This study was supported by the Chinese National Science Foundation Grant (No. 32072820, 31702242), grants from Jiangsu Government Scholarship for Overseas Studies (JS20190246) and High-level Talents of Yangzhou University Scientific Research Foundation, a project founded by the Priority Academic Program of Development Jiangsu High Education Institution.

All animal experiments in this study were approved by the Yangzhou University Institutional Animal Care and Use Committee (SYXK20200041).
1. Preparation of porcine APN protein antigen
NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells pEGFP-C1-APN-IPEC-J2 were constructed in a previous study11.
<ol>
	<li>Recover bacteria from a frozen glycerol stock and streak onto Luria-Bertani (LB) plates containing 50 &#181;g/mL kanamycin (Km+) for single colony isolation.</li>
	<li>Select a single colony from the freshly streaked plate, culture in 4 mL of LB medium (10 g/L tryptone, 10 g/L sodium chloride (NaCl) and 5 g/L yeast extract, pH 7.2) supplemented with Km+ (50 &#181;g/mL), and leave to grow overnight (12-16 h) with agitation (178 rpm) at 37 &#176;C.</li>
	<li>Dilute the prepared bacteria at 1:100 in fresh Km+ LB broth and incubate at 37 &#176;C with shaking for 2-3 h until the OD600 reaches 0.4-0.6.</li>
	<li>Add isopropyl &#946;-d-1-thiogalactopyranoside (IPTG) to the medium to a final concentration of 0.4 mM, and incubate the cultures for an additional 10 h at 16 &#176;C.</li>
	<li>Consequently, centrifuge and harvest the bacteria using IPTG induction (10,000 &#215; g, 4 &#176;C 15 min).</li>
	<li>Resuspend the cell pellet using 5 mL of LEW (Lysis/Equilibration/Wash) buffer (50 mM anhydrous sodium phosphate monobasic (NaH2PO4) and 300 mM NaCl, pH 8.0) containing 1 mg/mL lysozyme. Stir the bacterial suspension for 30 min on ice and sonicate completely (15 s pulse and 20 s off, 15 min) using an ultrasonic homogenizer.</li>
	<li>Centrifuge the crude cell lysate at 4 &#176;C and 10,000 &#215; g for 30 min to remove cellular debris. Transfer supernatant into a pre-equilibrated column and incubate 1-2 min before gravity drainage. Repeat this step three times.</li>
	<li>Wash the column using 20 mL of LEW buffer and drain using gravity. Elute the histidine-tagged APN protein using 9 mL of elution buffer (50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole, pH 8.0) and collect into dialysis tubing.</li>
	<li>Dialyze the protein solution overnight at 4 &#176;C in sodium carbonate-sodium bicarbonate (PBS, 135 mM NaCl, 4.7 mM potassium chloride, 2 mM NaH2PO4, and 10 mM dodecahydrate sodium phosphate dibasic, pH 7.2) buffer.</li>
	<li>Analyze using a 12.0% SDS-PAGE gel and western blotting to assess the purity of the APN protein.
