgdorferi in patient confirmed with Lyme disease.8

93

66

58

41

39

30

28

23

41

39

23

Host-Adapted OspA during Inflammation Borrelia burgdorferi in Mice Expresses

Helena Crowley and Brigitte T. Huber, Infection and Immunity, (2003), pp 4003-4010

Antibody responses to outer surface protein A (OspA) of Borrelia burgdorferi may occur during periods of arthritis late in the clinical course of untreated Lyme disease. A prominent late manifestation of Borrelia burgdorferi is Lyme arthritis. Antibody reactivity to OspA typically develops near the beginning of prolonged episodes of arthritis. These patients may progress to autoimmune state by developing a cross-reactive response between OspA and a self antigen. Therefore, treatment-resistant Lyme arthritis is associated with immune reactivity to OspA of Borrelia and the major histocompatibility complex II allele DRB1. The amino acid sequence of OspA is shown below:

Lyme OspA, SYVLEGTLTAEKTTL 15 MER 11

Analysis of the OspE Determinants Involved in Binding of Factor H and OspE-Targeting

Antibodies elicited during Borrelia burgdorferi Infection in Mice

Metts et al., Infection and Immunity, (2003), pp 3587-3596

Peptides from Outer surface protein-E (OspE) are involved in immune evasion. The ability of the Lyme spirochete to maintain chronic infection indicates that the organism is capable of immune evasion. This is done by either antigenic variation or through the binding of Complementary Regulatory Protein Factor H. The OspE is surface-exposed and is expressed in both ticks and mammals, and elicits a strong antibody response. Analysis of the specificity of the antibody response to different OspE variants suggests that it is the hypervariable regions that are targeted by antibody response during infection. On the other hand, it is the conserved regions of OspE that are involved in Complement Regulatory Protein Factor H. Therefore, it is important to assess the specificity of the antibody response to OspE epitopes that are exposed during infection. Two different OspE peptides that span the C terminal of BBL39 are shown below:

OspE-pep1, INNSAGGDKIAEYAISLEELK RNLK

OspE-pep2, IKTKIEKINDTEYITFLGDKINNSA

12

Identification of LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme

ArthritisGross et al., Science, (1998), 281:703

Treatment-resistant Lyme arthritis is associated with immune reactivity to Outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A hormology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly-related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease. The amino acid sequence of peptides selected from leukocyte function-associated antigens is shown below:

Lyme LFA, ELQKKIYVIEGTSKQDLTSF 20 MER13

An Immunodominant Conserved Region within the Variable Domain of VlsE, the Variable Surface Antigen of Borrelia burgdorferiFang Ting Liang et al., Journal of Immunology, (1999), 163:5566-5573

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid

immune destruction. Variable Ags such as the spirochete that causes Lyme disease express a surface lipoprotein called Variable Major protein (VMP) or Variable Major Protein Like Sequence (VlsE). A 19-MER peptide (C2 peptide) and 25-MER (C6 peptide) are clearly conserved across both genospecies and strains of B. burgdorferi, Sensu lato, Sensu stricto and B. garinii. The amino acid sequences of these peptides are shown below:

Lyme immunodominant C2 peptide, DAASVNGIAKGIKGIVDAA 19 MER

Lyme immunodominant C6 peptide, KKDDQIAAAMVLRGMAKDGQFALK 25 MER

Antigenicity of this peptide was confirmed in humans, monkeys and mice. In fact, sera from all these hosts reacted with C2 and C6 peptides early and persistently in the course of infection, thus indicating that these peptides contain one or more epitopes that are broadly antigenic.

Over all, this peptide-based ELISA was found to have diagnostic sensitivities of 100% in late-stage Lyme disease and 74 % in acute or early stage Lyme disease.

14

Evidence that the Variable Regions of the Central Domain of VlsE are Antigenic during

Infection with Lyme Disease SpirochetesMcDowell et al., Infection and Immunity, (2002), pp 4196-4203

Infection-induced sequence changes that alter the antigenic properties of VlsE

contribute to immune evasion. In an experimental model, a strong anti-VlsE IgG response was shown by the fourth week of infection. All variants of B. burgdorferi were recognized by antibodies that developed against VlsE. Most Lyme disease patients develop anti-VlsE response. Based on this, researchers employed recombinant VlsE in ELISA format and demonstrated diagnostic sensitivities of 63% for culture-confirmed erythema migrans cases, and 92% for late stage infections. The following peptides selected from antigens expressed during the early stage of infection were used in this assay:

Variable Major Protein Like Sequence-1: VR1 ANDNAAKAADKDSVK

Variable Major Protein Like Sequence-2: VR2 GGSEKLKAVAAAKENNK

15

Species-Specific Serodiagnosis of Lyme Arthritis and Neuroborreliosis Due to Borrelia

burgdorferi sensu stricto, B. afzelii, and B, garinii by Using Decorin Binding Protein A.

