Laboratory of Immunobiochemistry
Allergenic Products Advisory Committee, April 8, 2003
Allergenic Products Advisory Committee, April 8, 2003
Lab overview Staffing Lot release Reference replacement
Operational issues Sheep serum replacement issues ISO certification report
Research/regulatory update Endotoxin studies Cockroach antigens and antibodies
Lab overview Staffing Lot release Reference replacement
Principal Investigators Jay E. Slater, MD, Lab Chief – Supervisory
Medical Officer (4) Ronald Rabin, MD - Senior Staff Fellow (2)
Post Doctoral Fellows
Jonny Finlay, PhD - IRTA (2) Bo Chi, MD - Visiting Associate (<1)
Research Technicians Albert Gam – Biologist (2) Mona Febus – Microbiologist (3) Marc Alston – Biologist (2) Cherry Valerio – Biologist (2) Katia Dobrovolskaia – Visiting associate (2)
LIB staffing 1998-2003
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“Routine” regulatory activities Lot release Reference distribution Reference maintenance
semiannual checks replacement
Lot release activities 357 protocols submitted and
reviewed 1 withdrawn
Reference distribution 2002: 1978 vials in 107 shipments
sent to manufacturers
Lot release protocols submitted
Year Protocols
1999 4762000 4012001 4062002 357
Reference distribution
Year Shipments Vials
1999 104 19832000 146 24762001 101 21512002 107 1978
Laboratory of Immunobiochemistry
Operational issues
Operational issues Replacement of cat and ragweed
antisera
Transition to ISO compliance
Ragweed and cat antisera need to be replaced
S2a cat Released in 1998
S6 ragweed Released in 2000
Replacement programs for both initiated spring 2002
Immunization protocol
Allergen: Ragweed CatEwe #: 6747 7777
14-Jun-2002 Pre-bleed Plasmapheresis17-Jun-2002 Dose 1: 25 mcg Amb a 1 in CFA Dose 1: 25 mcg rFel d 1 in CFA
15-Jul-2002 Dose 2: 25 mcg Amb a 1 in IFA Dose 2: 25 mcg rFel d 1 in IFA
7-Aug-2002 Bleed Bleed12-Aug-2002 Dose 3: 25 mcg Amb a 1 in IFA Dose 3: 25 mcg rFel d 1 in IFA
4-Sep-2002 Bleed Bleed9-Sep-2002 Plasmapheresis Plasmapheresis
6/1 7/1 7/31 8/30 9/29
Immunization protocol
plasmapheresis
Immunization doses
6/1 7/1 7/31 8/30 9/29
6/1 7/1 7/31 8/30 9/29
Transmissible spongiform encephalopathies Animal
scrapie (sheep and goats) chronic wasting disease (mule deer,
elk) transmissible mink encephalopathy bovine spongiform encephalopathy
feline spongiform encephalopathy
Transmissible spongiform encephalopathies Human
kuru Creutzfeldt-Jakob disease
classic sporadic familial iatrogenic new variant (a/w bovine TSE)
Gerstmann-Sträussler-Scheincker fatal familial insomnia sporadic fatal insomnia
Scrapie In Europe for >250 years In US since 1947
>1000 flocks Vertical transmission Horizontal transmission, presumably by
contamination with placenta and blood during lambing season
Long incubation period No human transmission
Prion polymorphisms associate with different susceptibility, especially at codon 171 QQ (glutamine/glutamine) susceptible RR (arginine/arginine) resistant QR (glutamine/arginine) intermediate
Scrapie eradication program Preclinical testing and surveillance
Live animal test (third eyelid biopsy) May take months to years to turn positive
Tracking of infected and exposed animals
Cleanup strategies: identify and genotype exposed animals Destroy QQ exposed QR or RR exposed are tracked but may be
slaughtered for human consumption
Not believed to pose any risk to humans No recognized human transmission in three
centuries of exposure in Europe This serum is safe to use because
No documented transmission to humans Contact with affected sheep was limited
Not in adjoining pens; >30 feet distant Normal BSL 2 precautions in place for all work
with animal sera However, in order to maintain a serum
reagent that is as safe as possible…
Current approach… Begin immunizing two new sheep Process plasma from ewes 6747
and 7777 now Conserve current stocks
Current approach… If new sera are available before we
run out, sera from 6747 and 7777 will be saved frozen for possible future use
If we have a shortfall, sera from 6747 and/or 7777 will be used until the new sera are available
Current approach… Advantages
