Name: dynamic monitoring of seroconversion using a multianalyte immunobead assay for covid-19
Uploaded: 2022-02-16T15:00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19 at JoVE.com

The authors wish to thank the Rush Biomarker Development Core for the use of their facilities, the Rush Biorepository for subject enrollment and biospecimen processing, and the Walder Foundation&#39;s Chicago Coronavirus Assessment Network (Chicago CAN) Initiative grant numbers SCI16 (J.R.S. and J.A.B.) and 21-00147 (J.R.S. and J.A.B.) and an award from the Swim Across America Foundation (J.A.B.).

All subjects were enrolled with written informed consent with full Institutional Review Board (IRB) approval of Rush University Medical Center under protocol ORA 20101207 with all institutional guidelines for ethical research conduct observed. Blood was collected via conventional phlebotomy into lavender vacutainers (K2EDTA) and processed with recommended protocols. The resulting plasma was archived at -80 &#176;C until assessments were performed.
1. Preparation of antigen-conjugated microspheres
<ol>
	<li>Select three different vials of magnetic microspheres with unique bead regions, recording bead ID and lot information for each vial used. 
	NOTE: The following steps will be followed for each distinct microsphere region. Microspheres are light sensitive and should be protected from prolonged exposure to light. During wash steps, be careful not to disturb the microspheres. If disturbed, allow a second 60 s separation.</li>
	<li>Vortex the microsphere stock for 60 s and sonicate for 5 min prior to use to dissociate aggregated beads.</li>
	<li>Transfer 1.0 x 106 beads to a 1.5 mL low-protein binding microcentrifuge tubes.</li>
	<li>Insert the tube into a magnetic separator and allow separation to occur for 60 s. With the tube still in the magnetic separator, carefully remove the supernatant without disturbing the bead pellet.</li>
	<li>Remove the tube from the magnetic separator, resuspend the beads with 100 &#181;L of HPLC-grade water, and vortex for 30 s. Place the tube back into the magnetic separator for 60 s, and subsequently remove the supernatant. Repeat this wash protocol twice.</li>
	<li>Remove the tube from the magnetic separator and resuspend the washed microspheres in 90 &#181;L of 100 mM Monobasic Sodium Phosphate, pH 6.2 (Activation Buffer) by vortex for 30 s.</li>
	<li>Add 10 &#181;L of 50 mg/mL Sulfo-NHS (diluted with Activation Buffer) to the microspheres and vortex gently for 10 s. Add 10 &#181;L of 50 mg/mL EDC solution (diluted with Activation Buffer) and vortex gently for 10 s. Incubate microspheres for 20 min at room temperature (RT) with a gentle vortex every 10 min.</li>
	<li>Repeat wash steps 1.4-1.5 with 50 mM MES, pH 5.0 (Coupling Buffer) in lieu of LC/MS-grade water for a total of two washes.</li>
	<li>Remove the tube from the magnetic separator and resuspend the beads with 100 &#181;L of Coupling Buffer by vortex for 30 s followed immediately by the addition of the desired quantity of protein.</li>
	<li>Bring the total volume to 150 &#181;L with Coupling Buffer. Mix coupling reaction by vortex for 30 s and incubate for 2 h by rotation at RT. 
	NOTE: For assays defined herein, conjugations were performed with the following protein concentrations: Spike S1: 5 &#181;g; Nucleocapsid: 5 &#181;g; Membrane: 12.5 &#181;g.</li>
	<li>Repeat wash step 1.4 with Phosphate Buffered Saline (PBS)-1% Goat Serum Albumin, 0.01% Polysorbate-20 (Quench Buffer) in lieu of LC/MS-grade water for a total of two washes. Resuspend the washed microspheres in 100 &#181;L of Quench Buffer containing 0.05% sodium azide by vortex for 30 s. 