Heikkilä et al., Journal of Clinical Microbiology, (2002), pp 453-460

In the United States Lyme borreliosis is caused by a subspecies, B. burgdorferi sensu stricto. In

Europe, however, three different Borrelial subspecies, B. burgdorferi sensu stricto, B. afzelii, and B. garinii, are known etiologic agents of Lyme disease. Among individual Borrelial proteins from different species, sequence heterogeneity varies up to 40%. Decorin binding protein A, a Borrelial outer surface protein, is one of the key proteins in B. burgdorferi. This antigen elicits a strong antibody response during experimental murine Borreliosis and has been suggested as a potential vaccine protein. This protein was cloned and sequenced from the three pathogenic Borrelia species common in the U.S. and in Europe.

16

• Decorin binding protein-peptide from B. sensu stricto: CGLTGATKIRLERSAKDITDEIDAIKKDAA

• Decorin binding protein-peptide from B. garinii: EKTPPTTAEGILAIAQAMEEKLNNVNKKQQ

• Decorin binding protein-peptide from B. afzelii: SGIYDLILNAAKAVEKIGMQGMKQAVEEAA

100% of patients with neuroborreliosis (NB) and 93% of patients with Lyme arthritis (LA) reacted

positively. Sera from the majority of patients reacted with one rDbpA only and had no or low cross-reactivity to the other two variant proteins. In patients with culture-positive erythema migrans (EM), the sensitivity of rDbpA immunoglobulin G (IgG) or IgM ELISA was low. The DbpA seems to be a sensitive and specific antigen for the serodiagnosis of LA or NB, but not EM.

Identification and Characterization of PutativeSecreted Antigens from Babesia microti

Homer et al., Journal of Clinical Microbiology, (2003), pp 723-729

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing 7 genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.The following species-specific Babesia peptides were used in the ELISA assay:

17

• Babesia microti: IVEFNAIFSNIDLNNSSTVKNEIIK

• Babesia bovis: VEAPWYKRWIKKFRDFFSKNVTQ

• Babesia equi: DFFHPEDVVAPHSGITTPK

Highly Conserved Regions of the Immunodominant Major Surface Protein 2 of the

Genogroup II Ehrlichial Pathogen Anaplasma marginale are rich in Naturally Derived CD4+ T Lymphocyte Epitopes that Elicit Strong Recall

ResponsesBrown et al., Journal of Immunology, (2001), 166:1114-1124

18

Members of the order Rickettsiales constitute a diverse group of obligatory intracellular bacteria of eukaryotic cells, including Rickettsia, Orientia, Anaplasma, Ehrlichia, Neorickettsia, Bartonella and Coxiella. In the U.S., Ehrlichia has been shown to be the agent of human monocytic granulocytic ehrlichiosis. Ehrlichia expresses a markedly imunodominant outer membrane protein designated Major Surface Protein-2 (Msp2). Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of Msp2 that enhances the primary Ab response against newly emergent variants. Therefore, measurements of IgG and IgM antibody against these highly conserved regions of Msp2are the best method for assessing humoral immune response against ehrlichial pathogens.Overlapping peptides that span the N and C termini of Anaplasma marginale and A. ovis were used in this study:

• Ehrlichial N-terminal (AA 1-30)

• MSAVSNRKLPLGGVLMALVAAVAPIHSALLA

•Ehrlichial C-terminal (AA 272-301)

• VAGAFARAVEGAEVIEVRAIGSTSVMLNAC

• In 1998, during pregnancy, developed serious tension headaches of muscular originPhysical exam was normal

• In 2001 developed muscle and joint painsCognitive difficultiesSleep deprivationRecommended for treatment for Lyme disease

