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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L., 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+microsphere-based+immunoassay+increases+the+sensitivity+of+siv-specific+antibody+detection+in+serum+samples+and+mucosal+specimens+collected+from+rhesus+macaques+infected+with+SIVmac239.">A multiplex microsphere-based immunoassay increases the sensitivity of siv-specific antibody detection in serum samples and mucosal specimens collected from rhesus macaques infected with SIVmac239.</a> BioResearch Open Access. 2 (3), 171-178 (2013).</li><li>Volpetti, F., Garcia-Cordero, J., Maerkl, S. 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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 ...

dynamic-monitoring-seroconversion-using-multianalyte-immunobead-assay

Research

JoVE Journal

Medicine

2.7K Views.  Rush University Medical Center. 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. 

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

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

Evaluation of Capnography Sampling Line Compatibility and Accuracy when Used with a Portable Capnography Monitor

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment of end-tidal CO2 (ETCO2), its accuracy is commonly validated using commercial kits composed of a capnography monitor and its matching disposable nasal cannula sampling lines. The purpose of this study was to assess the compatibility and accuracy of cross-paired capnography sampling lines with a single portable bedside capnography monitor. A series of 4 bench tests were performed to evaluate the tensile strength, rise time, ETCO2 accuracy as a function of respiratory rate, and ETCO2 accuracy in the presence of supplemental O2. Each bench test was performed using specialized, validated equipment to allow for a full evaluation of sampling line performance. The 4 bench tests successfully differentiated between sampling lines from different commercial sources and suggested that due to increased rise time and decreased ETCO2 accuracy, not all nasal cannula sampling lines provide reliable clinical data when cross-paired with a commercial capnography monitor. Care should be taken to ensure that any cross-pairing of capnography monitors and disposable sampling lines is fully validated for use across respiratory rates and supplemental O2 flow rates commonly encountered in clinical settings.

The goal of this study was to evaluate the accuracy of capnography sampling lines used in conjunction with a portable bedside capnography monitor. Sampling lines from 7 manufacturers were evaluated for tensile strength, rise time, and ETCO2 accuracy as a function of respiratory rate or supplemental oxygen flow rate.

Capnography is commonly used to monitor patient&#8217;s ventilatory status. While sidestream capnography has been shown to provide a reliable assessment ...

Real-Time Dynamic Navigation System for the Precise Quad-Zygomatic Implant Placement in a Patient with a Severely Atrophic Maxilla

Zygomatic implants (ZIs) are an ideal way to address cases of a severely atrophic edentulous maxilla and maxilla defects because they replace extensive bone augmentation and shorten the treatment cycle. However, there are risks associated with the placement of ZIs, such as penetration of the orbital cavity or infra-temporal fossa. Furthermore, the placement of multiple ZIs makes this surgery risky and more difficult to perform. Potential intraoperative complications are extremely dangerous and may cause irreparable losses. Here, we describe a practical, feasible, and reproducible protocol for a real-time surgical navigation system for precisely placing quad-zygomatic implants in the severely atrophic maxilla of patients with residual bone that does not meet the requirements of conventional implants. Hundreds of patients have received ZIs at our department based on this protocol. The clinical outcomes have been satisfactory, the intraoperative and postoperative complications have been low, and the accuracy indicated by infusion of the designed image and postoperative three-dimensional image has been high. This method should be utilized during the entire surgical procedure to ensure ZI placement safety.

Here, we present a protocol to achieve precise quad-zygomatic implant placement in patients with severely atrophic maxilla using a real-time dynamic navigation system.

Zygomatic implants (ZIs) are an ideal way to address cases of a severely atrophic edentulous maxilla and maxilla defects because they replace extensive ...