	<ol>
		<li>Load 5 &#181;g of protein into each well of the gel and allow to run at 110 V for 1.5 h. Then, transfer protein onto a PVDF membrane for 50 min at 15 V. Determine the concentration of the purified protein using a BCA assay.</li>
	</ol>
	</li>
</ol>
2. Animal immunization
<ol>
	<li>Subcutaneous (s.c) inject female BALB/c mice, 6-8 weeks of age, with 50 &#181;g of APN protein or PBS (negative control) mixed with adjuvants once every 2 weeks. Use complete Freund&#39;s adjuvant that contains the heat-killed Mycobacteria for initial immunization, and incomplete Freund&#39;s adjuvant for booster immunizations. Mix equal volumes of APN protein (or PBS) and Freund&#39;s adjuvant or incomplete Freund&#39;s adjuvant, respectively.</li>
	<li>Detect antibody titers against APN in the sera of these mice by indirect enzyme-linked immunosorbent assay (ELISA) using a microtiter plate coated with 5 &#181;g/mL APN protein diluted in 0.05 M PBS (pH 9.6). .&#160;</li>
</ol>
3. Hybridoma technology to produce monoclonal antibodies against APN
<ol>
	<li>Intraperitoneally (i.p.) inject 100 &#181;g of APN protein into the selected mice for a final antigen boost.</li>
	<li>Three days later, euthanize the mice using pentobarbital sodium (50 mg/kg, v/v, intraperitoneal) and cervical dislocation.</li>
	<li>Collect spleens, and wash with DMEM twice to remove blood and fat cells. Filter the spleen-cell suspension using a 200-mesh copper grid to remove tissue debris, and harvest spleen cells using centrifugation (1500 &#215; g, 10 min) to remove the membrane of the spleen.</li>
	<li>Seed mouse myeloma SP2/0 cells in a 25 cm2 flask containing 5 mL of DMEM supplemented with 6% fetal bovine serum (FBS) and culture at 37 &#176;C, 6% CO2 atmosphere to maintain cell viability. After 5-6 days of culture, the cells reach 80%-90% confluence post-resuscitation and are in growth log phase. Under the microscope, the cells are round, bright, and clear.</li>
	<li>One day before hybridization, collect macrophages from peritoneal cavities of the mice according to a previously published method12,19.</li>
	<li>Seed peritoneal macrophages at a density of 0.1-0.2 &#215; 105/mL in 96-well plates, each well containing 100 &#181;L of HAT medium (DMEM supplemented with 10% FBS and 1x HAT Supplement), and incubate at 37 &#176;C, 6 % CO2 humidified atmosphere overnight.</li>
	<li>For hybridization, gently aspirate SP2/0 cells with a pipette from 8-10 bottles, and suspend in 10 mL of serum-free DMEM medium. Wash cells with fresh DMEM, centrifuge (1500 &#215; g, 10 min) twice, and then re-suspend in 10 mL of DMEM.</li>
	<li>Mix the quantified spleen cells with SP2/0 cells at a ratio of 10:1 and transfer into 50 mL tubes. Centrifuge (1500 &#215; g, 10 min) and discard the supernatant. Collect the cell pellets at the bottom of the tubes and tap with palm to loosen the pellets prior to hybridization.</li>
	<li>Add 1 mL of polyethylene glycol 1500 (PEG 1500), pre-warmed to 37 &#176;C, dropwise using a dropper to the loosened cell pellet over the time period of 45 s while gently rotating the bottom of the tube.</li>
	<li>Slowly add 1 mL of DMEM pre-warmed to 37 &#176;C to the above mixture over the period of 90 s, followed by another 30 mL of fresh DMEM. Place the fusion tube into a 37 &#176;C water bath for 30 min.</li>
	<li>After incubation in the warm bath, harvest the cells and re-suspend in HAT medium. Then culture in a 96-well plate inoculated with peritoneal macrophages.</li>
	<li>Five days later, add 100 &#181;L of fresh HAT medium to each well, and incubate the plate for an additional 5 days, after which replace the medium with HT medium (DMEM supplemented with 10% FBS and 1x HT Supplement).</li>
	<li>Use a microtiter plate coated with 5 &#181;g/mL APN protein diluted in 0.05 M PBS (pH 9.6) to analyze monoclonal antibodies in the hybridoma supernatant using ELISA assay.
	<ol>
		<li>When the medium in the wells of the 96-well plate turns yellow (due to cell growth and metabolite release, pH in the medium decreases to 6.8, and phenol red turns from fuchsia to yellow) or cell clusters are observed, acquire 100 &#181;L supernatant from the selected wells and add to the wells of the coated ELISA plate. Use a microplate reader to measure the OD450 values.</li>
		<li>Use the polyclonal antibodies against APN and non-infected mouse serum as positive and negative control, respectively, and use PBS as blank control. In this study, OD450 ratio of sample to negative control (P/N) &#8805; 2.1 was recognized as positive selection standard.</li>
	</ol>
	</li>
	<li>After three consecutive positive selection rounds, select the hybridoma showing increased serology response against the APN protein for a limited dilution assay.
	<ol>
		<li>Prepare peritoneal macrophages and seed in 96-well plates as described previously.</li>
		<li>Suspend hybridoma cells in HT medium at an average of 0.5-2 cells per well and culture in a 37 &#176;C, 6% CO2 incubator. Repeat this step three or four times until the positive rate indicated by ELISA immunoassay reaches 100%.</li>
	</ol>
	</li>
	<li>Under the pressure of continuous freezing and thawing, select the positive hybridoma cells able to stably secrete anti-APN antibodies and proliferate normally.
