Name: standardization of a cytometric bead assay based on egg-yolk antibodies
Uploaded: 2023-05-19T12:50:00
Description: Watch this Scientific Journal Video about Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies at JoVE.com

We would like to thank the FIOCRUZ (&#34;Programa de excel&#234;ncia em pesquisa b&#225;sica e aplicada em sa&#250;de dos laborat&#243;rios do Instituto Le&#244;nidas e Maria Deane - ILMD/Fiocruz Amaz&#244;nia-PROEP-LABS/ILMD FIOCRUZ AMAZ&#212;NIA&#34;), the Post-graduate Program in Biotechnology (PPGBIOTEC at the Universidade Federal do Amazonas - UFAM), the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel (CAPES), and the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Amazonas (FAPEAM) for providing the scholarships. Figure 2&#160;and Figure 4&#160;were created with biorender.com.

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
<ol>
	<li>Hygienization of the eggs
	<ol>
		<li>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...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Sousa, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+polyclonal+antibody-based+sandwich+ELISA+for+detection+of+Plasmodium+vivax+developed+from+two+lactate+dehydrogenase+protein+segments.">A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments.</a> BMC Infectious Diseases. 14 (1), 49 (2014).</li><li>Klimentzou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+immunochemical+evaluation+of+antibodies+Y+for+the+poorly+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha.</a> Peptides. 27 (1), 183-193 (2006).</li><li>Redwan, E. M., Aljadawi, A. A., Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+protocol+for+immunoglobulin+Y+purification+from+chicken+egg+yolk.">Simple and efficient protocol for immunoglobulin Y purification from chicken egg yolk.</a> Poultry Science. 100 (3), 100956 (2021).</li><li>Lee, H. Y., Abeyrathne, E. D. N. S., Choi, I., Suh, J. W., Ahn, D. U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+separation+of+immunoglobulin+Y+and+phosvitin+from+chicken+egg+yolk+without+using+organic+solvents.">Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents.</a> Poultry Science. 93 (10), 2668-2677 (2014).</li><li>Pauly, D., Chacana, P., Calzado, E., Brembs, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgY+Technology:+Extraction+of+chicken+antibodies+from+egg+yolk+by+polyethylene+glycol+(PEG)+precipitation.">IgY Technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation.</a> Journal of Visualized Experiments. (51), e3084 (2011).</li><li>Chang, H. M., Lu, T. C., Chen, C. C., Tu, Y. Y., Hwang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+immunoglobulin+from+egg+yolk+by+anionic+polysaccharides.">Isolation of immunoglobulin from egg yolk by anionic polysaccharides.</a> Journal of Agricultural and Food Chemistry. 48 (4), 995-999 (2000).</li><li>Ko, K. Y., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+immunoglobulin+Y+from+egg+yolk+using+ammonium+sulfate+precipitation+and+ion+exchange+chromatography.">Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography.</a> Poultry Science. 86 (2), 400-407 (2007).</li><li>Polson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+IgY+from+the+yolks+of+eggs+by+a+chloroform+polyethylene+glycol+procedure.">Isolation of IgY from the yolks of eggs by a chloroform polyethylene glycol procedure.</a> Immunological Communications. 19 (3), 253-258 (1990).</li><li>Simonova, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=xMAP-based+analysis+of+three+most+prevalent+staphylococcal+toxins+in+Staphylococcus+aureus+cultures.">xMAP-based analysis of three most prevalent staphylococcal toxins in Staphylococcus aureus cultures.</a> Analytical and Bioanalytical Chemistry. 406 (25), 6447-6452 (2014).</li><li>Sharma, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+assay+for+detection+of+staphylococcal+and+streptococcal+exotoxins.">A multiplex assay for detection of staphylococcal and streptococcal exotoxins.</a> PLoS One. 10 (8), e0135986 (2015).</li><li>Merbah, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+a+cytometric+p24-capture+bead-assay+for+the+detection+of+main+HIV-subtypes.">Standardization of a cytometric p24-capture bead-assay for the detection of main HIV-subtypes.</a> Journal of Virological Methods. 230, 45-52 (2016).</li></ol>

