This work was funded by the Austrian Science Fund (FWF) grant P18820-B13.
Ethics statement:
The study was approved (PLUS_Ethik_090514) by the Ethics Committee for Experiments Involving Humans and/or Animals at the University of Salzburg complying with the Helsinki Declaraction as revised in 1983 and all patients participating gave their written informed consent.

1. Preparation of drug conjugates
<ol>
 <li>Dissolve 10 mg DF in 1 ml dH2O in a 1.5 ml reaction tube. In the further proceeding use 95 &mu;l of this DF solution. To conjugate PP to HSA dissolve 30.4 mg PP derivative (304 g/mol) in 500 &mu;l 0.3 M sodium hydroxide (NaOH).</li>
 <li>Add 430.6 &mu;l dH2O and 100 &mu;l 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.5 to 95 &mu;l of dissolved DF. Add 33.1 &mu;l dH2O and 140 &mu;l of 2 M MES buffer pH 2.9 to the PP sample.</li>
 <li>Add 57.5 &mu;l of a freshly prepared N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) stock solution dissolved in dH2O with a concentration of 100 mg/ml to the DF sample, or 10 &mu;l EDC solution to the PP sample. Vortex the samples for 1 minute.</li>
 <li>Add 16.9 &mu;l of HSA solution with a concentration of 118 mg/ml to each sample.</li>
 <li>Incubate the samples for 2 hours at room temperature while shaking at 300 rounds/min.</li>
 <li>Dialyze the DF and PP samples 3 times against 2 liters of phosphate-buffered saline (PBS) pH 7.4 for several hours each. Perform all dialyzing steps at 4&deg;C.</li>
 <li>Centrifuge the sample 10 minutes at 14,000 x g and collect the supernatant containing DF or PP conjugated to HSA.</li>
 <li>Check supernatant for proteins by performing a SDS-PAGE, using 12% gels. Load about 12 &mu;g of HSA conjugate into one gel slot.</li>
</ol>
2. Determine coupling rate of drug conjugates (Figure 2)
<ol>
 <li>For the analysis of the coupling rate of DF and PP conjugates by HPSEC use a 100 mM sodium phosphate buffer pH 6.5 containing 150 mM NaCl and 0.05% sodium azide. Use a 7.8 x 300 mm TSK-Gel-G2000SWXL column.</li>
 <li>Perform detection of signals by using an online-coupled RI and UV detector system.</li>
 <li>Prepare a HSA standard sample with a concentration of 1 mg/ml in dH2O. For detector calibration inject 50 &mu;l HSA standard solution.</li>
 <li>Calculate the RI constant kRI and the UV constant kUV for the detector. Use the formulas areaRI=kRI*[(dn/dc)HSA*c(HSA)] and areaUV=kUV*[(dA/dc)HSA*c(HSA)] with (dn/dc)HSA=0.185 and (dA/dc)HSA=0.5185.</li>
 <li>Prepare DF and PP derivative standards for HPSEC that 5, 10, and 30 nmol amounts of both standards can be easily injected.</li>
 <li>Calculate mean values for (dn/dc)drug and (dA/dc)drug for both standards. For DF, a (dn/dc) of 0.235 &plusmn; 0.010 and a (dA/dc) of 39.000 &plusmn; 0.493 was determined. For PP, a (dn/dc) of 0.328 &plusmn; 0.016 and a (dA/dc) of 53.155 &plusmn; 2.464 was determined.</li>
 <li>Inject drug conjugates on HPSEC and determine their RI and UV peak areas.</li>
 <li>Calculate the concentration of DF, PP derivative, and HSA by solving the two equations areaRI=kRI*[(dn/dc)drug*c(drug)+(dn/dc)HSA*c(HSA)] and areaUV=kUV*[(dA/dc)drug*c(drug)+(dA/dc)HSA*c(HSA)] for the unknowns.</li>
</ol>
3. BAT using drug conjugates
<ol>
 <li>Prepare seven flow cytometry vials and label them depending on their content: unstained control, negative control, positive control, and drug conjugates.</li>
 <li>Pipette 50 &mu;l of B-CCR-STB stimulation buffer (Flow2 CAST, B&uuml;hlmann Laboratories, Sch&ouml;nenbuch, CH) into the vials reserved for the unstained and negative control. As a positive control, use 50 &mu;l of an anti-Fc&#949;RI solution provided by the Flow2 CAST kit. Add the appropriate amounts of conjugate solution to the vials reserved for the drug conjugates by creating a 1:10 dilution series from 0.02 -20 &mu;g/ml DF conjugate and PP conjugate (final concentrations in an assay volume of 200 &mu;l). These titration experiments must be performed to determine the concentration of optimal basophil activation for each patient sample.</li>
 <li>Add 100 &mu;l stimulation buffer to each tube and 50 &mu;l patient's EDTA whole blood. Mix the samples carefully.</li>
 <li>Pipette 20 &mu;l Flow2 CAST staining reagent containing anti-CCR3 antibodies labeled with phycoerythrin (PE) and anti-CD63 antibodies labeled with fluorescein isothiocyanate (FITC) to each tube and mix again. Do not pipette staining reagent into the unstained sample!</li>
 <li>Incubate the samples for 45 minutes in a 37&deg;C water bath.</li>
 <li>Stop reaction on ice for 5 minutes.</li>
 <li>Lyse erythrocytes by adding 2.0 ml Flow2 CAST lysing reagent to each vial and vortex gently. Incubate samples in the dark for 10 minutes at room temperature.</li>
 <li>Centrifuge samples for 5 minutes at 500 x g and carefully decant supernatant.</li>
