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

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.