ISL No. 156557

42-year-old female

19

ISL No. 156557 by IgeneX Western Blot

20

IgG

+

+

+ -

IgM

+

+

+

+

+ + -

+ +

ISL No. 156557 by CDC Western BlotIgG

+

IgM

+

+

Ehrlichia IgG

1.91.9

Normal Abnormal1.0

1.0

6.0Antigen

Index Value 4.02.0 3.0

1.0

1.0

1.0

5.0

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG

Unrelated Spirochete IgG

Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

B. Afzeli IgG

Babesia IgG

1.0

1.0B.B. Sensu Stricto IgG

1.01.0

1.0

1.0

2.4

1.0

1.0

2.4

1.0

1.0

1.0

1.0

1.2 1.2

1.0

1.0

IgG Antibodies to Borrelia burgdorferi and Cross Reactive Antigens:

1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive

>5.0 Highly Positive

Index Values

ISL No. 156557

21

ISL No. 156557

1.0

1.0

1.0

1.0 1.0

1.4

1.0

1.0

1.0

1.0

1.0

1.0

1.0

B. Afzeli IgM

Babesia IgM

1.4

1.0B.B. Sensu Stricto IgM

3.63.6

2.1

Unrelated Spirochete IgM

Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM

5.02.0 3.0

2.1

1.0

1.0

6.0Antigen

Index Value 4.0

Ehrlichia IgM

1.01.0

Normal Abnormal1.0

1.0

IgM Antibodies to Borrelia burgdorferi and Cross Reactive Antigens:

1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

22

• Headaches

• Stiffness in the neck

• Difficulty speaking

• Change in smell

• Blurred vision

• Ringing in the ears

• Nausea

• Joint Pain

• Loss of reflexes

• Loss of muscle tone in the legs

• Shortness of breath

• Night sweats

• Diminished exercise tolerance

• Burning sensations in the body

ISL No. 156834

Spect Scan: Abnormal Diagnosis: CNS Lyme

• Weakness in thighs

• Pressure in head

• Poor balance

• Increased motion sickness

• Encephalopathy

• Depression

• Personality change (becomes quiet when in pain)

• Anxiety and panic attacks

• Bipolar disorder

• Dementia

• Overemotional

• Disturbed sleep

In 2000, gradually developed the following symptoms:

Comments: I have been to 45 doctors - every specialty you can imagine. Please help!23

ISL No. 156834 by CDC Western Blot

24

IgG

+

+

IgM

+

+

++

ISL No. 156834 by IgeneX Western BlotIgG

+ -+ -

+ -

IgM

+

+ -

++

++

ISL No. 156834

1.1

2.3

1.8

2.6 2.6

1.2

4.4

1.1

3.5

1.0

1.1

3.5

1.0

B. Afzeli IgG

Babesia IgG

1.2


4.54.5

1.4


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG

5.02.0 3.0

1.4

1.8

1.1

6.0Antigen

Index Value 4.0

Ehrlichia IgG

3.93.9

Normal Abnormal1.0

2.3


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

25

ISL No. 156834

Ehrlichia IgM

2.12.1

Normal Abnormal1.0

1.0

6.0Antigen


1.1

1.3

1.0

5.0

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

B. Afzeli IgM

Babesia IgM

1.0


2.92.9

1.1

1.0

1.8

1.0

1.0

1.8

1.0

1.0

1.0

1.3

1.1 1.1

1.0

2.2


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

26

• In 1999, developed:HeadachesLightheadednessAbdominal discomfortNausea and painMuscle spasm

• 3 months later:Blurred visionMemory impairmentDifficulty concentratingChronic fatiguePoor sleepJoint aches and painsFreezing sensations in feet, legs and forearms

ISL No. 156955Athletic 48-year-old man with suspected Lyme

arthritis and vaccinated with Lymerix. Neuropathy possibly induced by Lymerix.

This resulted in difficulty walking or keeping his arms up, lasting for several weeks. Lyme titers and Western Blot were repeatedly negative. He was treated with the antibiotics Rociphin and Plaquinil without obvious benefits.

EMG/Nerve conduction - normal

MRI of brain-normal

Mild-moderate neuropathy was detected.

He was vaccinated with Lymerix.