 <li>Resuspend cells in 500 &mu;l Flow2 CAST wash buffer and store on ice until flow cytometry.</li>
 <li>Adjust the flow cytometer's voltage settings for forward and side scatter (FSC, SSC) while acquiring the unstained sample.</li>
 <li>Gate the cells predicted as lymphocytes from the SSC-FSC plot to the SSC-PE plot. Basophils are identified by a PE-labeled anti-CCR3 antibody as CCR3hi SSClo and gate them into the FITC-PE plot. The anti-CD63 antibody detecting activated basophils is labeled with FITC.</li>
 <li>Set the cut-off to maximum 2.5% of basophils as CD63hi in the negative control. A sample is considered positive for basophil activation if &gt;5% basophils are CD63hi and the mean fluorescence index (MFI) of the sample divided by the MFI of the negative control exceeds 2 (Figure 3).</li>
</ol>
4. Representative Results:
This experiment evaluates BAT as a beneficial tool to detect IgE-dependent drug hypersensitivity. Protein conjugates of NSAIDs are used for activation of basophils. By HPSEC with RI and UV detection aggregation state, coupling degree, and effective yield of the conjugates are determined. As Figure 4 shows, 43.5% of the DF conjugates remained monomeric with a coupling degree of 5.0 DF/HSA. For the aggregates a coupling degree of 9.5 was determined. The propyphenazone conjugate was shown to consist of 68.5% monomers with a coupling degree of 21.2. The coupling degree of the aggregates was 27.9. In total, the determined coupling degree of DF conjugates was 7.6 DF and that of PP conjugates was 23.2.
BAT performed with conjugates under optimized conditions (concentration of conjugates, stimulation time) allowed investigation of IgE-dependent reactions in NSAID hypersensitivity. As shown in Figure 5, only the PP conjugate was able to trigger basophil activation visualized by an upregulation in CD63 surface expression: 20.6% of basophils were activated characterized by a log-shift in fluorescence intensity. The MFI increased by a factor of 4.9 from 386 (negative control) to 1893. In contrast, using the DF conjugate only 3.1% of the basophils were activated which was below the 5% cut-off. Also the MFI increased only by a factor 1.1 (limit 2.0) from 197 (negative control) to 210. Assay validity is given by (i) &lt;5% basophil activation in the negative control, (ii) a log-shift in fluorescence intensity of a basophil subpopulation, and (iii) &gt;50% activated basophils (positive control).
<img alt="Figure 1" src="/files/ftp_upload/3263/3263fig1.jpg" /> 
Figure 1. Coupling of DF and PP derivative to HSA. After dissolving the drugs, water, MES buffer, and EDC (coupling reagent) are added. The samples are vortexed for 1 minute before HSA is pipetted to the drug solutions. Within 2 hours shaking at room temperature the drugs covalently bind to HSA. Monomers as well as aggregates may result.
<img alt="Figure 2" src="/files/ftp_upload/3263/3263fig2.jpg" /> 
Figure 2. Calculation of coupling degrees. First, the constants kRI and kUV are determined. Therefore, HSA standard with known concentration is injected into the HPSEC system. (dn/dc) and (dA/dc) of HSA are given values of 0.185 and 0.518, respectively 5. The peak areas are used for calculating the constants. Second, for each drug three standards are injected to determine drug- and concentration-specific RI and UV areas. Since the kRI and kUV constants are known from the steps above, the drug-specific (dn/dc) and (dA/dc) derivatives can be calculated. Third, drug-HSA conjugates are injected into HPSEC. The resulting peak areas are used to calculate the concentrations of drug and HSA per peak by solving the two equations with two unknown variables.
<img alt="Figure 3" src="/files/ftp_upload/3263/3263fig3.jpg" /> 
Figure 3. Basophil gating. Cells predicted as lymphocytes are gated side scatter versus forward scatter (SSC-FSC plot). The basophils are identified as CCR3hi SSClo from the gated lymphocytes (SSC-CCR3-PE plot). Basophils are analyzed for activation (CD63hi) in the CD63-FITC-CCR3-PE plot. The negative control is used to set the cut-off of maximum 2.5% basophils remaining CD63hi.
<img alt="Figure 4" src="/files/ftp_upload/3263/3263fig4.jpg" /> 
Figure 4. Determined coupling degrees of drug conjugates. RI and UV signals show two peaks representing aggregates (eluting at low retention volume) and monomers (eluting at high retention volume) of drug-HSA conjugates. Percentages of monomers and aggregates (determined from RI signals) and their coupling degrees are depicted (n=1).
<img alt="Figure 5" src="/files/ftp_upload/3263/3263fig5.jpg" /> 
Figure 5. Basophil activation test. Results of drug conjugate-activated samples, negative controls, and positive controls are shown in CD63-FITC-CCR3-PE plots (n=1).