Highest degree of safety Large supplies for future
Disadvantages Possibility of two serum switches in
short period Time Expense
CBER Laboratory Quality Management Initiative
Seek ISO-17025 compliance for official product testing ISO – International Organization for
Standardization, Geneva, Switzerland 17025: “General requirements for the
competence of testing and calibration laboratories” (1999)Set of guidelines for labs that do testing and calibration to show operation of a quality system to assure technical competence and production of valid results
CBER Laboratory Quality Management Initiative
Establish policy with Center level Quality Manual
Audit for compliance with ISO-17025
Obtain Test/Lab Accreditation where appropriate “Accreditation – successful 3rd party audit
Why is CBER becoming ISO compliant? Establish recognized competence, assure
the trust and value of our data and processes
We require the manufacturers’ labs to be in GMP compliance International efforts at harmonization are
attempting to equate GMP and ISO requirements
ISO is an internationally recognized standard
Why is CBER becoming ISO compliant? (continued) Implement Laboratory Quality
Management policies and practices for official testing activities To document a high level of training,
competence, and proficiency To establish a consistent product
testing process
Elements of ISO compliance Management of
People Equipment Documents Processes
Elements of ISO compliance Management of People
Defined roles Appropriate training Demonstrated proficiency
Initial and ongoing Authorization process
Elements of ISO compliance Management of Equipment
Program for equipment calibration Maintenance Process to assure that only calibrated
and maintained equipment is used Assurance of measurement
traceability to accepted standards
Elements of ISO compliance Management of Documents
Includes policies, procedures, specifications, equipment manuals, certifications
Must be reviewed, issued and controlled
Approved and issued prior to use
Elements of ISO compliance Management of Processes
Approval of testing materials Environmental specifications and monitoring Handling of samples Validation and suitability Handling of data (including non-conforming
data) Corrective action, Preventive action systems Internal Audits Management review Recording and handling complaints
CBER’s commitment to implementation of a Lab Quality System
Establishment of CBER Quality Board Development of Quality Assurance
structure within CBER Appointment of Center Level Quality Manager Hiring Office quality Managers Appointment and hiring of Division Quality
Coordinators Preparation of CBER Quality Manual Purchase of Integrated Quality
Management computer software
What will this mean to LIB? No substantive protocol revisions Documentation
Formatting Substance
Formal separation of research and regulatory equipment
Internal audits External audits
Timetable (tentative) Implementation in stages over the
next 3 years… Seek accreditation in 2005 Software operational during 2003 Policies and Quality Manual issued 2003 Initiation of compliance audits (ongoing) Training and process development
(ongoing)
Laboratory of Immunobiochemistry
Research/regulatory update
Active research projects PI Slater
Cockroach allergen standardization Determination of optimal surrogate test Depletion analysis of CR extracts
Cockroach IgE combinatorial library Endotoxin in allergen vaccines
PI Rabin MDR proteins in T cell activation RSV responses in human tonsil
Publications
Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen vaccines. Clin Exp Allergy 2002;32:721-727
Sutherland MF, Drew A, Rolland JM, Slater JE, Suphioglu C, O'Hehir RE. Specific monoclonal antibodies and human IgE show Hev b 5 is an abundant allergen in high protein powdered latex gloves. Clin Exp Allergy 2002;32:583-589
Trivedi B, Valerio C, Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003 (in press).
Publications (reviews)
Lockey RF, Slater JE, Esch R. Preparation and standardization of allergen vaccines. In Middleton’s Allergy: Principles and Practice, 6th ed. St. Louis: Mosby (in press).
