an assay to measure influenza neuraminidase inhibition antibody titers

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Heat and activate the serum samples in a water bath at 56 degrees Celsius for 45 to 60 minutes. Thaw a vial of virus, vortex, and resuspended in the sample diluent at the dilution that was selected. Prepare at least 5 milliliters of virus for each assay plate.
Keep the diluted virus on ice, until the plates are washed, and serum samples have been added to the plate. Prepare dilutions of the serum samples and any controls, by making serial two-fold dilutions in a round-bottom 96-well plate, beginning with a 1-in-10 10 dilution in sample diluent.
Use a multichannel pipette to transfer 50 microliters of each serum control or sample dilution from the dilution plate into duplicate wells of a washed fetuin-coated plate. The sample dilution should be added in columns 2 to 11. Add 50 microliters of diluted virus to all wells, except for the negative control in column 12.
Add 50 microliters of sample diluent to wells in column 1, and add 100 microliters of sample diluent to column 12. Cover the wells with a plate sealer, and then mix by gently tapping the sides of the plate or by placing it on a plate shaker at moderate speed for 10 seconds.
Place the plate in a humidified incubator at 37 degrees Celsius for 16 to 18 hours. When the overnight incubation is complete, wash the plate before adding PNA-HRPO, and complete the assay as before. Read the optical density at 490 nanometers, and confirm that the assay results are valid. Determine the 50% endpoint titer, as described in the text protocol and the results section.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and Starting Material
Note: Refer to the Materials Table to obtain the source of all reagents.
<ol>
	<li>Fetuin-coated Plates
	<ol>
		<li>Prepare the coating buffer by mixing 10 ml of the 10x coating buffer with 90 ml of deionized H2O.</li>
		<li>Prepare a stock solution of fetuin at 25 mg/ml in 1x coating buffer and store in 500 &#181;l aliquots at -20 &#176;C.</li>
		<li>Prepare a working solution of fetuin (25 &#181;g/ml) immediately before coating plates by diluting the stock solution 1,000-fold in 1x coating buffer.</li>
		<li>Use a multichannel pipette to dispense 100 &#181;l of the working solution of fetuin into all wells of a 96-well plate with high protein-binding capacity.</li>
		<li>Cover each plate with a plate sealer, stack the plates in groups of 10, and wrap them in foil.</li>
		<li>Place the plates in a refrigerator (2-8 &#176;C) for at least 18 hr before performing an assay. 
		Note: Plates may be coated in advance and stored with the coating solution at 2-8 &#176;C for up to 2 months.</li>
	</ol>
	</li>
	<li>Diluents
	<ol>
		<li>To prepare the diluent to make virus and sample dilutions (2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, 20 mM CaCl2, 1% bovine serum albumin (BSA) and 0.5% Tween 20), mix 94.2 ml 1X MES, pH 6.5 with 2 ml CaCl2 (10 mg/ml), 3.3 ml 30% BSA and 0.5 ml Tween 20.</li>
		<li>To prepare the diluent for Peanut agglutinin-horseradish peroxidase conjugate (PNA-HRPO) (MES, pH 6.5 with 20 mM CaCl2 and 1% BSA), mix 94.7 ml 1x MES, pH 6.5 with 2 ml CaCl2 (10 mg/ml), and 3.3 ml 30% BSA.</li>
	</ol>
	</li>
	<li>Neuraminidase (NA) 
	Note: H6Nx virus reassortants (these have HA of the H6 subtype and the NA of interest) are generated. Request vials of inactivated reassortant virus from a collaborator if they are unavailable in-house. When using inactivated virus, confirm that the preparation is suitable for use in the ELLA through demonstration that the inactivation process did not significantly impact NA activity.
	<ol>
		<li>Culture a stock of H6Nx virus in embryonated chicken eggs and store 0.5 ml aliquots of neat allantoic fluid at -80 &#176;C.</li>
		<li>Use a new aliquot of virus for each assay. Do not freeze and thaw the aliquots.</li>
	</ol>
	</li>
	<li>Serum Samples and Controls
	<ol>
		<li>Heat-inactivate all sera (test samples, e.g., pre- and post-vaccination sera, and controls, e.g., sample with known NI titer) in a water bath at 56 &#176;C for 45-60 min. Store the sera at -20 to -70 &#176;C before or after the heat treatment. If the samples are to be tested repeatedly, make several aliquots before freezing so that the sample is not repeatedly frozen and thawed.</li>