<ol>
	<li>Himly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE-mediated+immediate-type+hypersensitivity+to+the+pyrazolone+drug+propyphenazone.">IgE-mediated immediate-type hypersensitivity to the pyrazolone drug propyphenazone.</a> J Allergy Clin Immunol. 111, 882-888 (2003).</li><li>Demoly, P., Pichler, W., Pirmohamed, M., Romano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+questions+in+Allergy:+1--drug+allergy/hypersensitivity.">Important questions in Allergy: 1--drug allergy/hypersensitivity.</a> Allergy. 63, 616-619 (2008).</li><li>Ebo, D. G., Hagendorens, M. M., Bridts, C. H., Schuerwegh, A. J., Clerck, L. S., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+in+vitro+activated+basophils,+specific+IgE+and+skin+tests+in+the+diagnosis+of+pollen-associated+food+allergy.">Flow cytometric analysis of in vitro activated basophils, specific IgE and skin tests in the diagnosis of pollen-associated food allergy.</a> Cytometry Part B (Clinical Cytometry). 64B, (2005).</li><li>Harrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diclofenac+hypersensitivity:+antibody+responses+to+the+parent+drug+and+relevant+metabolites.">Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.</a> PLoS One. 5, e13707-e13707 (2010).</li><li>Wen, J., Arakawa, T., Philo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-exclusion+chromatography+with+on-line+light-scattering,+absorbance,+and+refractive+index+detectors+for+studying+proteins+and+their+interactions.">Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions.</a> Anal Biochem. 240, 155-166 (1996).</li><li>De Weck, A. L., Sanz, M. L., Gamboa, P. M., Aberer, W., Bienvenu, J., Blanca, M., Demoly, P., Ebo, D. G., Mayorga, L., Monneret, G., Sainte Laudy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+based+on+human+basophils:+more+potentials+and+perspectives+than+pitfalls.+II.+Technical+issues.">Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.</a> J Investig Allergol Clin Immunol. 18, 143-155 (2008).</li><li>Ebo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-assisted+allergy+diagnosis:+current+applications+and+future+perspectives.">Flow-assisted allergy diagnosis: current applications and future perspectives.</a> Allergy. 61, 1028-1039 (2006).</li><li>Poulsen, L. K., Quan, S., Kragh, C., Platzer, M. H., Skov, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basophil+granulocyte+in+allergic+reactions:+experimental+models+and+their+use+for+the+identification+of+drugs+with+effects+or+side+effects+on+basophils.">The basophil granulocyte in allergic reactions: experimental models and their use for the identification of drugs with effects or side effects on basophils.</a> Curr Med Chem - Anti-inflammatory &#38; Anti-Allergy Agents. 3, 167-180 (2004).</li><li>Sanz, M. L., Gamboa, P., de Weck, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+combined+test+with+flowcytometric+basophil+activation+and+determination+of+sulfidoleukotrienes+is+useful+for+in+vitro+diagnosis+of+hypersensitivity+to+aspirin+and+other+nonsteroidal+anti-inflammatory+drugs.">A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs.</a> Int Arch Allergy Immunol. 136, 58-72 (2005).</li></ol>

