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

1
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.

2
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.
3

• 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.
4
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.
5
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

6
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

7
Final Conclusions

• Correlation 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

8
PCR-based methods are insensitive for routine lab
testing due to the absence or low number of
bacteria in clinical samples.
4
5
9
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.
93 66 58 41 39 30 28 23
41 39 23
IgG and IgM antibody pattern with different
peptides of Borrelia burgdorferi in patient
confirmed with Lyme disease.
8
10
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
11
Analysis of the OspE Determinants Involved in
Binding of Factor H and OspE-Targeting Antibodies
elicited during Borrelia burgdorferi Infection in
MiceMetts 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
12
Identification of LFA-1 as a Candidate
Autoantigen in Treatment-Resistant Lyme
ArthritisGross et al., Science, (1998), 281703

• 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 DRB10401. 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 MER

13
13
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),
1635566-5573

• 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
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
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.

• 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.
16
16
Identification and Characterization of
PutativeSecreted Antigens from Babesia
microtiHomer 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

• Babesia microti
• IVEFNAIFSNIDLNNSSTVKNEIIK

• Babesia equi
• DFFHPEDVVAPHSGITTPK

• Babesia bovis
• VEAPWYKRWIKKFRDFFSKNVTQ

17
17
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), 1661114-1124

• 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 C-terminal (AA 272-301)
• VAGAFARAVEGAEVIEVRAIGSTSVMLNAC

• Ehrlichial N-terminal (AA 1-30)
• MSAVSNRKLPLGGVLMALVAAVAPIHSALLA

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ISL No. 156557
42-year-old female

• In 1998, during pregnancy, developed serious
  tension headaches of muscular origin
• Physical exam was normal
• In 2001 developed muscle and joint pains
• Cognitive difficulties
• Sleep deprivation
• Recommended for treatment for Lyme disease

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ISL No. 156557 by IgeneX Western Blot
ISL No. 156557 by CDC Western Blot
IgG
IgM
IgG
IgM








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-
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ISL No. 156557
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ISL No. 156557
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ISL No. 156834
Spect Scan Abnormal Diagnosis CNS Lyme
In 2000, gradually developed the following
symptoms

• 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

• 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

Comments I have been to 45 doctors - every
specialty you can imagine. Please help!
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23
ISL No. 156834 by CDC Western Blot
ISL No. 156834 by IgeneX Western Blot
IgG
IgM
IgG
IgM
-




-







-
-
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24
ISL No. 156834
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25
ISL No. 156834
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ISL No. 156955
Athletic 48-year-old man with suspected Lyme
arthritis and vaccinated with Lymerix. Neuropathy
possibly induced by Lymerix.

• In 1999, developed
• Headaches
• Lightheadedness
• Abdominal discomfort
• Nausea and pain
• Muscle spasm
• 3 months later
• Blurred vision
• Memory impairment
• Difficulty concentrating
• Chronic fatigue
• Poor sleep
• Joint aches and pains
• Freezing sensations in feet, legs and forearms

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
27
ISL No. 156955 by CDC Western Blot
ISL No. 156955 by IgeneX Western Blot
IgG
IgM
IgG
IgM









-

-
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28
ISL No. 156955
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ISL No. 156955
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30
ISL No. 155842
Female diagnosed with Lyme disease.Doxycycline
for two months. Symptoms returned.

• Diagnosed in January 2003. Went
• to Lyme/specialist/neurologist,
• who gave her
• Biaxin Keflex for - 2 months
• Biaxin Plaquenil - 5 months
• She felt better for two weeks but
• milder symptoms returned. Tried
• to go off antibiotics. All symptoms
• returned.
• Major symptoms
• Sore throat, swollen glands
• Red eyes
• Sound sensitivity
• Unexplained weight loss
• Joint pain, swelling or stiffness

• 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.

31
31
ISL No. 155842 by CDC Western Blot
ISL No. 155842 by IgeneX Western Blot
IgG
IgM
IgG
IgM
-
-

-


-
-




-
-



32
32
ISL No. 155842
33
33
ISL No. 155842
34
34
ISL No. 156569
Diagnosed with Lyme disease in 2001Main symptom
Muscle pain,fatigue, sensitivity to light

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

35
35
ISL No. 156569 by CDC Western Blot
ISL No. 156569 by IgeneX Western Blot
IgG
IgM
IgG
IgM



-














-
-


-



-
36
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ISL No. 156569
37
37
ISL No. 156569
38
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
39
ISL No. 156073 by CDC Western Blot
ISL No. 156073 by IgeneX Western Blot
IgG
IgM
IgG
IgM
-
-




-

-
-
40
40
ISL No. 156073
41
41
ISL No. 156073
42
42
ISL No. 157460
Mycoplasma Ab and PCR positive Mold Ab
(Stachybotrys) 5549 IgG, 5760 IgA
51
43
ISL No. 157460 by CDC Western Blot
ISL No. 157460 by IgeneX Western Blot
IgG
IgM
IgG
IgM
-
-

All neg
-

-
-

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44
ISL No. 157460
53
45
ISL No. 157460
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46
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 diagnosis No treatment Many MDs
Positive MBP Antibody
60
47
ISL No. 161409 by CDC Western Blot
ISL No. 161409 by IgeneX Western Blot
IgG
IgM
IgG
IgM
-
-







-





-

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-
-






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ISL No. 161409
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ISL No. 161409
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ISL No. 172642 TD-14 CAP by CDC WB
ISL No. 172642 TD-14
IgG
IgM
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













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ISL No. 172642
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ISL No. 172642
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Comparison of all methodologies on East Coast
specimens
71
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Comparison of all methodologies on West Coast
specimens
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55
Immunosciences Igenex CDC
Symptoms
Spect scan ID IgG IgM
IgG IgM IgG IgM
187801 1 0 3 2 0 0 Tic, 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 0 Palate Paralysis, Cog, Fatigue  
190173 3 0 3 2 0 0 Tic, 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  
56
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

57
(No Transcript)
58
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
  shouldnt it?

59
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