		<li>Use the ELLA to measure the NI titers of human sera available in reasonable quantity (&#62;5 ml) to identify at least one with a low titer and another with a high titer. Store 100 &#181;l aliquots at &#8804;-20 &#176;C so that the same sample can be used as a control in many assays to track assay performance.</li>
	</ol>
	</li>
	<li>Peanut Agglutinin (PNA)-Horseradish Peroxidase (HRPO)
	<ol>
		<li>Prepare the PNA-HRPO diluent (MES, pH 6.5 with CaCl2 and 1% BSA).</li>
		<li>Prepare a PNA-HRPO stock solution by dissolving 1 mg of PNA-HRPO in 1 ml diluent. Store 20-200 &#181;l aliquots at -20 &#176;C.</li>
		<li>Before using a new PNA-HRPO lot, test 1:500, 1:750, 1:1,000, and 1:2000 dilutions of this reagent to identify the amount that results in maximal optical density (OD) with the positive control (virus only) and a background (no virus) that is &#60;10% of the positive signal.
		<ol>
			<li>Make quadruplicate virus dilutions as described in section 2.1.</li>
			<li>Transfer the dilutions to a fetuin-coated plate as described in section 2.2 and incubate the plate at 37 &#176;C.</li>
			<li>Wash the plate 16-18 hr later and add 1:500, 1:750, 1:1,000, and 1:2,000 dilutions of PNA-HRPO (100 &#181;l/well) to duplicate rows of the plate.</li>
			<li>Complete the assay as described in steps 2.3.4 to 2.3.9.</li>
			<li>Review the data to select the dilution resulting in a maximum signal of&#62;10 times the background.</li>
		</ol>
		</li>
		<li>Immediately before use, prepare the optimal dilution of PNA-HRPO identified in 1.5.3.</li>
	</ol>
	</li>
	<li>Prepare a large volume of Wash buffer (0.01 M phosphate-buffered saline (PBS), pH 7.4, 0.05% Tween 20 (PBS-T)). Store at RT.</li>
	<li>Prepare the peroxidase substrate (o-Phenylenediamine dihydrochloride (OPD)): Note: alternative peroxidase substrates such as 3,3&#8242;,5,5&#8242;-Tetramethylbenzidine (TMB), may be used
	<ol>
		<li>Prepare phosphate-citrate buffer by dissolving 1 capsule in 100 ml dH2O on the day of assay.</li>
		<li>Dissolve 1 OPD tablet (10 mg) in 20 ml of phosphate-citrate buffer immediately before use.</li>
	</ol>
	</li>
	<li>Prepare the stop solution (1 N H2SO4): add 27.2 ml stock 98% H2SO4 to 973 ml dH2O. Mix and then store at RT.</li>
</ol>
2. Determination of the Amount of NA to Use in ELLA
<ol>
	<li>Prepare Virus Dilutions in a 96-well Plate
	<ol>
		<li>Dispense 120 &#181;l sample diluent (section 1.2) into columns 1-11 of the dilution plate.</li>
		<li>To column 1, add an additional 96 &#181;l sample diluent.</li>
		<li>Thaw and then vortex the virus vial before adding 24 &#181;l to duplicate wells in column 1. This gives a 1:10 dilution of the virus.</li>
		<li>Make serial 2-fold dilutions of virus in sample diluent by transferring 120 &#181;l from one well to the next using clean pipette tips for each dilution.</li>
	</ol>
	</li>
	<li>Transfer Virus Dilutions to a Fetuin-coated Plate
	<ol>
		<li>Wash the fetuin-coated plate 3 times with PBS-T (section 1.6) and then blot each plate onto an absorbent paper towel to remove any excess wash buffer.</li>
		<li>Add 50 &#181;l of sample diluent (section 1.2.1) to each well in columns 1 to 11 of the fetuin-coated plate.</li>
		<li>Add 100 &#181;l of sample diluent to column 12 (these wells are the negative control).</li>
		<li>Transfer 50 &#181;l of diluted virus from columns 1-11 of the dilution plate to the corresponding wells of the fetuin-coated plate.</li>
		<li>Cover the plate with a plate sealer and place it in a humidified incubator at 37 &#176;C.</li>
		<li>Note when the remaining steps will be performed (16-18 hr later).</li>
	</ol>
	</li>
	<li>Complete the NA Determination
	<ol>
		<li>When the incubation is complete (16-18 hr), transfer the plate to the bench and remove the plate sealer.</li>
		<li>Wash the plate 6 times with PBS-T (section 1.7), then invert and pat on absorbent paper towels to ensure all liquid has been removed from the wells.</li>
		<li>Add 100 &#181;l/well PNA-HRPO solution (at the dilution determined in 1.5.3) to all wells and incubate the plate for 2 hr at RT.</li>
		<li>Less than 15 min before the incubation ends, prepare the OPD solution as described in section 1.7.</li>