No conflicts of interest declared.

BAT is a well-established although not yet routinely used method for diagnosis of IgE-mediated allergic disease 6,7. For drug hypersensitivity, however, its applicability is compromised as low molecular weight compounds are not able to crosslink IgE receptors, a prerequisite for basophil activation 8. Therefore, drugs under investigation need to be covalently coupled to suitable carrier proteins (e.g. HSA). Importantly, the coupling degree (i.e. number of drug molecules per carrier protein) needs to be controlled to guarantee immunological activity (i.e. IgE receptor cross-linking) of conjugates. Theoretically, two haptens per carrier molecule should be sufficient for IgE receptor cross-linking and as few as five DF molecules per HSA have been shown to trigger mediator release in a cell-based assay4. Both conjugates, PP-HSA and DF-HSA, have been determined to display a sufficiently high coupling degree (HPSEC) and to be immunologically active making them suitable reagents for use in BAT. Another issue may be that drug metabolites may play a role, as a metabolite instead of the parent drug may cause the hypersensitivity reaction. In case of DF, this possibility has been evaluated in detail previously using five major Phase I metabolites and one linkage variant4. Important factors for mastering the described technique include quality of blood sample (&lt;12 hours since blood collection, number of detected basophils &gt;500) and optimization of stimulation conditions (time, dose). In addition, so-called non-responders who do not even react with the positive control (antibody directed against the IgE receptor) have to be identified. Therefore, validation criteria have to include an anti-Fc&#949;RI antibody as positive control. An important advantage of using drug conjugates is the fact that they appear non-toxic in contrast to pure drugs, as shown for DF-HSA conjugate by co-stimulation with anti-Fc&#949;RI antibody in BAT4. Pure DF, in contrast, displays cytotoxic effects at a concentration of 1.25 mg/ml causing problems with the interpretability of BAT9. Generally, testing for potential cytotoxicity is strongly recommended for every newly produced drug conjugate.
Here, we have shown the potential of PP-HSA conjugate used in BAT to reveal IgE-mediated hypersensitivity. In contrast, DF hypersensitivity is not associated with IgE demonstrated by the lack of basophil activation in response to DF-HSA conjugate. Importantly, in case of uncertainty concerning an IgE-mediated mechanism, conjugates have to be evaluated for immunological activity by in vitro cell-based assay systems as has been shown for DF4.
Using the described setup of conjugating low molecular weight drugs covalently to suitable protein carrier molecules a number of hypersensitivity reactions against drugs including antibiotics, other NSAIDs, radiocontrast media, muscle relaxants, anesthetics, etc. may be investigated for an involvement of an IgE-mediated mechanism. Hence, BAT may serve as an addition to the existing methods for diagnosis (skin test, oral provocation testing).