AbstractsRabin RL, Alston MA, Huang H, Slater JE. Cytokine secretion by
activated T cells is dependent on multidrug resistance protein-1 (MRP-1). J Allergy Clin Immunol 2003; 111:S153.
Valerio C, Slater JE. The effects of lipopolysaccharide (LPS) on
immune responses in C57Bl/6 mice. J Allergy Clin Immunol 2003; 111:S166.
Slater JE, Valerio C, Trivedi B. Endotoxin in standardized
allergen vaccines. J Allergy Clin Immunol 2003; 111:S243. Finlay WJJ, Rabin RL, Slater JE. Analysis of IgE heavy chain V-
gene usage in human tonsil. J Allergy Clin Immunol 2003; 111:S313.
Endotoxin content of allergen vaccines Allergenic extracts are not
required to undergo evaluation for the presence of pyrogens (21CFR 610.13(b))
Prior studies confirmed variable endotoxin content (Siraganian, et al. J Allergy Clin Immunol 1979; 64:526-533)
Products exempted in 21 CFR 610.13(b) Blood products Horse serum Bacterial, rickettsial and viral
vaccines Toxoids Toxins Allergenic extracts
LAL gel-clot method
LAL gel-clot method
Factor C Act. factor C
Factor B
Proclotting enzyme
Clotting enzyme
Factor GAct. factor B Act. factor G
Coagulogen Coagulin
ENDOTOXIN
–D- glucan
Endotoxin content of allergen vaccines – possible interference
Non-endotoxin (1,3)--D-glucans may induce clotting by an alternative pathway in the standard LAL assays
Proteases (especially in cat and mite extracts) may also induce clotting
Endotoxin content of allergen vaccines - approach Determine endotoxin content using
gel-clot method
Assess the contribution of non-endotoxin components ((1,3)--D-glucans, enzymes)
Study design Standardized allergen vaccines LAL gel-clot assay Adsorption of selected allergens with
ENP-silica resin, followed by LAL assay Endotoxin neutralizing protein (ENP) is a
~12 kDa, cationic, amphipathic protein that binds to and neutralizes the biological activity of lipopolysaccharide.
Pre-treat selected allergens at 95°C for 15 min, followed by LAL assay
Extract ManufacturerTotal gel-clot
activityDepleted gel-clot
activity
Corrected endotoxin content
(EU/mL)
Bermuda A 6--12 <6 8Bermuda E 12--24 Not tested 17Bermuda F 6--12 <6 8
June Grass A 12--24 <6 17June Grass B 60-120 6--12 76June Grass J 120-240 0 170
Meadow Fescue A 6--12 <6 8Meadow Fescue C 12--24 <6 17Meadow Fescue D 24-48 0 34Meadow Fescue F 12--24 0 17
Orchard A 60-120 <6 85Orchard C 12--24 <6 17Redtop E 24-48 <6 34Redtop G 24-48 <6 34
Ryegrass A 120-240 <6 170Ryegrass C 600-1200 320-600 410Ryegrass E 480-960 0 679Ryegrass F 12--24 Not tested 17Ryegrass G 1200-2400 96-192 1561
Sweet Vernal A 0.6-12 <6 3Sweet Vernal D 24-48 <6 26Sweet Vernal F 240-480 0 339
Timothy A 60-120 6--12 76Timothy D 6--60 6--12 10
GP
GP
GP
GP
GP
GP
GPGP
GP
GG
GG
G
G
G
G
G
G
GG
GG
P
Extract ManufacturerTotal gel-clot
activityDepleted gel-clot
activity
Corrected endotoxin content
(EU/mL)
Ragweed Mix A 48-60 12--24 37Ragweed Mix B 60-120 32-60 41
Short Ragweed A 6--12 Not tested 8Short Ragweed B 60-120 32-60 41Short Ragweed C 600-1200 24-48 815Short Ragweed D 240-480 48-96 339Short Ragweed E 60-96 32-60 76Short Ragweed F 6--60 Not tested 19Short Ragweed J 1200-2400 0 1697
