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Laboratory of Immunobiochemistry
Research review
Jay E. Slater, MDOVRR/DBPAP
18 March 2009
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LIB Research Program
Projects Publications Support
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Projects
Rabin Characterization of innate immune
responses to respiratory syncytial virus
Slater Endotoxin in mite extracts Multiplex allergen extract potency
assay
Bacterial endotoxin and DNA in house dust mite cultures and extracts
Cherry ValerioLarry Arlian, PhD
Patrick Murray, PhDBhavini Trivedi, MD
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Initial studies Endotoxins are present in many
standardized allergen extracts Cat and mite > pollens Cat pelt > cat hair D. farinae >> D. pteronyssinus Next step:
Investigate differences between D. farinae and D. pteronyssinus using live mite cultures
Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:777-783.
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Can we detect bacterial DNA in live mite cultures?
Extract genomic DNA from fresh, washed mites
Amplify with 16S rRNA sequences Quantify using internal standards Sequence after high fidelity
amplification Identify predominant organisms
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DNA from mites D. farinae D. pteronyssinus
EcoR1 digests
undigested DNA
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0
20
40
60
80
100
120
0.1 1 10 100DNA (ng)
Ba
nd
de
ns
ity
Df
Dp
TM = 42
Dp Df
slope 0.02 0.02
int 0.57 -0.55
r^2 0.97 0.98
TM equiv (ng) 19.57 1.45
TM number (copies) 1500 1500
copies/ng 77 1033
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16S rRNA sequences recovered
Bartonella species B. henselae B. quintana B. vinsonii B. elizabethae
E. coli Pseudomonas species Acinetobacter species
Uncharacterized -proteobacteria endosymbionts from
Ixodes scapularis Vestimentiferan
tubeworms Brevipalpus
phoenicis Metaseiulus
occidentalis Aspidiotus nerii
Valerio CR, Murray P, Arlian LG, Slater JE. Bacterial 16S ribosomal DNA in house dust mite cultures. J Allergy Clin Immunol. 2005; 116:1296-300.
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Bartonella organisms Gram-negative
rods 0.6 by 1.0 m facultative intracellular fastidious
Harbored by Lice Fleas Ticks Hippoboscidae
flies (house dust mites)
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Bartonella-associated diseases Zoonotic: cat-scratch disease (B. henselae) Louse-borne (Pediculus humanus) (B. quintana)
trench fever urban trench fever
Sandfly-borne (Phlebotomus) (B. bacilliformis) Oroya fever (Carrion’s disease) verruga peruana
Uncertain transmission (B. henselae and B. quintana)
bacillary angiomatosis bacillary peliosis culture-negative endocarditis
Emerg Infect Dis 1995; 1(1):16-21.N Engl J Med 1997; 337(26):1916-7.
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Conclusions (1) D. farinae allergen extracts contain more
endotoxin than D. pteronyssinus extracts No evidence of adverse events associated with
endotoxin in allergen extracts Culture data uninformative Analysis of amplified mite DNA suggests
the presence of about 10-fold more bacterial DNA in D. farinae than in D. pteronyssinus
Sequence analysis of recovered bacterial DNA indicates the presence of Bartonella species as well as other Gram-negative organisms No evidence of iatrogenic infection
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Next questions
Are the bacterial DNA sequences detectable in commercial allergen extracts?
Are the bacterial DNA sequences detectable wild mite species?
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Are the bacterial DNA sequences detectable in commercial allergen extracts? Methods
DNA isolation by QIAamp
PCR
Sequence
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Are the bacterial DNA sequences detectable in commercial allergen extracts?
DNA present? fD1 fD2D farinae (13) 12/13 5/13 6/13D pteronyssinus (14) 12/14 0/14 0/14Cat hair (2) 0/2 0/2 0/2Cat pelt (2) 0/2 0/2 0/2German roach (3) 2/3 1/3 1/3Honeybee venom 0 0 0Bermuda grass 0 0 0Ryegrass 0 0 0
PCR
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Are the bacterial DNA sequences detectable wild mite species?
Chortoglyphus arcuatus Lepidoglyphus destructor Euroglyphus maynei Acarus siro Tyrophagus putrescentiae
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Are the bacterial DNA sequences detectable wild mite species?
