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 using caprylic acid. (A) Separation of the yolk, recovery in a conical tube, and freezing. (B) Yolk dilution, pH adjustment to 5. (C) Centrifugation of the solution and filtration of the supernatant. (D) Addition of caprylic acid and centrifugation of the solution. (E) Supernatant antibodies isolated in the previous step. Please click here to view a larger version of this figure.
In this work, the antibodies before (lane 1, Figure 3) and after the caprylic acid separation (lane 2, Figure 3) showed similar electrophoretic profiles, with characteristic bands of 65 kDa and 27 kDa, which refer to the light and heavy chains of the IgY, respectively. In addition to these, other bands were also visible, likely due to the C-terminal fragment egg yolk protein vitellogenin II precursor10.
Figure 3: SDS-PAGE (12% gel) demonstrating the electrophoretic profile of the IgY-PfHRP2 antibody before and after separation using caprylic acid. Lane M: protein molecular marker; lane 1: IgY-PfHRP2 antibody before caprylic acid separation; lane 2: IgY antibody after caprylic acid separation. Please click here to view a larger version of this figure.
Figure 4 is a schematic representation of the proposed test, including the initial step of coupling the IgY antibodies to the latex beads and the other steps for the detection of the antigen and reading in a flow cytometer.
Figure 4: Schematic representation of the cytometric bead assay proposed in the present study. (A) Incubation of IgY PfHRP2 antibodies with latex beads. (B) IgY PfHRP2 antibodies coupled to the latex beads. (C) Latex beads coupled to the IgY anti-PfHRP2 bound to the rPfHRP2 protein, mouse IgG primary antibody anti-PfHRP2, and fluorescent anti-mouse secondary antibody. (C1) Dot plot graph from the latex bead samples analyzed by flow cytometry. The pink line delimitation represents the analysis gate. (C2)Single-cell analysis. (C3)Fluorescence analysis of single cells. Please click here to view a larger version of this figure.
The evaluation of the saturation concentration of the antibodies in the beads is a crucial step for the development of an immunoassay using latex beads. Figure 1 shows a curve of the percentage fluorescence of different concentrations of antibodies coupled to each microliter of commercial latex beads used. Increasing percentage values were observed with increasing concentration, and the percentage values reached a plateau at an antibody concentration of 2 µg; this was then considered the best coupling concentration for performing the immunoassay with the target antigen. This assay showed negligible standard deviations between the replicates (Supplemental Table S1).
The detection limit was determined by the evaluation of the fluorescence percentage of the reactivity of the latex beads (containing the anti-PfHRP2 antibody in the saturating concentration) against different concentrations of the rPfHRP2 protein (Figure 5). After development using a sandwich immunoassay, it was possible to observe an increase in the fluorescence from an amount of 0.01 µg of the rPfHRP2 protein, and the percentage fluorescence reached a plateau at an amount of 0.1 µg. When comparing the amounts of 0 µg and 0.01 µg, there was no significant difference in fluorescence between them. However, when comparing the amount of 0.01 µg with 0.1 µg, 1 µg, and 10 µg, a significant difference in fluorescence was observed: p < 0.0048 (Kruskal-Wallis test) (Figure 5). This test also showed minimal standard deviations among the replicates (Supplemental Table S2).
Figure 5: Graph of the flow cytometry analysis to determine the limit of detection of the rPfHRP2 protein in the proposed sandwich assay. The x-axis represents the concentration of the rPfHRP2 protein, and the Y-axis represents the percentage fluorescence obtained. Kruskal-Wallis test, p < 0.0048. Please click here to view a larger version of this figure.
Supplemental Figure S1: Fluorescence analysis of the egg yolk antibody-based bead assay by flow cytometry. (A) Morphometric analysis of size by complexity (FSC/SSC), (B) analysis of single cells by complexity, (C) analysis of single cells by size, (D,E) analysis of the percentage fluorescence of Alexa Fluor 488 in spheres based on egg yolk antibodies at different concentrations. Abbreviations: FSC = forward scatter; SSC = side scatter. Please click here to download this File.
Supplemental Table S1: Percentage means and standard deviations of the saturation curve test of the IgY-PfHRP2 antibody coupling to the latex beads. Please click here to download this File.
Supplemental Table S2: Percentage means and standard deviations of the limit of detection of the rPfHRP2 protein. Please click here to download this File.
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 those using PEG precipitation12,13, sodium sulphate14,15, and chloroform16, among others, because these involve toxic reagents or reagents that are difficult to remove from the sample. In this work, the electrophoretic profiles of the antibody samples extracted before and after the lipid separation using caprylic acid were similar and presented the same profile. However, after the separation using caprylic acid, the antibodies were concentrated in a step that was efficient in the removal of lipids without having any negative effects on the removal of contaminating proteins from the samples.
The standardization of the method of coupling IgY antibodies to latex beads is essential for maximizing the analysis capacity of flow cytometry in each assay. In the tests carried out in our laboratory, it was observed that molecules of different sizes presented different saturation concentrations depending on the number of beads to be used (data not shown). For this reason, for each assay to be performed, the bead saturation concentration should ideally be verified, as demonstrated in the present study. In the assay described here, the saturation coupling condition was 2 µg of IgY antibody for each microliter of beads, as this presented a fluorescence percentage close to 100%, and a similar percentage was maintained when doubling the antibody concentration.
The proposed cytometric bead assay uses commercial latex beads, which implies the use of centrifugation and resuspension cycles for the washing and incubation steps of the analysis components. The washings are a crucial step for the success of the test, and, therefore, it is important not to use a greater centrifugation force than proposed here, since a higher speed leads to changes in the morphology of the beads, which impairs the reading in the flow cytometer. However, if the centrifugation is less powerful than that proposed here, there is a greater risk of loss of beads during the process, since they can be easily resuspended from the walls of the microtubes or the bottom of the plates. For this reason, the process of removing the supernatant must be carried out carefully to avoid these problems.
When applying this methodology to the development of immunoassays for other targets, adjustments in relation to the buffers, temperature, and incubation time can be made, taking into account the specific characteristics of the antibodies and antigens to be used in the new test. The analysis of the antigen saturation curve in the present study allowed the determination of the detection limit of the test. As shown here, using the conformation of the sandwich format, we identified a detection limit of 0.01 µg, and this value similar to the lowest detection limit obtained by conventional ELISA. However, this test has a much higher level of reliability than classical ELISA methodologies due to the high accuracy of the assays performed in flow cytometry, as can be seen by the detection limit obtained from several previous studies, such as Simonova et al.17 (5 pg/mL). Sharma et al.19 showed a sensitivity 4-fold to 80-fold greater than conventional ELISA, and Merbah et al.19 obtained a test that was 91% more sensitive than conventional ELISA. It can also be observed that these CBA assays have high reproducibility, since the triplicate analyses presented very low standard deviations in this work.
The main limitations of the method described here are the necessity of a flow cytometer and the fact that this method has several steps and takes some time to be performed. The steps described represent an important standardization, which is necessary to ensure the reliability of diagnostic tests using latex beads that are evaluated via flow cytometry. Therefore, the methodology proposed here is an alternative to the classic sandwich test models. This method can be applied to diverse types of assays and may be suitable for coupling several molecules to the surface of the beads, as well as for the detection of different analytes.
The authors have nothing to disclose.
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.
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 |