27


28

IgG

+ +

IgM

+

+

+++ +


+

IgM

+ -

+ -

+++ +

ISL No. 156955

1.0

3.2

3.6

1.7 1.7

2.0

3.0

3.5

4.1

3.5

3.5

4.1

3.5

B. Afzeli IgG

Babesia IgG

2.0


3.63.6

3.2


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG

5.02.0 3.0

3.2

3.6

1.0

6.0Antigen

Index Value 4.0

Ehrlichia IgG

4.04.0

Normal Abnormal1.0

3.2


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

29

ISL No. 156955

Ehrlichia IgM

1.91.9

Normal Abnormal1.0

1.0

6.0Antigen


1.6

1.5

1.0

5.0

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

B. Afzeli IgM

Babesia IgM

1.3


1.21.2

1.6

1.2

2.2

1.1

1.2

2.2

1.1

1.0

1.0

1.5

1.5 1.5

1.3

1.7


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

30

Diagnosed in January 2003. Wentto Lyme/specialist/neurologist,who gave her

Biaxin + Keflex for - 2 months Biaxin + Plaquenil - 5 months

She felt better for two weeks butmilder symptoms returned. Triedto go off antibiotics. All symptomsreturned.Major symptoms• Sore throat, swollen glands• Red eyes • Sound sensitivity• Unexplained weight loss• Joint pain, swelling or stiffness

ISL No. 155842

Female diagnosed with Lyme disease.Doxycycline for two months. Symptoms returned.

31

• Shortness of breath • Night sweats or unexplained chill • Diminished exercise tolerance• Lightheadedness• Malaise• Mood swings• Depression• Forgetfulness• Attention deficit• Difficulty with concentration

SPECT scan of brain: No perfusion abnormalities were identified.


32

IgG

+ +

IgM

++

+ ++ +


+ -

++ -

IgM

+ -+ -+ -

+

+ ++ +

+ - +

-+ +

ISL No. 155842

Ehrlichia IgG

2.12.1

Normal Abnormal1.0

4.3

6.0Antigen


1.1

1.2

1.5

5.0

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

B. Afzeli IgG

Babesia IgG

1.3


1.31.3

1.1

4.8

3.8

4.1

4.8

3.8

4.1

1.5

4.3

1.2

1.9 1.9

1.3

3.9


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

33

ISL No. 155842

2.0

3.9

2.2

2.3 2.3

4.7

5.9

1.6

4.2

2.1

1.6

4.2

2.1

B. Afzeli IgM

Babesia IgM

4.7


1.91.9

1.6


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM

5.02.0 3.0

1.6

2.2

2.0

6.0Antigen

Index Value 4.0

Ehrlichia IgM

5.65.6

Normal Abnormal1.0

3.9


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

34

• Doxycycline 3 weeks• Tetracycline 7 weeks• Rocephin 10 weeks• Biaxin 5 weeks• Paxil 6 weeks

ISL No. 156569

Diagnosed with Lyme disease in 2001Main symptom: Muscle pain,

fatigue, sensitivity to light

35


36

IgG

+

+

IgM

+ +

+

+ +

+

+ +

+


+ -+ +

+

IgM

+

+ - +

+

+

+

+ -

+

+ - + -

+ +

+ +

ISL No. 156569

1.0

1.7

1.3

2.5 2.5

3.5

2.6

4.0

6.0

1.0

4.0

6.0

1.0

B. Afzeli IgG

Babesia IgG

3.5


2.52.5

2.5


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG

5.02.0 3.0

2.5

1.3

1.0

6.0Antigen

Index Value 4.0

Ehrlichia IgG

7.97.9

Normal Abnormal1.0

1.7


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

37

ISL No. 156569

Ehrlichia IgM

5.35.3

Normal Abnormal1.0

5.2

6.0Antigen


6.4

5.3

3.1

5.0

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

B. Afzeli IgM

Babesia IgM

5.3


6.46.4

6.4

5.2

5.4

5.1

5.2

5.4

5.1

3.1

5.2

5.3

3.4 3.4

5.3

5.7


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

38

ISL No. 156073

In the summer of 2003, patient had episodes of Lyme bites with Erythema migrans on his arms. He was put on antibiotics for 2 days. His symptoms were fatigue, falling asleep during

the day, and numbness in the arms and legs. He was put on anti-psychotic agents, which made his situation worse and nearly killed him.