Extract genomic DNA (DNAzol) from fresh, washed mites
Amplify with 16S rRNA sequences Sequence after high fidelity
amplification (pfx) Identify predominant organisms
(BLAST)
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16S rRNA sequences recovered
Forward Reverse Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD
A. siro Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella australis strain Aust/NH1 Bartonella sp.
C. arcuatus Bartonella clarridgeiae strain M9HN-SHQ Bartonella clarridgeiae strain M9HN-SHQ Bartonella sp. Bartonella rattiaustraliensis strain AUST/NH14 Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD
E. maynei Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella henselae strain Houston-1 Bartonella henselae strain M40SHD Bartonella henselae strain M40SHD
L. destructor Bartonella henselae strain 882_ANT5 Bartonella henselae strain 882_ANT5 Bartonella henselae strain Houston-1 Bartonella henselae strain Houston-1
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Conclusions
D farinae endotoxin content is high, and associated with the presence of Bartonella DNA
Confirmed in Mites Mite extracts One wild mite species (C arcuatus)
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Next steps
Population analyses Bartonella culture Endotoxin analyses
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An Antibody-Based Multiplex Bead Assay to Determine the Potency and Composition of Allergen Extracts
Nicolette deVore, PhD
Jonny Finlay, PhD
Susan Huynh
Ekaterina Dobrovolskaia
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How do we measure potency?
Total protein (hymenoptera) Overall allergen (grasses, mites)
Pooled human antibody Specific allergen (cat, ragweed)
Sheep antibody
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Specific loss of a single allergen
0
0.1
0.2
0.3
0.4
0.5
-6-4-20
log dilution
resp
onse
(a
bsor
banc
e)
Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. The stability of house dust mite allergens in glycerinated extracts, J Allergy Clin Immunol 2000;105:482-488.
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The dilemma of these potency measures:
In order to measure specific allergens, we need to know which allergens are relevant
If we measure overall allergenicity, we are unable to detect the absence of specific (and potentially important) allergens
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Two possible solutions:
Divide the signal by
Separating the allergens, or
Separating the antibodies
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An assay that will do both
Identify currently known allergens And recognize potentially important allergens yet to be identified
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Aims To develop an multiplex antibody-
based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed
Apply this technique to German cockroach allergen standardization
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Aims To develop an multiplex antibody-
based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed
Apply this technique to German cockroach allergen standardization
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To produce recombinant antibodies
Step 1. Inject chicken with allergen mixture of interest.
Step 2. Once a strong immune response is detected, Remove bone marrow and spleen and purify total RNA
Step 3. PCR is performed to amplify antibody repertoire.
Step 4: PCR products are digested with Sfi I and ligated into a vector.
Step 5: Plasmid containing antibody library is then electroporated into F´ E. coli along with helper phage. The scFv is then expressed on the PIII coat protein attached to the phage
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scFvs are then screened at both protein and DNA level
1 7 8 9
Amb a 1 clones vs ragweed and cat hair extract
**
*
*
0
0.5
1
1.5
1 2 3 4 5 6 7 8 9 10 11 12
cont
rol
clone number
OD
450
nm
ragweed
cat hair
PCR and restriction digest of select clones
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Recombinant antibodies recognize specific allergens
F10 F11 F46 F118 F124
Anti-Fel d 1 clone number
Amb a 1
ragweed
Fel d 1
cat hair
0
0.5
1
1.5
2
2.5
F38 F 7 F 17
OD
450
(n
m)
3.0
Anti-Amb a 1 clone number
0
0.5
1
1.5
2
2.5
A8 A9 A23 A 24 A 55 A 107 A 119 A 113 A 121
OD
450
(n
m)
Amb a 1
ragweed
Fel d 1
cat hair
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Aims To develop an multiplex antibody-
based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed
Apply this technique to German cockroach allergen standardization
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The surface of each bead is coated with carboxylic acid groups.
Using EDC and sulfo-NHS, recombinant antibodies can be covalently bound to the bead surface via an amide bond.
Multiplex microbead technology
o
0
c N
O
OO
S
O
O
O-
C
O
C
O
NH2
0
EDC +Sulfo NHS
Sulfo-NHS estherCarboxy labeled bead scFv attached via amide bond
scFv
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Each bead type can be bound to recombinant antibodies with different specificities.
