An Interlaboratory Study to Evaluate the Equivalence of Milk Protein Allergen Measurement

Winnie Tran1, Marie-Alexandre Adom2, Melissa Phillips3, Ashley Beasley Green

4

, David Bunk

4

1Biochemistry Department, University of Maryland, College Park MD 2Chemistry Department, University of Virginia, Charlottesville, VA

3Chemical Sciences Division, NIST, Gaithersburg MD 4Biomolecular Measurement Division, NIST, Gaithersburg MD

Abstract:

• An interlaboratory study is planned by NIST to evaluate the equivalence of

milk protein allergen measurements.

• The study will include participation from the manufacturers of milk protein

ELISA kits, routine testing labs that utilize ELISAs, and users of liquid

chromatography-tandem mass spectrometry (LC-MS/MS) for milk protein

measurement.

• The study will utilize simple samples prepared from commercial non-fat

dry milk (NFDM), chosen to minimize the influence of sample extraction

and sample matrix on between-method measurement equivalence.

• Planned samples include solutions of NFDM, heat-treated NFDM,

irradiated NFDM, and NFDM spiked with both casein and whey proteins.

• These samples have been prepared at NIST and are being evaluated by

NIST for fitness for the study.

Materials and Methods:

Materials. Commercial non-fat dry milk (NFDM) was obtained from a local grocery

store. Casein (technical grade, product # C7078-500G) was obtained from Signa Life

Science. Certified reference materials of bovine alpha-lactalbumin (product # L-045-

100MG) and beta-lactoglobulin (product # L-046-100MG) were obtained from

Cerilliant. Candidate study materials were prepared in an aqueous phosphatebuffered saline (PBS), pH 8.2. All candidate study samples were prepared

gravimetrically. Treatment of the NFDM powder was done at 100 °C for 1 h and 175

°C for 30 min using a laboratory oven. An aliquot of NFDM powder was irradiated at

NIST (15 kGy of combined β- and γ-radiation from 60Co). Table 1 lists the candidate

study samples and their descriptions.

Methods. Characterization of the candidate study samples was performed using

SDS gel electrophoresis (Figure 1), LC-MS/MS analysis after trypsin digestion (Figure

2), and measurement using a commercial total milk ELISA (Figure 1).

Candidate Study Sample Description

NFDM ≈ 500 ppm NFDM (in PBS, pH 8.2)

Irradiated NFDM ≈ 500 ppm irradiated NFDM (in PBS)

100 °C heat-treated NFDM ≈ 500 ppm 100 °C heat-treated NFDM (in PBS)

175 °C heat-treated NFDM ≈ 500 ppm 175 °C heat-treated NFDM (in PBS)

NFDM + Casein ≈ 500 ppm NFDM + ≈ 100 ppm casein (in PBS)

NFDM + α-lactalbumin ≈ 500 ppm NFDM + ≈ 100 ppm α-LA (in PBS)

NFDM + β-lactoglobulin ≈ 500 ppm NFDM + ≈ 100 ppm β-LG (in PBS)

Table 1. Candidate interlaboratory study sample

1.00

1.21

1.060.96

0.44

1.14

1.38

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

NFDM in PBS

Irradiated...

100 °C NFDM

NFDM +...

175 °C NFDM

NFDM + 

α-LA

NFDM +

β- LG

Normalized ELISA [total milk]

Figure 1. (top) silver-stained SDS gel

electrophoresis image from the analysis of

candidate study samples; (bottom) normalized

total milk concentrations of candidate study

samples measured using a commercial total milk

ELISA

Irradiated NFDM

100 °C treated NFDM

NFDM + Casein

NFDM + α-lactalbumin

NFDM + β-lactoglobulin

Figure 2. Comparison of the targeted LC-MS/MS (QToF)

extracted ion chromatograms for 11 tryptic peptides from

bovine milk proteins for the candidate study samples.

Study Design:

• Multiple aliquots of each study sample will be shipped

to study participants on cold packs for preliminary

testing and final study measurement

• Study participants can use any platform (ELISA or LCMS/MS) available to them; ELISAs for specific milk

proteins or protein classes can also be used

• Participants will use their own extraction method (if

necessary) and diluents; extra sample aliquots will be

provided to optimize dilution for each measurement

platform

• Study participants will report measurement results in

the reporting units of their assays; additional proteinspecific quantitative information can also be reported

• Study results will be uploaded through a reporting

website; NIST will perform statistical analysis on the

study data and will produce a study report for

publication

• If requested, participating laboratories and assay

platform identification will be blinded in the study

report

• Future studies, using more complex food-matrix

samples, are also planned

Study Timeline:

• Participant sign-up deadline = November 22, 2019

• Study sample shipment = mid-December 2019

• Result reporting deadline = March 16, 2020

• Preliminary study results reporting = September 2020

(@ AOAC Annual Meeting, Orlando, FL)

• Study report publication = December 2020

For more information or to participate in the Milk 

Interlaboratory Study, please 

email: nistfoodallergens@nist.gov

Please provide:

• Names of platforms you intend to use

• Contact information

• Shipping information

https://www.nist.gov/programsprojects/nist-food-proteinallergen-program

NFDM

β-Casein

αS2-

Casein

αS1-

Casein

κ-Casein

β-LG

α-LA

β-Casein

αS2-

Casein

αS1-

Casein

κ-Casein

β-LG

α-LA

β-Casein

αS2-

Casein

αS1-

Casein

κ-Casein

β-LG

α-LA

β-Casein

αS2-

Casein

αS1-

Casein

κ-Casein

β-LG

α-LA

β-Casein

αS2-

Casein

αS1-

Casein

κ-Casein

β-LG

α-LA

β-Casein

αS2-

Casein

αS1-

Casein

β-LG α-LA κ-Casein