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This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.
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.
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 proteins2,3.
Different particles are used for the development of these assays, among them latex beads, which are an effective and low-cost input. These can present modifications on their surface, such as the presence of functional groups or proteins that allow the covalent or non-covalent coupling of certain molecules3,4,5.
These immunoassays use components such as antigens and antibodies to perform the detection of disease markers and commonly require antibodies from mammals such as mice, rabbits, and goats. This creates problems related to ethical issues, since the immunization of mammals generally requires many animals to obtain a good yield, as well as the frequent performance of procedures that lead to the suffering of the animals6,7. An alternative to this is the use of IgY antibodies isolated from the egg yolks of immunized chickens, since high concentrations of the specific antibodies against the inoculated antigens can be found in the yolks; the production of a chicken is equivalent to the production of 4.3 rabbits over the course of a year6,7.
Thus, the objective of this protocol is to provide a method for evaluating IgY antibodies obtained from chicken egg yolks using flow cytometry with latex beads. For this, we propose a standardization method for a cytometric bead immunoassay in sandwich format using latex beads. As a model, we used IgY antibodies directed to the Plasmodium falciparum histidine-rich protein II antigen (IgY-PfHRP2). We describe a method for extracting the antibodies, discuss the critical steps for defining the coupling concentration of these to the latex beads, and present an evaluation of the limit of detection of the antigen. The high accuracy of flow cytometry, coupled with the low cost of latex beads, make this technique applicable for the analysis of immunoassay tools, such as antibodies and antigens. This method can be used for the detection of diverse targets.
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
2. Saturation curve of IgY antibodies to latex beads
Figure 1: Graph of the flow cytometry analysis to determine the saturation point of the IgY-PfHRP2 antibody coupling to the latex beads. The x-axis represents the antibody concentration, and the Y-axis represents the percentage fluorescence obtained. Kruskal-Wallis test, p < 0.0033. Please click here to view a larger version of this figure.
3. Flow cytometry assay based on latex beads for antigen detection
4. Flow cytometer reading of the samples
5. Sample analysis using flow cytometry
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...
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...
The authors declare no potential conflicts of interest with respect to the research, authorship, and publication of this article.
We would like to thank the FIOCRUZ ("Programa de excelência em pesquisa básica e aplicada em saúde dos laboratórios do Instituto Leônidas e Maria Deane - ILMD/Fiocruz Amazônia-PROEP-LABS/ILMD FIOCRUZ AMAZÔNIA"), the Post-graduate Program in Biotechnology (PPGBIOTEC at the Universidade Federal do Amazonas - UFAM), the Coordenação de Aperfeiçoamento de Pessoal de Nível (CAPES), and the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for providing the scholarships. Figure 2 and Figure 4 were created with biorender.com.
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 |
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