Dynamic Navigation for Dental Implant Placement

In modern implantology, the application of surgical navigation systems is becoming increasingly important. In addition to static surgical navigation methods, a guide-independent dynamic navigation implant placement procedure is becoming more widespread. The procedure is based on computer-guided dental implant placement utilizing optical control. This work aims to demonstrate the technical steps of a new dynamic computer-aided implant surgery (DCAIS)&#160;system (design, calibration, surgery) and check the accuracy of the results. Based on cone-beam computed tomography (CBCT) scans, the exact positions of implants are determined with dedicated software. The first step of the operation is the calibration of the navigation system, which can be performed in two ways: 1) based on CBCT images taken with a marker or 2) based on CBCT images without markers. Implants are inserted with the aid of real-time navigation according to the preoperative plans. The accuracy of the interventions can be evaluated based on postoperative CBCT images. The preoperative images containing the planned positions of the implants and postoperative CBCT images were compared based on the angulation (degree), platform, and apical deviation (mm) of the implants. To evaluate the data, we calculated the standard deviation (SD), mean, and standard error of the mean (SEM) of deviations within planned and performed implant positions. Differences between the two calibration methods were compared based on this data. Based on the interventions performed so far, the use of DCAIS allows for high-precision implant placement. A calibration system that does not require labeled CBCT recording allows for surgical intervention with similar accuracy as a system that uses labeling. The accuracy of the intervention can be improved by training.

Dynamic computer-aided implant surgery (DCAIS) is a controlled implant surgical placement method performed without a surgical template using optical control. The real-time intraoperative control of movement and position of the surgical device simplifies the procedure and gives more freedom to the surgeon, providing similar precision as static navigation methods.

In modern implantology, the application of surgical navigation systems is becoming increasingly important. In addition to static surgical navigation ...

A Surgical Model of Heart Failure with Preserved Ejection Fraction in Tibetan Minipigs

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are limited for investigating the fundamental mechanisms of HFpEF and identifying potential therapeutic targets. This work provides a detailed description of the surgical procedure of descending aortic constriction (DAC) in Tibetan minipigs to establish a large animal model of HFpEF. This model used a precisely controlled constriction of the descending aorta to induce chronic pressure overload in the left ventricle. Echocardiography was used to evaluate the morphological and functional changes in the heart. After 12 weeks of DAC stress, the ventricular septum was hypertrophic, but the thickness of the posterior wall was significantly reduced, accompanied by dilation of the left ventricle. However, the LV ejection fraction of the model hearts was maintained at &gt;50% during the 12-week period. Furthermore, the DAC model displayed cardiac damage, including fibrosis, inflammation, and cardiomyocyte hypertrophy. Heart failure marker levels were significantly elevated in the DAC group. This DAC-induced HFpEF in minipigs is a powerful tool for investigating molecular mechanisms of this disease and for preclinical testing.

The present protocol describes a step-by-step procedure to establish a minipig model of heart failure with preserved ejection fraction using descending aortic constriction. The methods for evaluating cardiac morphology, histology, and function of this disease model are also presented.

More than half of heart failure (HF) cases are classified as heart failure with preserved ejection fraction (HFpEF) worldwide. Large animal models are ...

Dynamic Navigation in Endodontics: Guided Access Cavity Preparation by Means of a Miniaturized Navigation System

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common sequelae of dental trauma but can also occur with stimuli such as caries, bruxism, or after placing a restoration. In order to access the root canal as minimally invasive as possible in case of a necessary root canal treatment, dynamic navigation has recently been introduced in endodontics in addition to static navigation. The use of a dynamic navigation system (DNS) requires pre-operative cone-beam computed tomography (CBCT) imaging and a digital surface scan. If necessary, reference markers must be placed on the teeth before the CBCT scan; with some systems, these can also be planned and created digitally afterward. By means of a stereo camera connected to the planning software, the drill can now be coordinated with the help of reference markers and virtual planning. As a result, the position of the drill can be displayed on the monitor in real-time during preparation in different planes. In addition, the spatial displacement, the angular deviation, and the depth position are also displayed separately. The few commercially available DNS mostly consist of relatively large camera-marker-systems. Here, the DNS contains miniaturized components: a low-weight camera (97 g) mounted on the micromotor of the electric handpiece utilizing a manufacturer-specific connecting mechanism and a small marker (10 mm x 15 mm), which can be easily attached to an individually manufactured intraoral tray. For research purposes, a post-operative CBCT scan can be matched with the pre-operative one, and the volume of tooth structure removed can be calculated by the software. This work aims to present the technique of guided access cavity preparation by means of a miniaturized navigation system from imaging to clinical implementation.

Dynamic navigation systems (DNS) provide real-time visualization and guidance to the operator during endodontic access cavities preparation. The planning of the procedure requires three-dimensional imaging utilizing cone beam computed tomography and surface scans. After the export of the planning data to the DNS, access cavities can be prepared with minimal invasion.

In the case of teeth with pulp canal calcification (PCC) and apical pathology or pulpitis, root canal treatment can be very challenging. PCC are common ...