AOAC Guidance on FA Immunoassay Validation (August 2023)

5.2.2.2. Analyze at least four replicates of each concentration defined for the calibration curve. 5.2.2.3. Fit the calibration curve as described in method instructions and kit insert. Full descriptions must be provided with respect to performing the calibration function calculations, including any transformations conducted and the regression model used. Full calibration curve plots 5.2.2.4. From the calibration curve function, determine the calculated concentrations for each of the standards. Calculate the residuals for each non-zero concentration standard. Residuals are the difference between the observed value and the predicted value for each dependent variable in the calibration curve. (Residual = observed value - predicted value.) Residuals should be calculated from the instrument response. For most quantitative food allergen 5.2.2.6. Residuals should have random distributions and be centered on zero. If a non-random pattern is observed, the calibration function or measurement range may not be appropriate. 5.2.2.7. Residuals should generally also be <15% of the measured response, and up to 20% at the 297 The selectivity study is intended to provide information on potential sources of cross-reactivity and 298 interference. The information related to cross-reactivity and interference should be reported in the 299 validation report and in the package insert from the method developer. 300 5.2.3.1. Selectivity Panel Selection 301 5.2.3.1.1. Method developers must test their allergen detection method for cross-reactivity with 302 the target allergen in a variety of food commodities. Food commodities tested for 303 cross-reactivity should include a wide selection of foods and ingredients, particularly 304 those that are genetically similar to the target allergenic commodity and that are 305 likely to be analyzed for the presence of the target food allergen. (9) 306 5.2.3.1.2. For all food allergen analytes, all foods in Table 2 must be analyzed. For specific food 307 allergen analytes, additional matrices listed in Table 3 must be analyzed. Additional 308 foods and ingredients listed in Table 4 may be included in the study. 309 5.2.3.1.3. Method developers must make a good-faith effort to obtain the relevant foods listed 310 in Tables 2-4. If the method developer feels they are unable to secure any particular 311 food type, they should detail efforts to procure the food in the study report. 312 5.2.3.1.4. Method developers are advised to take extreme care in selecting, sorting, and 313 preparing samples for the cross-reactivity testing to avoid analyzing samples with 314 existing undeclared target food allergen residues. For example, it may be advisable 315 to obtain whole nuts, seeds, spices, etc. to facilitate hand sorting, washing/shelling, 316 and grinding/milling. 317 5.2.3.1.5. To identify possible allergen cross-contact, it is advisable to screen commodities with 318 an alternative method (ELISA, PCR or other). 319 5.2.3.2. Cross-Reactivity 320 5.2.3.2.1. Study Design 321 lowest non-zero calibration standard. 5.2.3. Selectivity Study 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 and calibration functions must be shown. methods, instrument response would be optical density (absorbance) values. 5.2.2.5. Plot the residuals versus concentration.