1. 1 Change of Potency Assay for Standardized Short Ragweed Pollen and Cat Allergen Extracts Ronald L. Rabin, MD Chief, Laboratory of Immunobiochemistry CBER/OVRR/DBPAP

2. 2 Allergen standardization (21 CFR 680.3(e)) Establish a US standard, and Establish a testing procedure Manufacturers may use the established procedure, or may develop equivalent procedures

3. 3 Standardized products are controlled for potency and stability House Dust Mites D. farinae D. pteronyssinus Cat Cat hair Cat pelt Hymenoptera Honey bee Wasp Yellow jacket Yellow hornet White-faced hornet Mixed vespid Short ragweed pollen Grass pollens Bermuda grass Red top June (Kentucky blue) Perennial rye Orchard Timothy Meadow fescue Sweet vernal

4. 4 Unitage for standardized allergens Venom - µg protein based on the activity of allergenic enzymes Ragweed - units Amb a 1/mL based on the concentration of the major allergen Mite - AU/mL; Cat & Grass - BAU/mL based on the correlation of skin testing to an in vitro assay

5. 5 Biological potency by ID 50 EAL testing (Intradermal Dilution for 50 mm Sum of Erythema Determines Bioequivalent Allergy Units) Highly allergic individuals Serial 3-fold dilutions Establish dilution at which  E = 50 mm (D 50 )

6. 6 Biological potency by ID 50 EAL testing Screen: puncture test (bifurcated needle) with concentrates Intradermal testing with serial 3-fold dilutions Record wheal and erythema size Calculate  E

7. 7 Calculation of D 50 A D50 of 14 (dilution factor 5 x 10 6 ) is 100,000 BAU

8. 8 Surrogate Assays for Potency Current tests D. farinae and D. pteronyssinus Competition ELISA Protein Cat pelt and cat hair Fel d 1 (RID) IEF Protein Grasses Competition ELISA IEF Protein Short ragweedAmb a 1 (RID) Hymenoptera Hyaluronidase Phospholipase Allergen(s) Protein

9. 9 Surrogate Assays for Potency Current tests D. farinae and D. pteronyssinus Competition ELISA Protein Cat pelt and cat hair Fel d 1 (RID) IEF Protein Grasses Competition ELISA IEF Protein Short ragweedAmb a 1 (RID) Hymenoptera Hyaluronidase Phospholipase Allergen(s)

10. 10 Radial Immunodiffusion (RID) Procedure antibodies specific to the major allergen added to agar (1% agar + 1% azide) solidify agar glass slide punch wells in agar add equal amounts of antigen to wells incubate 72 hrs in humidified chamber (antigen complexes with antibody) immerse in 10% acetic acid for 2 min measure diameter

11. 11 Preparation of agarose slides

12. 12 Developing slides in acetic acid

13. 13 Mounting slides on reader

14. 14 Reading the slides

15. 15 Precipitant rings

16. 16 Radial Immunodiffusion (RID)

17. 17 Passing Values Short Ragweed: no limits or target range vial labeled: Amb a 1, X units / mL Cat: Radial Immunodiffusion (RID)

18. 18 Enzyme Linked Immunosorbent Assay All ELISAs have a revealing step in which an enzyme coupled to a revealing antibody (or streptavidin) converts a substrate into a detectable and quantifiable signal. Signals may be: Colorimetric easy relatively inexpensive instrumentation Fluorescent broader dynamic range instrumentation more expensive Luminescent most sensitive transient signal expensive instrumentation

19. 19 Potential Advantages of ELISA vs. RID Much less time consuming RID readers are obsolete and no longer in production ELISA plates much easier to set up than gel slides Automated reader Specifications and reproducibility (e.g. correlation coefficient) much higher for ELISA Less reagent used per test Broader dynamic range

20. 20 Sandwich ELISA Coat plate with a capture antibody (scFv) then add blocking buffer. Add sample (allergenic extract.) Antigen binds to the capture antibody. Add detection antibody (sheep anti-cat/ragweed antiserum.) Detection antibody binds to the antigen. Add revealing antibody (rabbit anti-sheep antiserum.) Revealing antibody binds to the detection antibody. Add Substrate followed by Stop solution (H3PO4).

21. 21 Development of Sandwich ELISA for measurement of Fel d 1 and Amb a 1 Capture with one of three specific single chain variable fragments (scFv, 20  g/mL)* Detect with sheep polyclonal antiserum (1:1000) Reveal with HRP conjugated anti-sheep IgG (1:1000)

22. 22 Sandwich ELISA for measurement of Fel d 1 2.5 2.0 1.5 1.0 0.5 0 OD 450 1 10 100 1000 native Fel d 1 (ng/mL) Concentration

23. 23 Sandwich ELISA for measurement of Fel d 1 from Cat Hair Extract OD 450 Dilution of Cat Hair Extract Concentration

24. 24 Sandwich ELISA for measurement of Amb a 1 OD 450 Dilution of Short Ragweed Extract Concentration

25. 25 Next Steps for FDA to Adopt the Sandwich ELISA for testing of standardized Cat (Fel d 1) and ragweed (Amb a 1) allergenic extracts Select one of three scFv Western blot (cat scFv) Basophil stimulation assay (both) Prepare a validation protocol LIB perform the validation Invite manufacturers participate in the assay validation Summarize validation in a report Prepare at least 20g of each scFv