Multiplex microbead technology
www.bio-rad.com
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Up to 100 different bead types can be combined into a single well of a 96-well plate
Multiplex microbead technology
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Assay design
•Each well contains the same mixture of six different beads bound to six different anti-Feld 1 recombinant antibodies
•12 2-fold dilutions of each extract are added to each well of each row
Extract dilutions Streptavidin – RPE
Anti-rabbit biotin
Fel d 1 specific rabbit sera
Fel d 1 in allergenic extract
scFv bound to
Carboxy labeled bead
E4 standardCat hair
Company ACat hair
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The beads are drawn up single file into the detection chamber
Here the sample is hit with two lasers:
• A 635nm laser excites the dyes within the bead.
• The dyes emit distinct photons. • Photons are detected and the
ratioof photon wavelengths emitted is calculated to determine the bead
type.• A 532 nm laser detects the RPE
bound to the bead.• Output consists of median
fluorescence index (MFI) of each bead-type in each well.
Multiplex array technology
Luminex 200, Luminex Corp.
635 nm
532 nm
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Analyzing dose response curves
-5 -4 -3 -2 -1 0
0
10000
20000
30000
maximum
minimum
EC50
slope
MF
I
Allergen extract (log dilution)
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Relative potencies
Relative potency = EC50 standard / EC50 sample
Log EC50
-5 -4 -3 -2 -1 00
5000
10000
15000
20000
25000
30000
35000Standard cat hairSample cat hair
standard sample LOGEC50 -2.46 -2.74 EC500.0034 0.0018
rp = .0034/.0018 = 1.8
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Aims To develop an multiplex antibody-
based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed
Apply this technique to German cockroach allergen standardization
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Summary of anti-Amb a 1 data
The average calculated potencies of ragweed extract vary greatly when anti-Amb a 1 scFvs are used alone or in groups
The potency of some ragweed extracts can be accurately computed from extracts with known potencies using the microbead method.
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Potencies of ragweed extracts obtained using bead assay are consistent with manufacturer data
Extract RID Microbead assay
I 111 28 109 20
II 205 51 248 9
III 209 52 152 12
IV 107 27 100 1
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Summary of anti-Fel d 1 data
When anti-Fel d 1 scFvs are used alone or in groups the average calculated potencies of cat hair extracts are similar
Potency of cat hair extracts can be accurately computed from extracts with known potencies using the microbead method.
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Comparing microbead data to RID data for cat hair extracts
Extract RID Microbead assay
V 4 1 2 0
VI 6 2 6 0
VII 18 5 16 2
VIII 7 2 5 1
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Aims To develop an multiplex antibody-
based method for profiling complex allergen mixture Antibodies Assay development Apply to cat and ragweed
Apply this technique to German cockroach allergen standardization
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test new anti-cockroach scFvs
0.000
0.500
1.000
1.500
2.000
2.500
6A1
6A2
6A3
6B11
6B12
6G2
4B7
6E1
2A1
1E11
1D9
clone number
OD
450
(n
m)
positive
negative
•Selection of 250 positive clones•DNA sequencing of 250 clones: 150 unique clones •Select 85 clones to express in a soluble form and analyze by ELISA•Select 50 best clones to purify
Summary of the work performed by Millegen
Data from Millegen Labege, France
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Future experiments
Binding of soluble scFv’s to bead-bound known allergens
Inhibition assays using known allergens Analysis of scFv binding patterns in
Western blots Identification of scFv-recognized
antigens by N-terminal sequencing
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Publications: Rabin Le Nouën C, Munir S, Losq S, Winter CC, McCarty T, Stephany DA,
Holmes KL,Bukreyev A, Rabin RL, Collins PL, Buchholz UJ. Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3. Virology. 2009 Mar 1;385(1):169-82.
Mane VP, Heuer MA, Hillyer P, Navarro MB, Rabin RL. Systematic method for determining an ideal housekeeping gene for real-time PCR analysis.J Biomol Tech. 2008 Dec;19(5):342-7.
Chi B, Dickensheets HL, Spann KM, Alston MA, Luongo C, Dumoutier L, Huang J, Renauld JC, Kotenko SV, Roederer M, Beeler JA, Donnelly RP, Collins PL, Rabin RL. Alpha and lambda interferon together mediate suppression of CD4 T cells induced by respiratory syncytial virus. J Virol. 2006 May;80(10):5032-40.