	&#8203;NOTE: Permit the beads to quench fully for at least 6 h prior to proceeding to any other procedure.</li>
	<li>Count the number of recovered microspheres using an automated cell counter or a hemocytometer. Record the observed bead concentration.</li>
	<li>Refrigerate the coupled microspheres at 4 &#176;C in the dark.</li>
</ol>
2. Procedures
<ol>
	<li>Assay performance (Base protocol)
	<ol>
		<li>Resuspend the coupled microspheres by vortex for 30 s and sonicate for ~60-90 s.</li>
		<li>Remove the required quantity of each bead colloid from the respective tube and combine the bead colloids in a new 1.5 mL microcentrifuge tube.</li>
		<li>Insert the tube into a magnetic separator and allow separation to occur for 60 sec. With the tube still in the magnetic separator, carefully remove the supernatant without disturbing the bead pellet.</li>
		<li>Remove the beads from the separator, resuspend the beads with 100 &#181;L of PBS-1% bovine serum albumin, 0.01% Polysorbate-20 (Assay Buffer), and vortex for 30 s. Place the tube into a magnetic separator and allow separation to occur for 60 s. Repeat this washing protocol (i.e., steps 2.1.3-2.1.4) twice</li>
		<li>Adjust the concentration of the 3-plex working microsphere mixture by adding an appropriate volume of Assay Buffer to generate a final concentration of 100 microspheres per 1 &#181;L for each target.</li>
		<li>Aliquot 12.5 &#181;L of the microsphere mixture prepared in step 2.1.5 into each well of a 384-well plate or 25 &#181;L into each well of a 96-well plate.</li>
		<li>Dilute the plasma/serum specimens 500-fold in Assay Buffer. Prepare standard specimens according to titration desired.</li>
		<li>Add 12.5 &#181;L of Assay Buffer as the blank sample and add each of the diluted specimen or standard into each designated well of a 384-well specimen plate or 25 &#181;L of the blank, diluted specimen or standard into each well of a 96-well specimen plate.</li>
		<li>Cover the plate with an aluminum seal or foil and incubate for 1 h at RT on a plate shaker set to 700 rpm. 
		NOTE: Schematic for the dilution curves is provided in Table 1.</li>
		<li>Prepare a solution of anti-human detection antibodies (secondary antibody solutions) at 4 &#181;g/mL with Assay Buffer as specified in step 2.1.11.</li>
		<li>Prepare Goat-anti-human IgM, conjugated with Super Bright 436 (SB)/Goat-anti-human IgA, Phycoerythrin (PE) Conjugate detections antibodies at 4 &#181;g/mL; or Goat-anti-human IgM, SB Conjugate/Goat-anti-human IgG, PE Conjugate detections antibodies at 4 &#181;g/mL. 
		NOTE: For a 384-well plate format, 12.5 &#181;L/well of the prepared secondary antibody solution is required, and for a 96-well plate format, 25 &#181;L/well is required.</li>
		<li>Place the plate on a magnetic separator, wash rapidly, and forcefully invert over a biohazard container to remove liquid from the wells. With the plate still inverted, forcefully tap the plate against a thick wad of paper.</li>
		<li>Wash each well with 100 &#181;L of Assay Buffer and remove the liquid by forceful inversion over a biohazard container, as previously described. Repeat these steps (2.1.12-2.1.13) for a total of two washes. Discard all used wads of paper into a biohazard container.</li>
		<li>Add 12.5 &#181;L of the secondary antibody working solution to each well of a 384-well plate or 25 &#181;L to each well of a 96-well plate. Cover the plate with an aluminum seal or foil and incubate for 30 min at RT on a plate shaker set to 700 rpm.</li>
		<li>Repeat wash steps 2.1.12-2.1.13</li>
		<li>Add 75 &#181;L of Assay Buffer into each well of a 384-well plate or 100 &#181;L into each well of a 96-well plate. Cover the plate with an aluminum seal or foil and incubate for 5 min at RT on a plate shaker set to 700 rpm.</li>
		<li>Analyze 60 &#181;L via the instrument analyzer according to the system manual.</li>
	</ol>
	</li>
	<li>Optimization of specimen-capture incubation time
	<ol>
		<li>Perform the steps in section 2.1 using duration of incubation times of 30 min, 60 min, and 120 min in step 2.1.9. 