	<ol>
		<li>Administer a single i.p. injection of 0.3 mL of pristane to each mouse (8-10 weeks). At 10 days after receiving pristine, inject each mouse with 2-5 x 105 hybridoma cells in 0.5 mL of PBS (pH 7.2).</li>
		<li>Carefully collect peritoneal fluid from the peritoneal cavity of these mice 8 to 10 days after the injection.</li>
		<li>Harvest the supernatants by centrifugation at 5,000 &#215; g for 15 min, and purify antibodies in the supernatants using 33% saturated ammonium sulfate [(NH4)2SO4] precipitation and protein A agarose.</li>
	</ol>
	</li>
</ol>
4. Characterization of mAbs against APN protein
<ol>
	<li>Determine the immunoglobulin subtype of the collected mAbs using an SBA Clonotyping System-HRP20. Use SDS-PAGE and western blotting to assess mAb purity and specificity.</li>
	<li>Analyze mAb epitope specificity against the APN protein using ELISA21.&#160;Additivity value (AV) is the ratio of ODmAbs (a+b) to (ODmAbs-a+ODmAbs-b), which is used to evaluate whether mAbs recognize the same antigenic site; ODmAbs-a and ODmAbs-b represent the OD450 values of different monoclonal antibodies against APN alone, and ODmAbs (a+b) represent the OD450 values of a 1:1 mixture of two mAbs against APN.
	<ol>
		<li>Assess each sample at least four replicates, and repeat the whole experiment at least three times.</li>
	</ol>
	</li>
</ol>
5. Expression of rAbs against APN
<ol>
	<li>Extract total RNA from the above-mentioned hybridoma cells and spleens of APN-immunized mice (e.g., TRIzol)22. Synthesize complementary DNA (cDNA) using a cDNA synthesis kit per manufacturer&#39;s instructions.</li>
	<li>Amplify variable regions of mAbs using nested PCR and determine heavy chain (VH) and light chain (VL) sequences using sequencing. Analyze the genes encoding VH and VL using the IMGT mouse genome analysis tool (http://www.imgt.org/about/immunoinformatics.php).</li>
	<li>Combine the VH and VL genes with leader sequences and sequentially subclone them into the pET28a (+) and pIRES2-ZsGreen1 vectors, respectively, using seamless cloning technology to allow for scarless DNA fragment insertion. The specific primers are listed in Table 1.</li>
	<li>Grow the pET28a (+)-rAbs-APN-BL21-transformed bacteria in the presence of 0.4 mM IPTG in orbital shakers at 37 &#176;C for 10 h. Then induce, purify, and assess for the expression of the rAbs protein using routine protein purification.</li>
	<li>Seed 100 &#181;L 0.5 x 105 CHO cells per well into a 96-well plate and incubate at 37 &#176;C in a 6% CO2 atmosphere for 18-24 h. When the cells reach 80-90% confluence, dilute the pIRES2-ZsGreen1-rAbs-APN plasmid with Opti-MEM to a final concentration of 0.1 &#181;g/&#181;L, and incubate 5 min at room temperature before using for transfection.</li>
	<li>Gently mix 50 &#181;L of diluted pIRES2-ZsGreen1-rAbs-APN plasmid with 1 &#181;L of Lipofectamine&#160;2000 and 49 &#181;L of Opti-MEM, and incubate the mixture for an additional 20 min at room temperature. Add 100 &#181;L of mixture to each well of a 96-well plate containing CHO cells and incubate at 37 &#176;C in 6% CO2 atmosphere for 4-6 h.</li>
	<li>At 4-6 h post-transfection, replace the medium with DMEM-F12 medium supplemented with 10% FBS, and incubate the plate for another 48 h. Then, add 400 &#181;g/mL G418 to each well to select the stably transfected cells.</li>
	<li>After 10 days of selection using DMEM-F12 medium supplemented with 10% FBS and 400 &#181;g/mL G418, sort the cells (3.0 &#215; 107 cells/mL) by fluorescence-activated cell sorting. Approximately 10-15% of the cell population were positive.</li>
	<li>Serially dilute harvested positive cells, seed at an average of 0.5-2 cells per well in a 96-well plate, and culture in a 37 &#176;C, 6% CO2 incubator. Maintain the stably transfected pIRES2-ZsGreen1-rAbs-APN-CHO cells using selection with G418 (200 &#181;g/mL).</li>
	<li>FBS concentration in the above-described cell-culture medium decreases gradually from 10% to 0% during the logarithmic growth phase over the time period of 3 weeks. Then, adapt the adherent CHO cells to suspension growth in a serum-free medium.</li>