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-Chicken IgY (H+L), highly cross-adsorbed, CF&#160;488A antibody produced in donkey</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4600031</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG (H+L), F(ab&#8242;)2</td>
			<td>Sigma-Aldrich</td>
			<td>SAB4600388</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II</td>
			<td>BD Biosciences</td>
			<td>BF-FACSC2</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva CS&#38;T research beads (CS&#38;T research beads)</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva software 7.0</td>
			<td>BD Biosciences</td>
			<td>655677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad</td>
			<td>#5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Caprilic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O3907</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5702 R</td>
			<td>Eppendorf</td>
			<td>Z606936</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloride 37% acid molecular grade</td>
			<td>NEON</td>
			<td>02618 NEON</td>
			<td></td>
		</tr>
		<tr>
			<td>CML latex, 4% w/v</td>
			<td>Invitrogen</td>
			<td>C37253</td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 8R</td>
			<td>Thermo Scientific</td>
			<td>TS-HM8R</td>
			<td></td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide Hydrochloride Powder (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E7750-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>130672-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>1003335620</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Acs Cientifica</td>
			<td>P.10.0594.024.00.27</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>Acs Cientifica</td>
			<td>R09211000</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Mixer Heat/Cool</td>
			<td>KASVI</td>
			<td>K80-120R</td>
			<td></td>
		</tr>
	</tbody>
</table>

standardization of a cytometric bead assay based on egg-yolk antibodies

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.

Figure 2&#160;provides a graphical representation of the extraction process of IgY antibodies via acidification, followed by separation using caprylic acid (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65123/65123fig02.jpg" /> 
Figure 2: Schematic representation of the extraction step of IgY antibodies and separation...

Watch this Scientific Journal Video about Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies at JoVE.com

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

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 ...