GP
G
G
G
GG
P
P
P
Extract ManufacturerTotal gel-clot
activityDepleted gel-clot
activity
Corrected endotoxin content
(EU/mL)
Cat Pelt A 4800-6000 60-600 5177Cat Pelt A 24000-48000 96-192 33805Cat Pelt G 4800-6000 60-600 5177Cat Pelt G 4800-9600 6--12 6780Cat Hair B 240-480 0 339Cat Hair C 0.06-6 Not tested 1Cat Hair E 12000-24000 <6 16962Cat Hair F 600-1200 0 849Cat Hair H 240-480 6--60 339Cat Hair I 0.06-6 Not tested 1Cat Hair J 1200-2400 6--12 1689
GGG
GGG
G
GP
GP
Extract ManufacturerTotal gel-clot
activityDepleted gel-clot
activity
Corrected endotoxin content
(EU/mL)
D.farinae A 6000-12000 60-600 8296D.farinae B 1200-2400 <6 1697D.farinae D 6000-12000 0 8485D.farinae E 4800-9600 0 6788D.farinae G 4800-6000 6--60 5367D.farinae I 600-1200 <6 849D.farinae J 600-1200 0 849
D.pteronyssinus A 6--12 <6 8D.pteronyssinus B 0.6-6 Not tested 2D.pteronyssinus C 0 Not tested 1D.pteronyssinus D 0.6-6 Not tested 2D.pteronyssinus G 6--60 Not tested 19D.pteronyssinus I 6--12 <6 8D.pteronyssinus J 24--48 Not tested 34
GGGGG
G
GGGGG
G
GP
GP
0%
20%
40%
60%
80%
100%
Gel
-clo
t act
ivity
from
(1,
3)-
-D
-glu
cans
Heat inactivationHeat inactivated
Extract Manufacturer EU/ml EU/ml
Ryegrass G 1200-2400 1200-2400Short Ragweed J 1200-2400 1200-2400Cat Pelt A 24000-48000 24000-48000Cat Pelt G 4800-9600 4800-9600D. farinae D 6000-12000 6000-12000D. farinae E 4800-9600 4800-9600D.pteronyssinus G 24-48 24-48D.pteronyssinus J 24-48 24-48
0
1
10
100
1000
10000
100000C
orr
ecte
d e
nd
oto
xin
co
nte
nt
(EU
/mL
)
0
1
10
100
1000
10000
100000C
orr
ecte
d e
nd
oto
xin
co
nte
nt
(EU
/mL
)
p = 0.02 p = 0.0003
Conclusions The observed LAL gel-clot activity
probably represents real endotoxin content, not -glucan or protease activity
Is this amount of endotoxin physiologically significant? Mean endotoxin content: 1,900
EU/mL (allergen immunotherapy dose 0.5 – 2.0 mL per month) 40 – 50 EU/kg (2,800 to 3,500 EU)
(administered IV) can elicit a rise in temperature, heart rate, and white blood cell count
Wolff SM. J Infect Dis 1973; 128:Suppl-64.
Michie HR, et al. N Engl J Med 1988 Jun 9 318;1481-6.
Specific endotoxin limits Product-specific Generally based on
Drug dose 5.0 EU/kg limit
Specific endotoxin limits
Product Limits (EU/dose)Vaccines 25 - 3000Penicilloyl polylysine 583*Doxorubicin HCl 285*Fentanyl citrate 80*Insulin (human) 56*
* Based on USP limits and estimated maximum therapeutic doses
The clinical consequences of endotoxin in allergen vaccines have not been studied
No data that adverse events from IT are associated with endotoxin levels
No data to support a beneficial effect of endotoxins
Future studies of allergen IT should be controlled for endotoxin dose
Role of endotoxin in safety and efficacy of IT should be assessed
Conclusions Pollens < cat and mite Cat hair < cat pelt D. pteronyssinus << D. farinae
Bioburden? Endogenous heat-stable ENP-binding
LAL activator in D. farinae? Endogenous ENP in D. pteronyssinus?