		<li>Wash the test plates 3 times to remove the PNA-HRPO and blot dry before adding 100 &#181;l of the OPD substrate to each well.</li>
		<li>Incubate the plate for exactly 10 min at RT.</li>
		<li>Add 100 &#181;l/well of 1N sulfuric acid to stop the reaction.</li>
		<li>Use a plate reader to measure the Optical Density (OD) at 490 nm for 0.1 sec.</li>
		<li>Save and export all data files.</li>
	</ol>
	</li>
	<li>Select the Virus Dilution that will be used for Serology
	<ol>
		<li>Draw a graph that plots OD490nm values at each virus dilution. Inspect the titration curve to identify the maximum signal (this is usually the signal that corresponds to a plateau at the beginning of the titration curve), the minimum signal (wells that contain no virus in column 12 of the plate provide the background control), and the dilutions of virus that result in OD that is proportional to the input dilution (i.e., linear region of the curve).</li>
		<li>Select the virus dilution that gives approximately 90% of the maximum signal and is within the linear range. Confirm that the OD at the selected dilution is at least 10-fold greater than the background signal. Use the selected virus dilution for all assays employing this virus stock. 
		Note: Alternatively, measure the NA activity of the virus and use ~15-20 &#181;U NA activity/ml in the ELLA.</li>
	</ol>
	</li>
</ol>
3. Enzyme-linked Lectin Assay
Note: Figure 1 shows the setup of the dilution and assay plates.
<ol>
	<li>Make Sample Dilutions
	<ol>
		<li>Place the heat-inactivated samples on ice. 
		&#8203;Note: 8 sera can be diluted in each plate.</li>
		<li>For a setup using dilutions starting at 1:10 across the plate, add 120 &#181;l sample diluent to all wells in columns 3-11.</li>
		<li>To column 2, add 216 &#181;l of sample diluent and 24 &#181;l of each sample. Mix the sample in the well by pipetting up and down 3 times and then transfer 120 &#181;l to the next column.</li>
		<li>After changing pipette tips, mix the contents of the well by pipetting up and down and then transfer 120 &#181;l to the next column.</li>
		<li>Repeat step 3.1.4 until the sample has been transferred to column 11 and the remaining 120 &#181;l discarded.</li>
	</ol>
	</li>
	<li>Add Samples and Viruses to the Fetuin-coated Plate
	<ol>
		<li>Thaw a vial of virus, vortex, and resuspend the virus in the diluent (section 1.2) at the dilution selected in step 2.4.2. Prepare at least 5 ml of virus for each assay plate. Keep the diluted virus on ice until the plates are washed and serum samples are added.</li>
		<li>Decide on the number of fetuin-coated plates needed for the assay (generally apply 4 sera per plate). Wash the fetuin-coated plate 3 times with PBS-T, invert each plate, and blot onto an absorbent paper towel to remove excess wash buffer.</li>
		<li>Use a multichannel pipette to transfer 50 &#181;l of each serum control or sample dilution from the dilution plate into duplicate wells in columns 2-11.</li>
		<li>Add 50 &#181;l of diluted virus to all wells except for the negative control (column 12).</li>
		<li>Add 50 &#181;l of sample diluent to wells in column 1 and add 100 &#181;l of sample diluent to column 12.</li>
		<li>Cover the wells with a plate sealer and then mix by gently tapping the sides of the plate or placing them on a plate shaker at moderate speed for 10 sec.</li>
		<li>Place the plate in a humidified incubator at 37 &#176;C for 16-18 hr.</li>
	</ol>
	</li>
	<li>Add PNA-HRPO and complete the assay as described in section 2.3</li>
</ol>
4. Data Analysis
<ol>
	<li>Determine the Validity of the Assay Results
	<ol>
		<li>Confirm that the background values (no virus) are less than 10% of the positive control (virus and no serum).</li>
		<li>Confirm that the titers of control sera run in different assays using the same conditions are within 2-fold of the median titer.</li>
		<li>Confirm that OD measurements of control wells are consistent (&#8804;20% different) and that OD measurements of duplicate sample wells are consistent (&#8804;10% different).</li>
		<li>Determine the root cause of invalid results and repeat the assay if the criteria listed in 4.1.1, 4.1.2, or 4.1.3 are unmet. Consider the factors presented in&#160;Table 1&#160;when trying to troubleshoot.</li>