Among the immunoassay techniques aimed at diagnosing disease, the cytometric bead assay has emerged as a highly sensitive and reliable approach, analyzing thousands of particles in a single assay. The high accuracy of flow cytometry, coupled with the low cost of latex beads, make our technique applicable for the analysis of immunoassays tools, such as antibodies and antigens. IgY antibodies have a low cost, and the ethical advantages in their production, and can be used as a detection tool for rare disease.Begin by immersing the freshly laid or four-day-old eggs, from the Gallus gallus deckle white lineage, in a 0.2%diluted solution of sodium hypochlorite. Rinse them quickly under running water, and wipe gently. Break the egg carefully, separate the yolk from the white with the help of a yolk separator.And remove the excess white with filter paper. Pierce the yolk, and collect its interior into a 50 milliliter conical tube. Store the yolk at minus 20 degrees Celsius for at least 24 hours prior to use.After thawing the stored yolk, dilute it at a one to 10 ratio in one millimolar PBS. Adjust the pH of the solution to five, with one normal hydrochloric acid, and incubate the solution at four degrees Celsius for six to 24 hours. Centrifuge the solution at 3000 G for 40 minutes, and filter the recovered supernatant using a 0.7 millimeter cellulose filter, before readjusting the pH of the supernatant to five.Add caprylic acid to a final concentration of 8.7%under constant stirring, for 30 minutes at four degrees Celsius. After centrifuging the samples for 15 minutes, separate the supernatant from the precipitated material. Readjust the pH of the solution to 7.4 using one molar sodium hydroxide.And quantify the antibodies using the Bradford assay, before storing them at minus 20 degrees Celsius. Add 21 microliters of EDC, and 21 microliters of NHS, to 21 microliters of latex beads. And adjust to a final volume of 2.1 milliliters, with filtered 10 millimolar PBS.Incubate the solution at 22 degrees Celsius, with rapid shaking, for three hours. Add the previously extracted capture antibody, IgY PfHRP2, to different micro tubes containing 100 microliters of this solution. And incubate again for 16 hours.After centrifuging the samples and discarding the supernatants, wash the latex beads twice with 500 microliters of filtered 10 millimolar PBS, using centrifugation and resuspension cycles. For the blocking of the beads, resuspend the samples in one milliliter of blocking buffer, and follow the same procedure shown previously to incubate it for two hours. After washing the beads with PBS, resuspend them again in 10 millimolar PBS buffer.Add 100 microliters of fluorescent anti-chicken antibody, diluted to one to 2000 in 10 millimolar mixture of PBS and BSA, to each micro tube. Incubate the samples in the dark for 30 minutes. After washing the beads, resuspend them again with 250 microliters of filtered 10 millimolar PBS.Determine the limit of detection by distributing 100 microliters of the blocked bead preparation, into tubes containing different amounts of the diluted recombinant protein PfHRP2 in triplicate. Incubate the bead solutions with the protein at 22 degrees Celsius, with rapid shaking, for one hour. Wash the beads with 500 microliters of filtered 10 millimolar PBS, with centrifugation, as previously demonstrated.After discarding the supernatant add two micrograms of mouse IgG antibody against the recombinant protein, diluted in 10 millimolar PBS and BSA to each sample, and follow the same procedure for incubation. Next, add 100 microliters of the diluted fluorescent anti-mouse antibody to each sample. After incubation, resuspend the sample in 250 microliters of filtered 10 millimolar PBS.Turn on the computer and flow cytometer, and wait a few minutes for the equipment to connect to the computer. Click on the cytometry software installed on the computer and log into it. From the software workspace, select Cytometer, Followed by Fluidics Startup, Cleaning Modes, and SIT Flush.Define the gating strategy based on the negative control particle's morphometric and fluorescence characteristics. To determine the cell morphometry, choose dot plot plot graphic for the analysis of forward scatter A parameters using side scatter A.Determine the single cells by side scatter using dot plot plot graphic for the analysis of side scatter W in the Y axis, and side scatter H in the X axis. And determine the single cells by forward scatter, using dot plot plot graphic, for the analysis of forward scatter W in the Y axis, and forward scatter H in the X axis.For the fluorescence analysis, choose FL1 dot plot plots using forward scatter A.Use a stoppered polystyrene tube containing unlabeled samples, and samples previously labeled with single-color Alexa Fluor 488 And carefully mix the tube's contents. Attach the tube to the flow cytometer probe, and left click on Acquire. To set the power of the lasers, click on Parameters, and in the options below, adjust the voltage of the forward scatter to 182 volts, and the voltage of the side scatter to 236 volts.To set the threshold on cytometer, click on Threshold, and in the options below, adjust the FSC to 500 volts. Set the analysis gate to characterize particles by size and complexity, as well as to perform single cell analysis. Remove autofluorescence by analyzing the dye-free sample containing only the latex beads, and adjusting the voltage of the detector FL1 FITC to 332 volts.Set the fluorescence analysis gate in quadrangular format, from the negative point shown in the dot plot graph. After the morphometric and fluorescent analyses have been adjusted, configure the device for 50, 000 events, and click on Record. The electrophoretic profiles of the IgY PfHRP2 antibody, due to the separation using caprylic acid, are shown here.From this figure, it can be seen that the antibodies before and after the caprylic acid separation showed similar electrophoretic profiles, with characteristic bands of 65 kilodaltons and 27 kilodaltons, which refer to the light and heavy chains of the IgY. In addition to these, other bands are also visible, likely due to the C terminal fragment egg yolk protein vitellogenin II precursor. Assessing the antibody saturation concentration on latex beads, is vital for developing an immunoassay with the beads.A curve of the percentage fluorescence of different concentrations of antibodies, coupled to each microliter of commercial latex beads used, is shown here. As the concentration increased, the percentage values also increased, and plateaued at two micrograms of antibody concentration, establishing it as the optimal coupling concentration for the target antigen immunoassay. The detection limit was determined by assessing the fluorescence percentage of latex speed reactivity against varying rPfHRP2 protein concentrations.From a sandwich immunoassay, it was observed that the fluorescence was increased from 0.01 micrograms of PfHRP2 protein, and plateaued at 0.1 micrograms. There was no significant difference in fluorescence at zero micrograms and 0.01 micrograms. However, a significant difference in fluorescence was observed at 0.01 micrograms, 0.1 micrograms, and 10 micrograms.The washing step of this protocol is critical to avoid the loss of beads. The gauge must be well defined so that there are no mistakes in the result obtained. The steps described here represent an important standardization, which is necessary to ensure the reliability of the diagnostic tests using latex beads that are evaluated via full cytometry.Therefore, this methodology is an alternative to the classic sandwich test models. This method can be applied to diverse types of assays, and may be suitable for coping several molecules to the surface of the beads, as well as for the detection of different analytes.

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