		NOTE: Incubations may be performed either with distinct plates or by pausing at step 2.1.6 until the time to proceed to step 2.1.9. to attain the desired incubation times.</li>
		<li>Proceed with the plate-reading on the analyzer using 60 &#181;L of the assay mixture according to the manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
	<li>Optimization of secondary antibody concentration
	<ol>
		<li>Perform this procedure as detailed in section 2.1, with exception to the final concentrations of reagents in the secondary antibody working solution (prepared in Step 2.1.10-2.1.11) modified as follows: 
		Goat-anti-human (or rabbit) IgM, SB-conjugate detection antibodies at 8, 4, 2, 1 and 0.5 &#181;g/mL. 
		Goat-anti-human (or rabbit) IgA, PE-conjugate detection antibodies at 8, 4, 2, 1 and 0.5 &#181;g/mL. 
		Goat-anti-human (or rabbit) IgG, PE-conjugate detection antibodies at 8, 4, 2, 1 and 0.5 &#181;g/mL. 
		Goat-anti-human (or rabbit) IgG, PE-conjugate/Goat-anti-human (or rabbit) IgM, SB-conjugate detection antibodies at 8, 4, 2, 1 and 0.5 &#181;g/mL. 
		&#8203;Goat-anti-human (or rabbit) IgA, PE-conjugate/Goat-anti-human (or rabbit) IgM, SB-conjugate detection antibodies at 8, 4, 2, 1 and 0.5 &#181;g/mL.</li>
		<li>Proceed with the plate-reading on the analyzer using 60 &#181;L of the assay mixture according to the manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
	<li>Optimization of secondary antibody incubation time
	<ol>
		<li>Perform this procedure as detailed in section 2.1 with 15 min, 30 min, 60 min, and 120 min of incubation duration defined in step 2.1.14.</li>
		<li>Proceed with the plate-reading on the analyzer using 60 &#181;L of the assay mixture according to the manufacturer&#39;s recommendations.</li>
	</ol>
	</li>
	<li>Evaluation of subject specimens with optimized dual-channel assays
	<ol>
		<li>Collect subject plasma samples (n = 4) at days -21, -11, -1/0, +14, +28, +60, +90, and +120 relative to the completion of Covid-19 vaccine (i.e., second dose) administration. 
		NOTE: Day 0 represents the time point whereby vaccination was completed.</li>
		<li>Perform all assays using the Base Protocol (section 2.1.) as either an IgG/IgM or IgA/IgM dual-channel assay.</li>
		<li>Normalize the results to the maximal observed Median Fluorescent Intensity (MFI) value for each specific immunoglobulin and assay.</li>
	</ol>
	</li>
</ol>

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Dr. Borgia is the inventor of the serological test used in this manuscript which is patent pending. The article is presented in a descriptive manner only with no statistical analyses presented to avoid potential bias introduced by author conflict.

The appreciation of an immune response to SARS-CoV-2 exposure, in conjunction with RT-PCR-based surveillance of infection status, has been well described as a prescription to clarify the course of recovery from Covid-19, to serve as a means to identify convalescent plasma with potential therapeutic value, and to survey infection rates on a population-scale basis10,11. Different examples to understanding seroconversion in human subjects include protein (antigen) arrays12, immunoblots13, rapid immunochromatographic constructs14, and enzyme-linked immunosorbent assays (ELISAs)15,16,17. Each of these aforementioned techniques can assess multiple immunoglobulin isotypes individually with minor modifications. These procedures, however, are not capable of being practically repurposed to permit parallel analysis of multiple isotypes, as presented in this manuscript, rendering cost and throughput factors as limitations for their administration on a population-based scale testing strategy. Several of these applications also provide the opportunity to concatenate levels of multiple antigens in parallel either as presented or in altered formats, such as a multiplex ELISA18,19. Finally, the immunobead platform supplies the possibility to evaluate levels of multiple antigens in parallel20,21 but has been limited to a single epitope (i.e., immunoglobulin isotype) per assay unless, however, the recent instrument with &#39;dual-channel&#39; assay capabilities is employed.