Plan Expand study of endotoxin content
Additional standardized and non-standardized extracts
Different methods (GC mass spec) Investigate differences between D.
farinae and D. pteronyssinus
Cockroach allergen standardizationClinical studiesDeveloping the appropriate surrogate
CorrelationDepletion studies
IgE combinatorial library to cockroach
Cockroach allergen standardization Clinical studies Developing the appropriate surrogate
Correlation Depletion studies
IgE combinatorial library to cockroach
Cockroach allergen standardization Clinical studies Developing the appropriate surrogate
Correlation Depletion studies
IgE combinatorial library to cockroach
Current standardized allergens D. farinae D. pteronyssinus Cat hair Cat pelt Short ragweed pollen Hymenoptera
Honey bee Wasp Yellow jacket Yellow hornet White-faced hornet Mixed vespid
Grass pollens Bermuda grass Red top June (Kentucky
blue) Perennial rye Orchard Timothy Meadow fescue Sweet vernal
Allergen standardization Establish a US standard, and Establish a testing procedure
Manufacturers may use the established procedure, or may develop equivalent procedures
Which allergens should be standardized? Impact criteria… Availability of stable, preferably lyophilized
material for use as long-term reference extracts. Consistency of currently marketed product. Widespread use as a diagnostic and/or
therapeutic reagent in the U.S. Number of manufacturers producing the product. Potential use in immunotherapy or diagnostics. Public health impact of correct diagnosis and/or
adequate treatment.
Allergens and asthma
Indoor allergens dust mites* cat* cockroach molds dog
Outdoor allergens molds * = already standardized
Cockroaches
Why is cockroach allergy important?
Ubiquitous Difficult to control Associated with asthma
Why is cockroach allergen standardization important? To the patient
More accurate diagnosis Safer and more effective immunotherapy
To the physician/scientist Better science (if you can’t measure it, you
can’t study it…) Pathophysiology Epidemiology Environmental control
To the FDA Safer, more effective product
Phase I - Laboratory
Develop/adapt methods for allergen determination
Compare allergen content of different lots
Goals Determine consistency of available
US products: protein content specific allergen content overall allergenicity
Determine best lot release measures
Extracts used as reference E2-Cg and E2-Ca Previously characterized Limited skin test data Lyophilized; available in large
quantities
Cockroach extracts studied
From all nine allergen extract manufacturers
aqueous 50% glyAmerican 4 11German 2 9
Relative Potency of All Cockroach Extracts (determined by competition
ELISA)
RP RP (scaled)
American Mean 0.07 0.05SD 0.05 0.04
Range 0 to 0.15 0 to 0.12
German Mean 0.05 0.22SD 0.06 0.19
Range 0 to 0.18 0.01 to 0.46
Aqueous Mean 0.00 0.03
SD 0.00 0.05Range
Gly
ce
rin
ate
d
Relative Potency of All Cockroach Extracts (determined by competition
ELISA)
RP RP (scaled)
American Mean 0.07 0.05SD 0.05 0.04
Range 0 to 0.15 0 to 0.12
German Mean 0.05 0.22SD 0.06 0.19
Range 0 to 0.18 0.01 to 0.46
Aqueous Mean 0.00 0.03
SD 0.00 0.05Range
Gly
ce
rin
ate
d
Relative Potency of All Cockroach Extracts (determined by competition
ELISA)
RP RP (scaled)
American Mean 0.07 0.05SD 0.05 0.04
Range 0 to 0.15 0 to 0.12
German Mean 0.05 0.22SD 0.06 0.19
Range 0 to 0.18 0.01 to 0.46
Aqueous Mean 0.00 0.03
SD 0.00 0.05Range
Gly
ce
rin
ate
d
Relative Potency of All Cockroach Extracts (determined by competition
ELISA)
RP RP (scaled)
American Mean 0.07 0.05SD 0.05 0.04
Range 0 to 0.15 0 to 0.12
German Mean 0.05 0.22SD 0.06 0.19
Range 0 to 0.18 0.01 to 0.46
Aqueous Mean 0.00 0.03
SD 0.00 0.05Range
Gly
ce
rin
ate
d
Bla g 1 and Bla g 2 Levels in glycerinated German cockroach
Bla g 1 (U/mL) Bla g 2 (g/mL) Mean 3503 44 SD 1094 22 Range 2218 – 4854 8 - 66 E2 – Cg 13829 8
Correlation of protein concentrations and ELISA results
Protein concentration
Bla g 1 levels Bla g 2 levels
Relative potency
R2 = 0.83R
2 = 0
.92
R2 = 0.93
R2 =
0.9
6
R2 = 0.92
R 2 = 0.92
Conclusions Commercially available cockroach
allergen extracts: vary widely in protein content, Bla g 2
content, SDS-PAGE banding pattern, and overall allergenicity.