	</ol>
	</li>
	<li>Assign a 50% End-point Titer
	<ol>
		<li>For each assay plate, subtract the average background (no antigen added to wells) from all readings.</li>
		<li>Calculate the percent inhibition at each serum dilution using the formula: 100 x (ODvirus only control&#160;- ODtest sample)/ ODvirus only control.</li>
		<li>Identify the highest dilution that resulted in at least 50% inhibition of the maximum signal.</li>
		<li>Report the reciprocal of this dilution as the 50% end-point titer. 
		Note: If 50% inhibition was not achieved at any dilution, the titer is less than the first dilution tested,&#160;e.g., &#60;10 when 1:10 is the first dilution tested.</li>
	</ol>
	</li>
	<li>Calculate the 50% Inhibitory Concentration (IC50)
	<ol>
		<li>Use four parameter logistic regressions to determine the IC50&#160;titer as follows: subtract the average background from all readings and then transfer the results to a program that performs regression analysis.</li>
		<li>Use non-linear regression, with the maximum set to the virus-only control and the minimum set to zero, to determine the dilution corresponding to exactly 50% inhibition.</li>
		<li>Report the reciprocal of this dilution as the IC50&#160;titer.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coating buffer</td>
			<td>KPL</td>
			<td>50-84-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
			<td></td>
		</tr>
		<tr>
			<td>5X MES, pH 6.5</td>
			<td>KD-Medical</td>
			<td>PBS-0134</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>30% BSA</td>
			<td>Sigma</td>
			<td>A8327</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin PNA-HRPO</td>
			<td>Sigma</td>
			<td>L7759</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-T</td>
			<td>Sigma</td>
			<td>P3563-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>o-Phenylenediamine dihydrochloride</td>
			<td>Sigma</td>
			<td>P8287</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-citrate buffer</td>
			<td>Sigma</td>
			<td>P4922</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Maxisorp plates</td>
			<td>NUNC</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round bottom well plates</td>
			<td>NUNC</td>
			<td>267245</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealers</td>
			<td>Thermo Scientific</td>
			<td>14-245-192B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken eggs</td>
			<td>Charles River Laboratories</td>
			<td>9 day old embryonated specific pathogen free (SPF) chicken eggs</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette</td>
			<td>Variety of suppliers e.g., Rainin, Eppendorf</td>
			<td>8 or 12 channel manual pipettem 50-250 &#181;l volume</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Depends on pipettor brand</td>
			<td>Depends on pipettor brand</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader, with 490 nm filter</td>
			<td>Perkin Elmer</td>
			<td>Victor V</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath set to 37 &#176;C</td>
			<td>Variety of suppliers</td>
			<td>Variety of models</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath set to 56 &#176;C</td>
			<td>Variety of suppliers</td>
			<td>Variety of models</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator set to 4 &#176;C</td>
			<td>Variety of suppliers</td>
			<td>Variety of models</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator set to 37 &#176;C</td>
			<td>Variety of suppliers</td>
			<td>Variety of models</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Gao, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54573">Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay.</a> J. Vis. Exp. (2016).
This video demonstrates an assay measuring influenza neuraminidase (NA) inhibiting antibody titers in a serum sample. A serum sample containing antibodies against NA is added to a fetuin-coated microwell plate. Upon adding the virus, the antibodies prevent viral NA from cleaving fetuin and exposing its galactose moiety. A peroxidase-conjugated lectin is then added, which binds to the exposed galactose. The NA-inhibiting antibody titer is determined using a colorimetric test, where a peroxidase substrate gets cleaved by the immobilized lectin-conjugated peroxidase to produce color.