39


40

IgGIgM

+

+


+ +

IgM

+ -

+

+ - + -

+ - +

+ -

ISL No. 156073

Ehrlichia IgG

3.53.5

Normal Abnormal1.0

1.2

6.0Antigen


4.8

1.0

1.0

5.0

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

B. Afzeli IgG

Babesia IgG

2.6


2.42.4

4.8

2.6

5.1

1.0

2.6

5.1

1.0

1.0

1.2

1.0

2.1 2.1

2.6

1.1


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

41

ISL No. 156073

2.0

4.2

2.0

2.9 2.9

4.3

2.0

4.1

4.0

2.0

4.1

4.0

2.0

B. Afzeli IgM

Babesia IgM

4.3


7.67.6

7.8


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM

5.02.0 3.0

7.8

2.0

2.0

6.0Antigen

Index Value 4.0

Ehrlichia IgM

3.53.5

Normal Abnormal1.0

4.2


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

42

ISL No. 157460

Mycoplasma Ab and PCR positive

Mold Ab (Stachybotrys) 5549 IgG, 5760 IgA

51


52

IgGIgM

All neg


+ +

IgM

+ -

+ -

+ -

+ - + +

+ -

+

ISL No. 157460

Ehrlichia IgG

1.61.6

Normal Abnormal1.0

1.3

6.0Antigen


1.2

1.1

1.0

5.0

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

B. Afzeli IgG

Babesia IgG

1.0


1.01.0

1.2

1.0

1.2

1.1

1.0

1.2

1.1

1.0

1.3

1.1

1.8 1.8

1.0

1.4


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

53

ISL No. 157460

Ehrlichia IgM

1.61.6

Normal Abnormal1.0

1.3

6.0Antigen


1.2

1.1

1.0

5.0

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

B. Afzeli IgM

Babesia IgM

1.0


1.01.0

1.2

1.0

1.2

1.1

1.0

1.2

1.1

1.0

1.3

1.1

1.8 1.8

1.0

1.4


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

54

ISL No. 161409

In 2001 developed symptoms of weakness, stiffness, difficulty in walking, loss of balance in legs, sensitivity to light, decreased hearing.

Musculoskeletal system: muscle pains or cramps, poor muscle coordination, loss of muscle tone, muscle weakness.

Neurologic system: weakness, tremor, poor balance, disturbed sleep.

No diagnosisNo treatmentMany MDs

Positive MBP Antibody

60


61

IgGIgM

+


+

IgM

+ + + +

+ +

++ - + -

+

+

+ +

+

+ - +

++ +

+ - + +

+ -

+

+

+

+

+ + +

+ - +

+

+ -

ISL No. 161409

2.6

2.9

1.8

2.0 2.0

1.8

3.1

1.5

2.1

1.6

1.5

2.1

1.6

B. Afzeli IgG

Babesia IgG

1.8


4.74.7

2.2


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG

5.02.0 3.0

2.2

1.8

2.6

6.0Antigen

Index Value 4.0

Ehrlichia IgG

2.82.8

Normal Abnormal1.0

2.9


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

62

ISL No. 161409

1.9

1.5

1.2

2.1 2.1

1.3

1.8

1.8

1.3

1.5

1.8

1.3

1.5

B. Afzeli IgM

Babesia IgM

1.3


3.03.0

1.6


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM

5.02.0 3.0

1.6

1.2

1.9

6.0Antigen

Index Value 4.0

Ehrlichia IgM

2.12.1

Normal Abnormal1.0

1.5


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

63

ISL No. 172642 TD-14 CAP by CDC WB

67

IgGIgM

ISL No. 172642 TD-14

IgG ELISA 1.5 PositiveIgM ELISA 3.8 Positive

+

+

+

Positive

+

+ +

+ +

+

+ +

+ + ++

ISL No. 172642CAP TD-14 Patient presents with a history of fever and a “rash.” The mother reports that she had been playing outside the previous week, but that no ticks had been observed on the child.Babesia IgM - Positive

