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

Winnie Tran1, Marie-Alexandre Adom2, Melissa Phillips3, Ashley Beasley Green4, David Bunk4

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.

1.00

1.21

1.06 0.96

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 NFDM

100 °C NFDM

NFDM + Casein

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.

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

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 alphalactalbumin (product # L-045-100MG) and beta-lactoglobulin (product # L-046-

100MG) were obtained from Cerilliant. Candidate study materials were

prepared in an aqueous phosphate-buffered 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

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 LC-MS/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 protein-specific 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