Allergen Analysis : Customer Solution Case

Studies

ILSI Madrid 2018 – Adrian Rogers

Immunoassay Challenges

Every food is different and presents it’s own unique challenges with immunoassay analysis

If there is any doubt about how a food will perform VALIDATE

Knowledge is power, know your food

Case Study 1

Peanut Allergen Analysis in Confectionary

Peanut Allergen Analysis

The Challenge: Large multi-national confectionary company

Current ELISA discontinued

Very stringent validation procedure

Need Kit suppliers full support and complete transparency

in communication.

Romer’s Solution Use AgraQuant PLUS Peanut

To be as open and supportive as possible with the

customer.

To undertake the required validation quickly and thoroughly.

Peanut Allergen Analysis

Peanut Allergen Analysis Testing Criteria The method must be tested for peanut protein and the final

result must be expressed in peanuts.

Matrices: ice cream, chocolate and Chocolate Bar

Minimum size amount of sample to be analysed: 2g.

Spiking concentration: 1 ppm; 5 ppm and 15 ppm peanuts.

Peanut Allergen Analysis Testing Criteria Continued Range of peanut quantification should be at least

between 1 and 15 mg/kg; Recovery range: 60 – 130 %; Intermediate reproducibility < 40% at 95%

confidence level Linearity Limit of Detection (LOD) Limit of Quantitation (LOQ) Precision (2 operators over 8 days) Linearity of Dilution

Peanut Allergen Analysis

Outcomes Romer were able to satisfactorily meet the customers

validation criteria.

The AgraQuant PLUS Peanut kit was successfully beta tested at 3 manufacturing sites around the world.

Romer’s kit was adopted as the company’s peanut detection kit of choice world wide.

Case Study 2

Casein Allergen Analysis in Baby Formula

Casein Allergen Analysis

The Challenge: Large multinational baby formula company

Need to check for casein residue in hydrolysed whey based formula.

Complex problematic matrix

Issue with low recoveries of spiked casein

Casein Allergen Analysis

Romer’s Solution

Use AgraQuant PLUS Casein ELISA

Test fully and partially Hydrolysed Formulas for reactivity.

Add a known amount of casein (in the form of skimmed milk powder) to each formula to asses recovery.

Test at 2 levels, low level near LOQ of assay, High level in the middle of calibration curve.

Casein Allergen Analysis

Results Casein (ppm) % Recovery

Casein Low Spike No Matrix 1.3 Casein High Spike No Matrix 11.3 Formula 1 2.6 N/A Formula 1 Low Spike 0.4* 31 Formula 1 High Spike 5.0* 44 Formula 2 <LOQ N/A Formula 2 Low Spike <LOQ (0.6) 43 Formula 2 High Spike 9.7 86 Formula 3 <LOQ N/A Formula 3 Low Spike <LOQ (0.6) 48 Formula 3 High Spike 8.0 71

*Casein level in unspiked sample subtracted from spiked sample result

Casein Allergen Analysis

Outcomes Recoveries of High Spike were better then the

manufacturer expected.

Data to be reviewed by manufacturer.

Romer to work with the manufacturer to asses the rest of their infant formula range

Case Study 3

Almond and Peanut Allergen Analysis in Seasoning

Almond and Peanut Allergen Analysis

The Challenge: South American Seasoning producer.

Different ELISA kits from different manufactures

give different results.

How do they know which one is correct?

Almond and Peanut Allergen Analysis

Romer’s Solution Repeat all testing in Romer’s ISO17025 compliant

laboratory

Test using Lateral Flow Device which incorporates a different antibody

Test using an orthogonal method : LC-MS/MS that analyses for Almond and Peanut Peptide fragments.

Results Chile Results Austria

Manufacturer A ELISA kit

Romer Labs ELISA kit

Manufacturer A ELISA kit

Romer Labs

ELISA kit

Romer Labs LFD

LC-MS/MS

Almond 19.6 ppm <0.4 ppm 19.3 ppm <0.4 ppm neg neg

Peanut 31 ppm 1.1 ppm 9.7 ppm 3 ppm --- neg

Almond and Peanut Allergen Analysis Results

What would you decide ?

Almond and Peanut Allergen Analysis

Sometimes there is no clear cut answer

Need for an analytical tool box approach

Customer was grateful that Romer had “gone the extra mile” to find an answer.

Customer reviewed and changed their testing lab based on Romer’s findings

Assessing almond and peanut allergens using commercially available immunoanalytical kits and LC-MS/MS: a case study Daly et al : Journal of AOAC International Vol. 101, No. 1, 2018

Food allergens are extremely diverse

No certified reference material no common reference point for ELISA comparison

Close collaboration between customer and kit manufacturer necessary

Method must be suitable for the application

Customised solutions necessary

Summary

Thank you!

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