appear to be less potent and contain less Bla g 1 than the candidate reference extracts
Conclusions Established that cockroach
allergen vaccines need to be standardized
What we need now… New cockroach references Characterize the references ID50EAL testing (Intradermal Dilution for
50 mm Sum of Erythema Determines
Bioequivalent Allergy Units) Proceed to next phase
Phase II - Clinical
skin testing, histamine release, IT data
establish biological unitage and ideal dosing ranges
ID50EAL testing
Proficiency Recruitment Testing Analysis
ID50EAL testing
Recruitment Inclusion criteria
18 to 65 years of age history of allergic disease, such as
allergic rhinitis, related to exposure to the allergen of interest
puncture sum of erythema diameter responses (ΣE) to the allergen concentrate of ≥30 mm.
Recruitment Exclusion criteria
Asthma with use of systemic steroids in the past 12 months
Peak flow < 75% predicted at the time of testing Skin coloring or condition that would preclude the
measurement of erythema responses Dermographism (> 4 mm ΣE following saline skin test) Immunotherapy – past or present - with the test
allergen Current use of antihistamines, tricyclic
antidepressants, MAO inhibitors, and beta-blockers
How many study subjects?
22/11
2
2
zzn
n 1.0 0.05 26
0.20 181.5 0.05 59
0.20 392.0 0.05 104
0.20 69
Rabin et al., Sample Size Considerations for Establishing Clinical Bioequivalence of Allergen Formulations. Arb.Paul Ehrlich Inst.Bundesamt Sera Impfstoffe Frankf.A.M, in press
for D50 should be 10%
BAU/mL= 3-(14 - mean D50) * 100,000
D50 BAU/mL % change14 100000
12.6 21480 -79%15.4 465554 366%
10 12359 412 -67%
11 3704 200%8 137
7.2 57 -58%8.8 330 141%
for D50 is about 10-20%
Smith et al. Annals Allergy Asthma Immunol 1995; 75:317-323
for D50 is about 10-20%Using E no minimumGerman AmericanBAU E D50 BAU E D50
248892 82 14.83 8350 75 11.746928 74 11.57 440670 92 15.35
460467 107 15.39 111 77 7.811472 60 10.16 761 84 9.562723 36 10.72 1276 25 10.03980 47 9.79 218 31 8.42
1143 32 9.93 888 58 9.7213 60 8.4 13841 46 12.2
33333 67 13 1916 58 10.4639 46 9.4 88 61 7.6796 61 9.6
mean = 4429 mean = 11.16 mean = 1681 mean = 10.2895% LL 998 SD = 2.29 95% LL 342 SD = 2.3495% UL 19644 conf = 1.36 95% UL 8260 conf = 1.45
How many study subjects?