ISL No. 172642

Ehrlichia IgG

3.83.8

Normal Abnormal1.0

3.9

6.0Antigen


4.6

3.9

3.5

5.0

LFA IgG

C2 + C6 IgG

VR1+VR2 IgG

B. Garinii IgG


Borrelia Lysate IgG

OSPA IgG

OSPE 1+2 IgG

B. Afzeli IgG

Babesia IgG

1.0


4.14.1

4.6

4.2

4.1

4.1

4.2

4.1

4.1

3.5

3.9

3.9

3.5 3.5

1.0

3.5


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

68

ISL No. 172642

2.2

2.4

3.1

3.1 3.1

1.6

4.2

2.0

2.9

2.0

2.0

2.9

2.0

B. Afzeli IgM

Babesia IgM

1.6


5.55.5

4.4


Borrelia Lysate IgM

OSPA IgM

OSPE 1+2 IgM

LFA IgM

C2 + C6 IgM

VR1+VR2 IgM

B. Garinii IgM

5.02.0 3.0

4.4

3.1

2.2

6.0Antigen

Index Value 4.0

Ehrlichia IgM

3.23.2

Normal Abnormal1.0

2.4


1.0-2.0 Negative

2.1-3.0Intermediate

3.1-5.0 Positive


Index Values

69

IgG IgM IgG IgM IgG IgM156557 2+ 1+ 2+ 6+ Negative Negative Babesia IgG +156834 4+ 1+ 4+ 1+ 4+ 1+ Babesia IgG +156955 4+ 2+ 3+ Ń 6+ 1+ Babesia IgG +153842 1+ 4+ 1+ 4+ 6+ 6+157087 8+ 4+ 4+ 5+ 5+ 5+ Babesia IgG + IgM +156412 8+ 3+ 5+ 2+ 3+ 7+156659 6+ 5+ 4+ 6+ 6+ 10+ Babesia IgG + IgM +156073 1+ 1+ 1+ 1+ 5+ 7+ Babesia IgM +156080 1+ 1+ 9+ 1+ Ń 1+

154926 5+ 2+ 1+ 4+ 2+ 1+ B. garinii160890 3+ 3+ 3+ 4+ 1+ 5+154927 6+ 2+ 4+ 1+ 6+ 3+ Babesia IgG +155963 2+ 4+ 2+ 3+ 5+ 5+ Ehrlichia IgM +157460 Ń Ń 3+ Ń Ń Ń Mold + Mycotoxin +

161409 6+ 2+ 8+ 6+ 5+ 1+ Babesia IgG + IgM + Ehrlichia IgG +

*155961 3+ Ń 3+ Ń 4+ 2+

164873 Ń Ń 1+ 1+ Ń Ń

165054 1+ 1+ 3+ 3+ Ń ŃStachybotrys +

Mycotoxin + H. Pylori +

**156657 4+ 5+ 3+ 3+ 3+ Ń

*156074 2+ 1+ 3+ 5+ 4+ 3+ Babesia Ehrlichia IgG + IgM +

Multi-PeptideSpecimen #

Western Blot CDC Western Blot IgeneXComments

71

Comparison of all methodologies on East Coast specimens

IgG IgM IgG IgM166336 2+ 3+ 4+ 2+165661 3+ Ń 5+ 3+165846 5+ 1+ 4+ 4+165141 5+ 10+ 8+ 6+165148 5+ 3+ 5+ 7+ Babesia +165396 4+ 7+ 3+ 8+ Babesia +164740 2+ 2+ 1+ 3+ Ehrlichia +164967 3+ 4+ 2+ 6+ Babesia +, Ehrlichia +163805 Ń Ń 3+ 5+ Babesia +163959 4+ 3+ 2+ 7+ Babesia +163703 1+ 2+ 1+ 5+ Babesia +162536 Ń Ń 2+ 1+162449 2+ 2+ 1+ 1+ Cross-Reactive Ab162464 5+ 6+ 2+ 8+ Babesia +, Ehrlichia +162525 5+ 5+ 3+ 8+ Babesia +, Ehrlichia +162160 2+ 1+ 3+ 7+ Babesia +162044 5+ Ń 4+ 1+161705 5+ 1+ 2+ 1+ Cross-Reactive Ab159257 2+ Ń 4+ 6+ Babesia +159332 Ń Ń 1+ 5+ Cross-Reactive Ab159340 3+ 1+ 3+ 2+159349 5+ 1+ 2+ 1+159321 1+ Ń 1+ 3+ Babesia +, Ehrlichia +167426 1+ 1+ 1+ 3+