In this report, protocols are enumerated which establish the reliable measurement of multiple epitopes in an immunobead assay using the instrument in &#39;dual-channel&#39; mode in a case study of seroconversion of Covid-19-vaccinated individuals. The method demonstrated excellent precision (%CV values typically &#60;20%) using manual assay designs that may be improved with the integration of laboratory automated systems. Assay sensitivity and dynamic range for all selected analytes and epitopes were suitable for routine evaluations. Although the lack of commercially-available anti-antigen IgA and IgM immunoreagents limited quantitation to the IgG isotype, such a restriction does not preclude the capability to offer semi-quantitative assessments or assessments relative to a specific specimen as a calibrant22,23.
The validated immunobead assay was allocated to investigate seroconversion in a cohort of individuals immunized with the Covid-19 vaccine. Contrasted against other similar platforms, the family of instrumentation allows the prospecting of seroconversion in hundreds of individuals per day in a manual workflow and thousands per day in an automated scheme. Overall, these findings confirm that the &#39;dual-channel&#39; approach has appropriate sensitivity for the description of multiple epitopes in parallel for the identical assay and is viable in a multianalyte context. A difference in sensitivities to channel 1 and channel 2, as performed with phycoerythrin and Super Bright 436 fluorophores, respectively, may warrant the design of a specific experiment to ensure viable analytical results are acquired for a given experiment. That is, the reservation of channel 1 for epitopes or analytes of a lower prevalence may be necessary to maintain a dynamic range of the assay that includes the observed values of unknowns. Beyond this consideration, assay design was obvious and should be readily accessible to laboratories with limited analytical capabilities. Certainly, this consideration should be weighed when considering channel 1 to channel 2 interference, as we noted for Figure 5, whereby interference from higher abundance isotypes may cause misleading analytical errors for those in much lower abundance when measured in a dual-channel format. This potential for interference with situations with highly different analyte concentrations may represent a significant limitation to the approach if not properly handled in the assay design phase.
In conclusion, a method to rapidly measure antibody titers from the major immunoglobulin isotypes associated with an immune response to Covid-19 infection or vaccination was presented. Applying this approach in a longitudinal fashion to assess seroconversion may provide insights that could be better used to manage and/or monitor the course of the disease or, alternately, guide prospective Covid-19 booster vaccine programs.

Simultaneous measurement of multiple disease-related biomarkers in biological samples permits descriptive and predictive insights into pathological processes. While conventional single analyte immunological procedures, such as enzyme-linked immunosorbent assays (ELISAs), have been the cornerstone of quantitative analyses in both clinical and research settings, these techniques can have substantial limitations regarding throughput, the quantity of specimen required for each measurement, and cost-effectiveness that greatly limit the study of multiple biological elements that are frequently intertwined throughout the disease course1. Microsphere-based multiplexing technology has become an indispensable platform for both diagnostic and research facilities for its ability to combine assays to enhance laboratory throughput, mitigate sample scarcity, and reduce repetitive testing to maximize cost savings1,2,3,4. Recently, further augmentation of this multiplexing power has been introduced with instruments possessing dual-reporter capabilities. The dual-reporter feature implements two fluorescent channels for detection, supplying another dimension of multiplexing, permitting the detection of multiple epitopes on the same analyte.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic5. While RT-PCR testing is crucial for infection confirmation at the beginning of the disease course, serologic examinations of antibody titers have proved to be imperative for the accurate and complete critique of individuals regarding a previous exposure or recovery; a response to immunization; and/or an evaluation of Covid-19 vaccination efficacy6,7.