Zhang M, Drenkow J, Lankford CS, Frucht DM, Rabin RL, Gingeras TR, Venkateshan C, Schwartzkopff F, Clouse KA, Dayton AI. HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro. J Leukoc Biol. 2006 Jun;79(6):1328-38.
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Publications: Rabin Zhang J, Alston MA, Huang H, Rabin RL. Human T cell cytokine
responses are dependent on multidrug resistance protein-1. Int Immunol. 2006 Mar;18(3):485-93.
Song K, Rabin RL, Hill BJ, De Rosa SC, Perfetto SP, Zhang HH, Foley JF, Reiner JS, Liu J, Mattapallil JJ, Douek DC, Roederer M, Farber JM.Characterization of subsets of CD4+ memory T cells reveals early branchedpathways of T cell differentiation in humans. Proc Natl Acad Sci U S A. 2005 May 31;102(22):7916-21.
Gupta N, Arthos J, Khazanie P, Steenbeke TD, Censoplano NM, Chung EA, Cruz CC, Chaikin MA, Daucher M, Kottilil S, Mavilio D, Schuck P, Sun PD, Rabin RL, Radaev S, Van Ryk D, Cicala C, Fauci AS. Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy. Virology. 2005 Feb 20;332(2):491-7.
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Publications: Rabin (reviews) Rabin RL, Levinson AI. The nexus between atopic disease and
autoimmunity: a review of the epidemiological and mechanistic literature. Clin Exp Immunol. 2008 Jul;153(1):19-30.
Rabin RL. Recombinant and modified allergens: the U.S. perspective.Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2006;(95):191-3;discussion 193-4.
Rabin RL. Regulation of allergenic products in the United States: The promise and problem of adjuvants in allergen immunotherapy. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2009, in press.
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Publications: Slater Slater JE, James R, Pongracic JA, Liu AH, Sarpong S, Sampson HA,
Satinover SM, Woodfolk JA, Mitchell HE, Gergen PJ, Eggleston PA.Biological potency of German cockroach allergen extracts determined in an innercity population. Clin Exp Allergy. 2007 Jul;37(7):1033-9.
Soldatova LN, Tsai C, Dobrovolskaia E, Marković-Housley Z, Slater JE.Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells. Allergy Asthma Proc. 2007 Mar-Apr;28(2):210-5.
Padavattan S, Schirmer T, Schmidt M, Akdis C, Valenta R, Mittermann I,Soldatova L, Slater J, Mueller U, Markovic-Housley Z. Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab. J Mol Biol. 2007 May 4;368(3):742-52.
Finkelman MA, Lempitski SJ, Slater JE. beta-Glucans in standardized allergen extracts. J Endotoxin Res. 2006;12(4):241-5.
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Publications: Slater Dobrovolskaia E, Gam A, Slater JE. Competition enzyme-linked
immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture. Clin Exp Allergy. 2006 Apr;36(4):525-30.
Valerio CR, Murray P, Arlian LG, Slater JE. Bacterial 16S ribosomal DNA in house dust mite cultures. J Allergy Clin Immunol. 2005 Dec;116(6):1296-300.
Finlay WJ, deVore NC, Dobrovolskaia EN, Gam A, Goodyear CS, Slater JE.Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins. Clin Exp Allergy. 2005 Aug;35(8):1040-8.
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Publications: Slater (reviews) Slater JE. Standardized allergen vaccines in the United
States.Clin Allergy Immunol. 2008;21:273-81.
James R, Mitchell H, Gergen PJ, Eggleston PA, Slater JE. Analyzing of ID50EAL data for the standardization of German cockroach allergen extracts in the U.S.Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2006;(95):117-27;discussion 127, 155.
Slater JE. Characterization of allergen extracts. Dev Biol (Basel). 2005;122:145-52.
Slater JE. A global view of allergenic product potency. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2009; in press.
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LIB research support
FY source
2008 2009
Intramural $211,000 $275,000
Critical Path $178,000 $90,000
Extramural $290,000
Laboratory of Immunobiochemistry
site visits
January 2002
June 2006
[June 2010]
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LIB Research Program
Projects Publications Support