Estimate n = 40 to establish D50 for each extract (based on /= 1.5): 80
Geographic diversity: 80 x 3 = 240 Overlap between American CR and
German CR allergic subjects may permit reduction in n (150-200)
n 1.5 0.20 39
Conclusions Established that cockroach
allergen vaccines need to be standardized
Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
NIAID Inner City Asthma Consortium
“…established in FY 2002 to explore and evaluate promising new strategies for the treatment of asthma among minority children residing in the inner city. This consortium of basic scientists and clinical investigators will conduct clinical studies to elucidate the immunopathogenesis and natural history of asthma in this population.”
From the FY 2003 Budget Justification Narrative, NIAID, http://www.niaid.nih.gov/director/congress/2002/cj/narrative.htm
NIAID Inner City Asthma Consortium steering committee
Busse, William W., MD Chair
Adams, Kenneth, PhD Eggleston, Peyton A., MD Gruchalla
, Rebecca S., MD, PhD Kattan, Meyer, MD Kercsmar, Carolyn M., MD Liu, Andrew H., MD Malveaux, Floyd J., MD, PhD Mitchell, Herman, PhD
Morgan, Wayne, MD, CM
O'Connor, George T., MD, MS
Pongracic, Jacqueline A., MD
Sampson, Hugh A., MD Smartt, Ernestine, RN Strunk, Robert C., MD Szefler, Stanley J., MD
Timetable Steering committee approval - done Study centers identified Order extracts IRB approvals IND approval Distribute materials Proficiency testing Proceed with study
Endotoxin content of cockroach vaccines
Manufacturer glycerol EU/mLAmerican cockroach D yes 60000-120000
E yes 9600-19200B yes 24000-48000A yes 600-1200F yes 12000-24000G yes 1200-2400J 60-120E 240-480C 6-12C 600-1200G 120-240
German cockroach F yes 12000-24000A yes 60000-120000C 240-480E 24000-48000G yes 600-1200C 48000-96000
Conclusions Established that cockroach
allergen vaccines need to be standardized
Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
Endotoxin issue to be studied in depth
Will overall allergenicity measurements be sensitive to changes in specific allergen levels?• In mite stability study (1998-
1999), RP was stable at -20°C and 4°C for up to 12 months
Degradation of specific allergens (group 1 and 2; specific bands) was observed at 4°C
Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. J Allergy Clin Immunol 2000; 105:482-8.
RID with monospecific antiserum Examples: cat,
ragweed Advantages
quantitative monospecific
Disadvantages need to identify
relevant allergen(s)
Competition ELISA with pooled allergic human sera
Examples: mites, grasses Advantages
quantitative reflects spectrum of allergen
recognition does not require identification
of relevant allergens Disadvantages
use of pooled sera effects of fluctuations in
individual allergens difficult to measure
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)
Will overall allergenicity measurements be sensitive to changes in specific allergen levels?
Depletion analysis Raise specific antibodies to Bla g 1, 2,
4 and 5 Selectively adsorb Test for specific allergen levels Test for overall allergenicity
SDS-Page of Bla g 1 Absorbed Cockroach Antigen
Cr Sham Abs
Selective adsorption of Bla g 1
0
0.5
1
1.5
2
2.5
3
1 10 100 1000 10000
Dilution
Ab
sorb
ance
(45
0 n
m)
Bla g 1 ELISA
Sham adsorbedAdsorbed with anti-Bla g 1
Bla g 1 Bla g 2 Bla g 5Pre-immune 149.3 2.7 0.17anti-Bla g 1 14.8 4.1 0.07
Selective adsorption of Bla g 1 does not reduce the RP as measured by competition ELISA
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1 10 100 1000 10000 100000
Dilution
Ab
sorb
ance
(45
0nm
)
Conclusions Established that cockroach allergen
vaccines need to be standardized Need to establish the potency of
candidate US reference materials by bioassay (ID50EAL)
Endotoxin issue to be studied in depth
Surrogate test may not be the competition ELISA
Conclusions Standardized German and American
cockroach allergen vaccines will facilitate definitive studies on the role
of cockroach allergens in inner city asthma, and on the best methods for eradication and treatment
make for safer and more effective products