Table 1. Root cause analysis of assays that do not meet performance criteria. This table provides possible causes and solutions for problems that may occur when performing the ELLA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Possible cause(s)</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Weak signal or no plateau reached in virus titration</td>
			<td>i) low NA enzyme activity 
			ii) virus stock not stored under optimal conditions</td>
			<td>i) Confirm that the diluent has a pH that is optimal for NA activity; if pH is optimal, prepare a new virus stock or concentrate the virus 
			ii) Regrow and aliquot stock; snap-freeze vials on dry ice before storing at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Weak or no color in positive cell control wells</td>
			<td>i)&#160;&#160; Virus dilution incorrectly assigned 
			ii) Vial-to-vial variability in frozen virus aliquots 
			iii) PNA- HRPO denatured or diluted too much 
			iv) OPD incorrectly prepared</td>
			<td>i)&#160; Repeat virus titration 
			ii) Titrate several vials from the same batch to ensure no variability. If there is significant variability, prepare fresh aliquots 
			iii) Use optimum virus dilution to retitrate PNA- HRPO 
			iv) Repeat with a fresh preparation of OPD</td>
		</tr>
		<tr>
			<td>Weak or no inhibition by positive control sera</td>
			<td>i)&#160;&#160; Too much virus used in the assay 
			ii) Serum deteriorated</td>
			<td>i)&#160;&#160; Repeat virus titration 
			ii) Obtain new antisera. Check storage conditions</td>
		</tr>
		<tr>
			<td>Inhibition by negative control sera</td>
			<td>i)&#160; Inadequate heat treatment of serum 
			ii) Too little virus used in the assay</td>
			<td>i)&#160; Repeat heat-inactivation of serum 
			ii) Repeat virus titration</td>
		</tr>
		<tr>
			<td>High background</td>
			<td>i) Possible contamination of plates 
			ii) PNA- HRPO concentration too high/too low</td>
			<td>i) Repeat using freshly coated plates 
			ii) Titrate PNA-HRPO to identify the correct dilution to use</td>
		</tr>
		<tr>
			<td>Virus titration shows apparent inhibition of NA activity at low dilutions</td>
			<td>i) Allantoic fluid may contain a substrate for NA</td>
			<td>ii) Use a virus that has been pelleted through a sucrose cushion</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/22105/22105_Figure 1.jpg" /> 
Figure 1. Diagram to show the ELLA plate setup. Serial dilutions of serum samples are made in a dilution plate and then transferred to a fetuin-coated plate.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Reagents and ...

Take multi-well plate wells coated with a glycoprotein that acts as a substrate for neuraminidase or NA, surface proteins of the influenza virus.
Add serially diluted heat-inactivated human serum containing NA-inhibiting antibodies to test wells and media alone to the control well.
Introduce the virus and incubate.
In the control well, viral NA cleaves the glycoprotein&#39;s terminal sialic acid, exposing the penultimate galactose. In the test wells, antibody binding inhibits NA activity.
Remove the virus-serum mix. Add lectin-peroxidase conjugates, which bind to the exposed galactose.
Remove the unbound conjugates and add a chromogenic peroxidase substrate mixed with hydrogen peroxide.
Immobilized peroxidase utilizes hydrogen peroxide to oxidize the substrate into a colored product.
Add acid to lower the pH, stopping the reaction.
Measure the optical density and calculate the percent NA inhibition.
The reciprocal of the serum dilution needed for fifty percent NA inhibition is the NA-inhibiting antibody titer.

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Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

JoVE Journal

Immunology and Infection

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers

Transkrypcja

An Assay to Measure Influenza Neuraminidase Inhibition Antibody Titers