Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

Summary

We present a validated sandwich ELISA assay using novel anti-HER2 monoclonal antibodies. This assay enables precise quantification of cell-bound and released HER2 protein from in vitro cultured cells and other samples, including blood and tissues.

Abstract

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, especially in breast cancer and other HER2-expressing cancer types. In the clinic, the gold-standard immunohistochemical diagnostic methods employing the specific anti-HER2 antibodies are used to measure the expression level of the membrane-bound receptor. The soluble extracellular domain (ECD) of HER2 that is released from the overexpressing cells circulates in the blood and can reflect the tissue expression of the receptor. There is a need for accurate and validated assays to correlate the concentration of the circulating HER2 protein with disease clinical manifestations.

Our team has developed and validated the novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the membrane-bound and the released from cells ECD domain of HER2. The assay uses two unique monoclonal antibodies specific to HER2 developed previously. The quantitation range includes HER2 concentration from 1.56-100 ng/mL, which is expected for cancer cells cultured in vitro and shows sensitivity at the level of 0.5 ng/mL. The satisfactory intra- and inter-assay precision and accuracy of the method make it applicable for HER2 quantification in various types of biological samples, including cell culture medium, serum, and solid tumor tissue. Here, we focus on the comprehensive determination of the receptor-associated and secreted by the in vitro cultured cancer cells. The paper presents a step-by-step protocol for the quantification of HER2 protein that can be employed for testing a variety of cell lines, blood, and tissues.

Introduction

The success of modern therapeutics often relates to precision medicine that is based on accurate identification of therapy-sensitive patients¹. Among these therapies are the anti-HER2 drugs targeting the receptor overexpressed on a variety of tumors, including breast, endometrium, stomach, lung, and others. Several HER2-targeting agents are available with confirmed benefits in patients with HER2-positive cancers, including HER2-low type². Confirmation of the HER2-positive status is critical for the identification of the potential responding patients; however, it remains a challenge, especially in the HER2-low group.

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Protocol

1. Culturing of human cancer cells

1. Culture MDA-MB-231, SK-BR-3, and SK-OV-3 lines in Dulbecco's Modified Eagle's medium containing 4.5 g/L of ᴅ-glucose (DMEM-HG), supplemented with 2 mM ʟ-glutamine and 10% (v/v) heat-inactivated FBS. Incubate cultures at 37 °C in a humidified atmosphere of 5% CO₂.
2. When the culture reaches ~80% confluence, detach cells by trypsinization and collect them into separate 15 mL conical tubes.
3. Count the cells with an automatic cell counter and seed cells for the experiment into 12-well plates at a density of 3 × 10⁵ cells/per well in 1 mL of growth medium. Incubate cultur....

Results

Sandwich ELISA validation
The newly developed assay requires a validation procedure. The important validation parameters include linearity, precision, and detection limits, i.e., lower limit of detection (LLOD) and upper limit of detection. In the previous paper, we have performed thorough method validation. ELISA linearity was tested by using the mocked samples for low (2, 5, 10 ng/mL), medium-high (30 ng/mL), and significantly increased (50 ng/mL) concentrations of the antigen diluted in PBS and ot.......

Discussion

Among the critical components in constructing a sandwich ELISA, there are capturing antibodies that are immobilized on the plate and contribute to the assay specificity and sensitivity. In the presented assay, we have employed as the capturing antibody the novel monoclonal protein (HER2/70.27.58) generated and characterized in-house. The antibody had a unique sequence of the CDR (complementarity-determining region), and based on the affinity, it presented a sensitivity of ED50 at 0.0922 nM¹². The .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotin labeling kit-NH2	Abnova	KA0003
Blotting Grade, powdered milk, low in fat	Roth	T145.1
Cell Counting Slides for TC10/TC20 cell Counter, Dual-Chamber	Bio-Rad	145-0011
Cell Culture Plates	Biologix	07-6012
Cell Scrapers	Biologix	70-1250
Centrifuge	Ohaus	30130868
Class II Biological Safety Cabinet - Telstar Bio II Advance 6	Telstar	N/A
Clear Flat-Bottom 96-Well Plates	Thermo Fisher	442404
Culture Safe CO2 Incubators - Touch 190S	Leec	N/A
Dimethyl sulfoxide	Sigma Aldrich	D2650
DMEM - high glucose	Sigma Aldrich	D0822
ELISA plate reader	BioTek	800TSUVI
FBS Standard, fetal bovine serum	PAN Biotech	P30-19375
Forced circulation laboratory dryer	BINDER	9090-0018
HRP-Avidin	Thermo Fisher	43-4423
Human Her2 / ErbB2 Protein, Fc Tag, premium grade	AcroBIOSYSTEMS	HER2-H5253
Immunowash Microplate Washer	Bio-Rad	170-7009
L-Glutamine solution	Sigma Aldrich	G7513
mAb a-HER2 (clone 70.21.73.67)	SDS Optic	BIO-ABH-2
mAb a-HER2 (clone 70.27.58)	SDS Optic	BIO-ABH-1
MDA-MB-231 Cell line	ATCC	HTB-26
NaHCO3	POCH	810530115
NaOH	POCH	BA0981118
Protease Inhibitor Cocktail	Sigma Aldrich	P8340
RIPA Buffer	Sigma Aldrich	R0278
ROTI Fair PBS	Roth	1111.2
SK-BR-3 [SKBR3] Cell line	ATCC	HTB-30
SK-OV-3 [SKOV-3; SKOV3] Cell line	ATCC	HTB-77
Stop solution 1x	Abcam	ab210900
TC20 Automated Cell Counter	Bio-rad	1450102
TMB substrate 1x	Abcam	ab210902
Tween-20	Sigma Aldrich	P9416
Vortex	Ohaus	30392117
Wave motion shaker	Ohaus	30391968