This report delineates methods for gauging seroconversion for multiple SARS-CoV-2 viral antigens that utilize a flow-based, multiplex reader dual-reporting system. Specifically, concurrent detection of two antibody subtypes (configured as IgG/IgM or IgA/IgM) for a 3-plex SARS-CoV-2 antigens panel that includes assays for Spike S1, Nucleocapsid, and Membrane (aka Matrix) glycoproteins, are described. This approach provides an ideal enterprise for the capture of longitudinal seroconversion and contributes a valuable tool in the arsenal against the Covid-19 pandemic.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>384-well black side polystyrene microtiter plates</td>
			<td>Thermo Fisher</td>
			<td>12-565-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Activation buffer (0.1 M NaH2PO4, pH 6.2)</td>
			<td>Prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assay buffer (PBS, 1% Bovine Serum Albumin, 0.01% Polysorbate-20)</td>
			<td>Prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, heat shock fraction</td>
			<td>MilliporeSigma</td>
			<td>A-7888-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Coupling buffer (50 mM MES, pH 5.0)</td>
			<td>Prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>COVID 19 M Coronavirus Recombinant Matrix Protein (6xHis tag)</td>
			<td>MyBioSource</td>
			<td>MBS8574735</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)</td>
			<td>Thermo Fisher</td>
			<td>PIA35391</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Albumin, Fraction V Powder</td>
			<td>MilliporeSigma</td>
			<td>A2514-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>IgA Goat anti-Human, R-PE, Polyclonal</td>
			<td>Thermo Fisher</td>
			<td>OB205009</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Goat anti-Human, R-PE, Polyclonal</td>
			<td>Thermo Fisher</td>
			<td>OB204009</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Goat anti-Rabbit, R-PE, Polyclonal</td>
			<td>Thermo Fisher</td>
			<td>OB403009</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM Goat anti-Human, R-PE, Polyclonal</td>
			<td>Thermo Fisher</td>
			<td>OB202009</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM Mouse anti-Human, SB 436, Clone SA-DA4</td>
			<td>Thermo Fisher</td>
			<td>62999842</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument Analyzer</td>
			<td>Luminex Corp.</td>
			<td>INTELLIFLEX-RUO</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-Bind microcentrifuge tubes (1.5 mL)</td>
			<td>Thermo Fisher</td>
			<td>13-698-794</td>
			<td></td>
		</tr>
		<tr>
			<td>MagPlex-C Microspheres, Region XXX</td>
			<td>Luminex Corp.</td>
			<td>MC10XXX-01</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate (2-(N-Morpholino) ethane sulfonic acid hydrate)</td>
			<td>MilliporeSigma</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate aluminum sealing tape</td>
			<td>Thermo Fisher</td>
			<td>07-200-683</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate-20</td>
			<td>Thermo Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Quality Biological Inc. PBS, pH 7.2</td>
			<td>Thermo Fisher</td>
			<td>50-751-7328</td>
			<td></td>
		</tr>
		<tr>
			<td>Quench buffer (PBS, 1% Goat Serum Albumin, 0.01% Polysorbate-20)</td>
			<td>Prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Nucleocapsid Protein (His tag)</td>
			<td>Sino Biologicals</td>
			<td>40588-V08B</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His tag)</td>
			<td>Sino Biologicals</td>
			<td>40591-V08H</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, Rabbit pAb</td>
			<td>Sino Biologicals</td>
			<td>40592-T62</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody, Rabbit mAb</td>
			<td>Sino Biologicals</td>
			<td>40588-R0004</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (COVID-19) Membrane Antibody (IN), Rabbit pAb</td>
			<td>ProSci</td>
			<td>10-516</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Thermo Fisher</td>
			<td>PIA39269</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash Buffer (PBS, 0.01% Polysorbate-20)</td>
			<td>Prepared in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water, LC/MS grade</td>
			<td>Thermo Fisher</td>
			<td>W-64</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic monitoring of seroconversion using a multianalyte immunobead assay for covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

Watch this Scientific Journal Video about Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19 at JoVE.com

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

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