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 It was current when produced, but is no longer maintained and may be outdated.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. Ronald L. Rabin, MD Chief, Laboratory of Immunobiochemistry CBER/OVRR/DBPAP Possible Change of Potency Assay for Standardized Short Ragweed Pollen and Cat Allergen Extracts

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. Standardized products (19 so far) are controlled for potency and stability D. farinae D. pteronyssinus Cat hair Cat pelt Short ragweed pollen Hymenoptera oHoney bee oWasp oYellow jacket oYellow hornet oWhite-faced hornet oMixed vespid Grass pollens oBermuda grass oRed top oJune (Kentucky blue) oPerennial rye oOrchard oTimothy oMeadow fescue oSweet vernal

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. 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)

7. 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 Radial Immunodiffusion (RID)

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

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

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

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

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. 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. Precise but inaccurate Precise and accurate

26. Qualification: Specificity

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

29. Parameters Category I (Assay; dissolution; content/potency) Category II (Testing for Impurities) Category III (Performance Characteristics) Category IV (Identification) QuantitationLimit Accuracy ++--1 - Precision: Repeatability ++-+- Intermediate precision +2+2 +2+2 --1 - Reproducibility -1 Specificity +++++ Detection Limit --3-3 +-1 - Quantitation Limit -+--1 - Linearity ++--1 - Range ++--1 - 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 and precise Accurate but not precise

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

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

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. OD Log (Dilution) Upper Asymptote Lower Asymptote 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. 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 Parameters of Validation

38. ~~~~ 1 2 3 4 5 6 7 ABCDABCD Equipment, Reagents, Personnel, Procedure, etc Run Test Data/Analysis SSC Meets acceptance criteria established in the Robustness testing. System Suitability check (SSC) run with every test Valid/ Invalid √√√√ 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).

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