CommentsWestern Blot CDC Multi-Peptide

Specimen #

72

Comparison of all methodologies on West Coast specimens

187801 1+ 0 3+ 2+ 0 0Tic, tremors, Jt, HA, Fatigue

188581 0 0 1+ 3+ 0 0 Unilateral BP

187854 0 0 7+ 5+ 0 1+ BE, JT, Fatigue

188856 0 0 0 2+ Tic, JT, SOB, Fatigue

187714 0 0 1+ 3+ 0 0 Flu, HA, JT, Cog bilateral hypoperfusion

181282 1+ 2+ 2+ 1+ Neuro, Cog, Fatigue hypoperfusion RFL

190408 0 0 2+ 3+ 0 0Palate Paralysis, Cog, Fatigue

190173 3+ 0 3+ 2+ 0 0Tic, Fatigue, Cog, Vaccine

188083 0 2+ 1+ 2+ 0 0 JT, HA, Fagtigue, Cog

188084 2+ 0 6+ 2+ 0 0 JT, HA, Fagtigue, Cog

188374 2+ 0 2+ 0 0 JT, HA, Vision, Cog

189855 0 2+ 2+ 6+ 0 2+ HA, Fever, Fatigue, JT

188301 1+ 0 1+ 1+ 0 0 Anxiety, Fatigue, Tingling

188859 0 0 3+ 4+ 0 0 Muscle spasm, Fatigue

187802 0 0 0 10+ 0 0 Flu, HA, Jt. Pain, Fatigue

188990 0 0 0 3+ 0 0 Muscle spasm, Fatigue

Immunosciences Igenex CDC Symptoms Spect scan ID IgG IgM IgG IgM IgG IgM

189856 0 0 1+ 4+ 0 0 BE, JT, Numbness, Fatigue

179106 3+ 1+ 3+ 2+ JT, HA, Cog, Fatigue global hypoperfusion

188375 0 0 1+ 1+ 0 0 Tic, JT, Flu

189776 0 0 4+ 5+ 0 2+ Fatigue, HA, Cog, JT

188480 0 0 5+ 3+ 0 0 JT effusion, Fatigue, JT

188753 0 0 4+ 3+ 0 0 JT, Fatigue, Cog scattered bilateral defects

187491 0 2+ 5+ 6+ 0 0 Fever, Seizures, Tingling

188082 2+ 0 3+ 6+ 1+ 2+ Tic, Flu, Fatigue, HA

190101 0 1+ 1+ 4+ 0 2+ Fatigue, HA, Cog, JT frontal perfusion defect

Immunosciences Igenex CDC Symptoms Spect scan ID IgG IgM IgG IgM IgG IgM

Discussion Questions:• Why did you choose those particular peptides?

• How do you know that these peptides are expressed in humans?

• How were these applied to the wells? Where were they obtained?

• Is a negative IgG and IgM consistent with the exclusion of the Lyme diagnosis?

• How do you explain the discrepancy between the first group of data and the most recent group? Also, why do you think that there were primarily on B. Lysates showing up in the second group? Would the standard ELISA show the same results? Can this be tested and compared like the Western Blot?

• Are there any other peptide antigens that you have considered and not used? Why?

• Would you expect the values to change as the symptoms improve?

• Does the lack of expression of a particular peptide guide your clinical course? Can you prognosticate the neurological / MS picture?

• When reading the Western Blot, is that just individual ELISA tests?

• Does a positive IgG and IgM Babesia and / or Bartonella negate or contaminate a Western blot? Does it interfere with the peptide panel? Why shouldn’t it?

• What is more indicative of a Lyme diagnosis – a peptide antigen result on one peptide only (IgG or IgM) verses the MELIFA?

Conclusions:• Multi-peptide ELISA may be more specific than Western

Blot

• Multi-peptide ELISA measures antibodies against different peptides that are expressed during the life cycle of Borrelia.

• Multi-peptide ELISA is quantitative.

• Multi-peptide ELISA may exclude antibodies against unrelated Spirochete peptides.

• Multi-peptide ELISA may detect cross-infection with Babesia.

• Multi-peptide ELISA may detect cross-infection with Ehrlichia.

• Sensitivity and specificity of multi-peptide ELISA may be more than 85%

70

Documents

ELISA-BASED PEPTIDE ASSAY FOR SIMULTANEOUS MEASUREMENT OF ANTIBODIES AGAINST B. SENSU STRICTO, B. GARINII, B. AFZELII, AND DETECTION OF CROSS-REACTIVE