<table><tbody><tr><td>2-(N-Morpholino)ethanesulfonic acid</td><td>Sigma-Aldrich</td><td>M3671</td><td>-</td></tr><tr><td>7.8 x 300 mm TSK Gel&lt;sup&gt; &lt;/sup&gt;-G2000SWXL column</td><td>Tosoh Corp.</td><td>08540</td><td>-</td></tr><tr><td>BD FACSCanto II</td><td>BD Biosciences</td><td>338962</td><td>-</td></tr><tr><td>Bench top centrifuge</td><td>Sigma-Aldrich</td><td>-</td><td>-</td></tr><tr><td>Diclofenac sodium salt</td><td>Sigma-Aldrich</td><td>D6899</td><td>-</td></tr><tr><td>EDTA-Vacutainer</td><td>BD Biosciences</td><td>368589</td><td>-</td></tr><tr><td>Flow2 CAST Kit</td><td>B&amp;#252;hlmann Laboratories</td><td>FK-CCR</td><td>Effective June 14, 2011 Flow2 CAST kit is now Flow CAST</td></tr><tr><td>HP1100 HPLC system</td><td>Hewlett-Packard</td><td>-</td><td>-</td></tr><tr><td>Human Serum Albumin</td><td>Sigma-Aldrich</td><td>A9511</td><td>-</td></tr><tr><td>Laboratory centrifuge</td><td>Eppendorf</td><td>-</td><td>-</td></tr><tr><td>N-Ethyl-N&amp;rsquo;-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)</td><td>Sigma-Aldrich</td><td>E6383</td><td>-</td></tr><tr><td>PBS tablets</td><td>AppliChem</td><td>A9199</td><td>-</td></tr><tr><td>Propyphenazone derivative</td><td>University of Salzburg</td><td>-</td><td>-</td></tr><tr><td>Shaker</td><td>Eppendorf</td><td>-</td><td>-</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td>-</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S1679</td><td>-</td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma-Aldrich</td><td>90421C</td><td>-</td></tr><tr><td>Sodium hydroxid</td><td>Sigma-Aldrich</td><td>S5881</td><td>-</td></tr><tr><td>Sodium phosphate</td><td>Sigma-Aldrich</td><td>342483</td><td>-</td></tr><tr><td>Triple detector array</td><td>Viscotek</td><td>TDA302</td><td>-</td></tr><tr><td>Personal BioVortex V-1 plus</td><td>Peqlab</td><td>90-V-1</td><td>-</td></tr><tr><td>Water bath 1012</td><td>GFL</td><td>1012</td><td>-</td></tr></tbody></table>

basophil activation test for investigation of ige-mediated mechanisms in drug hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Watch this Scientific Journal Video about Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity at JoVE.com

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

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33.4K Views.  University of Salzburg. Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Video: Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (Fc&#949;RI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of &#946;-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Medicine

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Analyzing Basophil Activation in the Presence of a Test Allergen using Flow Cytometry

Source: Do&#241;a, I. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62600">Basophil Activation Test for Allergy Diagnosis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the test to evaluate basophil activation in the presence of a test allergen using flow cytometry.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sample preparation

	Collect ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

Humanized Mediator Enzyme Release Assay: An Immunochemical Method to Screen Allergic Potential of Test Antigen by Monitoring Basophil Degranulation Response In Vitro

Centrifuge the pre-incubated Ag8/serum suspension, and transfer 50 microliters of the centrifuged Ag8/serum suspension to each well containing humanized RBL cells without disturbing the Ag8 cell pellet.
Use sensitized, unstimulated, cells without antigen, as no-antigen control for indication of the bottom signal plateau or background, and do not sensitize the background and maximum lysis control wells. Cover the plate with the lid, and incubate overnight at 37 degrees Celsius and 5% to 7% carbon dioxide.
Aspirate the sera-containing cell medium. Invert and tap the plate on the absorbent paper to empty the plate for washing humanized RBL cells. Wash the cells three times by adding 200 microliters of Tyrode&#39;s buffer per well, and incubate for approximately 30 seconds per wash, for the first two washes. After adding Tyrode&#39;s buffer for the third time, aspirate the buffer and leave the solution in the wells, until ready to add the antigen dilution.
Transfer 100 microliters of antigen solution to each well containing the pre-sensitized, humanized RBL cells, but do not stimulate the maximum lysis and non-sensitized background cells with antigen.
Add 100 microliters of Tyrode&#39;s buffer in the maximum lysis control wells, non-sensitized background control wells, and sensitized no-antigen wells. Incubate the cells for one hour at 37 degrees Celsius and 5% to 7% carbon dioxide.
Treat the maximum lysis control wells with 10 microliters of 10% Triton X-100 per well, and mix properly to lyse the cells completely for 100% release of beta-hexosaminidase. Add 50 microliters of substrate solution into a new non-binding 96-well plate.
Transfer 50 microliters of supernatant from the wells of humanized RBL cells-containing plate into the new plate containing the substrate solution, and incubate the plate for one hour at 37 degrees Celsius to allow conversion of the fluorogenic substrate.
Add 100 microliters of stopping solution per well, and measure the fluorescence as described in the text manuscript.