Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Summary

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Abstract

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

Introduction

Among the immunoassay techniques aimed at diagnosing diseases, the cytometric bead assay has emerged as a highly sensitive and reliable approach, since it allows for the analysis of thousands of particles in a single assay¹. This technique, in addition to having high productivity and allowing the use of smaller volumes of samples, also presents great flexibility, since it allows for the detection of several molecules, such as cytokines, adhesion molecules, antibody isotypes, and proteins^2,3.

Different particles are used for the developme....

Protocol

NOTE: See the Table of Materials for details related to all the materials, reagents, and instruments used in this protocol.

1. Extraction of IgY from egg yolks

1. Hygienization of the eggs
   1. Immerse the eggs (freshly laid or until 4 days post-laid, from the Gallus gallus Dekalb White lineage) in a 0.2% diluted solution of sodium hypochlorite, rinse quickly under running water, and wipe gently for later or immediate use.
      NOTE: If not used immediately, keep at 4 °C for up to 15 days.
2. Separation of the yolks
   1. Break the egg carefully, a....

Results

Figure 2 provides a graphical representation of the extraction process of IgY antibodies via acidification, followed by separation using caprylic acid (Figure 2).

Figure 2: Schematic representation of the extraction step of IgY antibodies and separation.......

Discussion

The method of precipitation of the IgY antibody by pH reduction followed by lipid separation using caprylic acid is efficient in isolating total antibodies without any loss in functionality. The method proposed here is simpler and cheaper than that reported by Redwan et al.¹¹, which also used precipitation by acidification and caprylic acid but with a more complex and laborious protocol. This method also presents advantages over commonly used methodologies for IgY isolation from egg yolks, such as.......

Materials

Name	Company	Catalog Number	Comments
Anti-Chicken IgY (H+L), highly cross-adsorbed, CF 488A antibody produced in donkey	Sigma-Aldrich	SAB4600031
Anti-mouse IgG (H+L), F(ab′)2	Sigma-Aldrich	SAB4600388
BD FACSCanto II	BD Biosciences	BF-FACSC2
BD FACSDiva CS&T research beads (CS&T research beads)	BD Biosciences	655050
BD FACSDiva software 7.0	BD Biosciences	655677
Bio-Rad Protein Assay Dye Reagent Concentrate	Bio-Rad	#5000006
Bovine serum albumin	Sigma-Aldrich	A4503
Caprilic acid	Sigma-Aldrich	O3907
Centrifuge 5702 R	Eppendorf	Z606936
Chloride 37% acid molecular grade	NEON	02618 NEON
CML latex, 4% w/v	Invitrogen	C37253
Megafuge 8R	Thermo Scientific	TS-HM8R
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Hydrochloride Powder (EDC)	Sigma-Aldrich	E7750-1G
N-Hydroxysuccinimide (NHS)	Sigma-Aldrich	130672-25G
Phosphate buffered saline	Sigma-Aldrich	1003335620
Sodium hydroxide	Acs Cientifica	P.10.0594.024.00.27
Sodium hypochlorite	Acs Cientifica	R09211000
Thermo Mixer Heat/Cool	KASVI	K80-120R