U.S. Food and Drug Administration

1. U.S. Food and Drug Administration Notice: Archived Document The content in this document is provided on the FDA’s website for reference purposes only. It was current when produced, but is no longer maintained and may be outdated.

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

3. 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

4. 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 Standardized products (19 so far) are controlled for potency and stability

5. 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 • cat is also in Fel d1 units

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

7. Radial Immunodiffusion (RID) Procedure • antibodies specific to the major allergen added to 1% agar (+ 1% azide) • solidify agar glass slide • punch wells in agar • add equal amounts of antigen to wells • incubate 48-72 hrs in humidified chamber (antigen complexes with antibody) • immerse in 10% acetic acid for 2 min • measure diameter of precipitant ring

8. Preparation of agarose slides

9. Mounting slides on reader

10. Reading the slides

11. Precipitant rings

12. Measuring diameter of the precipitant rings

13. Radial Immunodiffusion (RID) Analysis: Compare precipitant rings of manufacturer’s lot to the CBER Standard Curve y = a(log x) + b corr coeff > 0.9

14. Radial Immunodiffusion (RID)

15. Radial Immunodiffusion (RID) Passing Values Short Ragweed: • no limits or target range • vial labeled only with units of Amb a 1/mL Cat:

16. Why consider a change of assay? RID • Time consuming • Labor intensive • Reader variability • Expensive; high reagent (anti-serum) use per assay Replacement assay • Quicker • Easier • Automated data collection and analysis • Use of less antiserum and other reagents • Greater precision • Better specifications: reproducibility (e.g. correlation coefficient), dynamic range, precision and accuracy

17. 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, which may be: • Colorimetric • easy • relatively inexpensive instrumentation • Fluorescent • broader dynamic range • instrumentation more expensive • Luminescent • most sensitive • transient signal • expensive instrumentation

18. ELISA Developmental Plan Phase 1 – Proof of concept • Feasibility-proof that a test system can work Phase 2 – Qualification and Validation • Validation-showing that test is ‘stable’ (evaluated over time and under different conditions) Phase 3 - Standardization • Standardization, quality control, establishment that test is precise and can be used by different workers in different laboratories. • Evaluate availability and inter-changeability of non-critical reagents

19. Phase 1 – Proof of concept Determine the type of ELISA • Direct • Indirect • Sandwich • Competition Evaluate commercially available ELISA Evaluate potential sources and types of: • Antibody - monoclonal, polyclonal (or combination) • Antigen • Enzymes • Conjugates

20. (1) (2) (3) Direct ELISA is the simplest type • Antigen passively attaches to plate • Add conjugated specific antibody • Add substrate to develop color

21. (1) (2) (3) (4) Indirect ELISA adds versatility and amplification • Antigen passively attaches to plate • Add unconjugated specific antibody • Add conjugated secondary antibody • Add substrate to develop color

22. Sandwich ELISA is more sensitive and does not require pure antigen • Capture antibody passively attaches to plate • Antigen is captured by plate-bound capture antibody • Add unconjugated specific antibody • Add conjugated secondary antibody • Add substrate to develop color Sandwich ELISA requires that the analyte has at least two epitopes (capture and detect)

23. Direct, indirect, or sandwich ELISA can be set up as competition ELISA

24. Phase 2 – Qualification and Validation Qualification • Precision (Intra-assay and Repeatability) • To determine acceptance criteria for validation phase • Specificity

25. Precise but inaccurate Precise and accurate Qualification: Precision • Closeness of agreement between a measurements obtained by one person repeating a method • Focus on intra-assay and inter-assay precision to establish acceptance criteria for validation.

26. Qualification: Specificity

27. Validation Validation • Snapshot of assay performance • Confirmation that the assay performs as you claim • Demonstrates that the assay is suitable for intended purpose Validation Plan • Includes a complete list of parameters to be evaluated. • Sets minimum acceptance specifications for each parameter • Describes in detail steps necessary to evaluate each parameter

28. Accuracy Precision -Repeatability -Intermediate precision -Reproducibility Specificity Detection Limit Quantitation Limit Linearity Range Robustness System Suitability Parameters of Validation

29. Parameters for Validation from USP and ICH 1 May be required, depending on the nature of the specific test. 2 In cases where reproducibility has been performed, intermediate precision is not needed. 3 May be needed in some cases.

30. = Validation includes Accuracy:A measure of trueness/bias Accurate but not precise Accurate and precise

31. Accuracy vs. Precision Accurate and Precise Accurate but not Precise Precise but not accurate Neither Precise nor accurate

32. Validation • Precision • Intra-assay = repeatability • Inter-assay variability (within the same lab) • Inter-laboratory = reproducibility

33. Limit of Detection is determined by blank + 3 x SD of the slope

34. Lower limit of quantitation is determined by baseline + 6 x SD of the slope

35. Upper Asymptote OD Lower Asymptote Log (Dilution) Upper Limit of Quantitation is determined by quantity of substrate and instrumentation

36. Linearity & Range Linearity Range where the response is proportional to the analyte added. Linear Range Accuracy +/- pre-determined variability (e.g. 1 SD of slope)

37. Parameters of Validation Robustness • Small, but deliberate, variations to measure the lack of internal influences on the test results. • Examples: vary temperature (37 +/- 3 C) vary incubation time (1 hr +/- 5 min) equipment source of reagents Standardization • System suitability • Collaborative study

38. 1 2 3 4 5 6 7 A B C D Parameter of Validation: System Suitability System suitability tracks and trends assay performance over time, and assesses the need for re-validation as a result of assay changes (e.g. change of source of reagent). Data/Analysis ~~~~ ~~~~ ~~~~ ~~~~ Equipment, Reagents, Personnel, Procedure, etc Run Test Valid/ Invalid √ √ √ √ System Suitability check (SSC) run with every test SSC Meets acceptance criteria established in the Robustness testing.

39. Summary RID is dependable and reproducible, but time consuming and relatively expensive ELISA may be a better surrogate assay for cat and ragweed allergen standardization, the two environmental allergen extracts that are standardized by the concentration of the major allergen, Fel d 1 and Amb a 1