	<li>Culture the seeded pIRES2-ZsGreen1-rAbs-APN-CHO cells in the logarithmic growth phase in serum-free medium at a density of 0.8-1.0 &#215; 105 cells/mL in shake flasks at 80-110 rpm shaking speed and 37&#176;C, 6% CO2.</li>
	<li>Collect the cell suspension every 12 h to determine changes in cell viability and vitality using a cell counting kit (e.g., CCK-8) per manufacturer&#39;s instructions.</li>
	<li>Antibody expression reaches peak levels when cell viability decreased to 80% and cell density reaches 1.0-2.0 &#215; 106 cells/mL. Harvest cell supernatants using centrifugation, filter using a 0.22 &#181;m polytetrafluoroethylene membrane filter, and purify using protein A agarose.</li>
	<li>Confirm production of APN-specific antibodies using indirect immunofluorescence assays (IFA).</li>
	<li>Determine antibody titers and binding affinities using ELISA assay as described previously.23 Calculate the equilibrium dissociation constant (KD value) of the antibodies with a four-parameter logistic equation using software.</li>
</ol>

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The authors declare no conflict of interest. All the authors approved and gave their explicit consent for publication of the manuscript.

Induction of mucosal immunity is one of the most effective approaches in counteracting pathogens and in prevention and treatment of various diseases. APN, a highly expressed membrane-bound protein in the intestinal mucosa, is involved in the induction of adaptive immune response and in receptor-mediated viral and bacterial endocytosis1,5,8. APN is used as antigen particulate in many formats of antigen loading and vaccine deliver...

Aminopeptidase N (APN), a moonlighting enzyme that belongs to the metalloproteinase M1 family, acts as a tumor marker, receptor, and signaling molecule via enzyme-dependent and enzyme-independent pathways1,2. In addition to cleaving the N-terminal amino-acid residues of various bioactive peptides for the regulation of their biological activity, APN plays an important role in the pathogenesis of various inflammatory diseases. APN participates in antigen processing and presentation by trimmed peptides that bind tightly to major histocompatibility complex class II molecules2,3. APN also exerts anti-inflammatory effects by binding with G protein-coupled receptors participating in multiple signal transduction, modulating cytokine secretion, and contributing to Fc gamma receptor-mediated phagocytosis in the immune response4,5,6,7.
As a widely distributed membrane-bound exopeptidase, APN is abundant in the porcine small intestinal mucosa and is closely associated with receptor-mediated endocytosis1,5,8. APN recognizes and binds the spike protein of the transmissible gastroenteritis virus for cell entry, and directly interacts with the FaeG subunit of enterotoxigenic Escherichia coli F4 fimbriae to affect bacterial adherence with host cells9,10,11. Thus, APN is a potential therapeutic target in the treatment of viral and bacterial infectious diseases.
Since the development of hybridoma technology and other strategies for monoclonal antibodies (mAbs) production in 1975, mAbs have been widely used in immunotherapy, drug delivery, and diagnosis12,13,14. Currently, mAbs are successfully used to treat diseases, such as cancer, inflammatory bowel disease, and multiple sclerosis12,15. Because of their strong affinity and specificity, mAbs can be ideal targets in the development of antibody-drug conjugates (ADC) or new vaccines16,17. The APN protein is critical for selectively delivering antigens to specific cells, and can elicit a specific and strong mucosal immune response against pathogens without any interference including low protein expression, enzyme inactivity, or structural changes5,8,18. Therefore, therapeutic products based on APN-specific mAbs show promise against bacterial and viral infections. In this study, we describe the production of APN-specific mAbs using hybridoma technology, and expression of anti-APN recombinant antibodies (rAbs) using prokaryotic and eukaryotic vectors. The result indicates that the APN protein was recognized by both rAbs expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and hybridoma-derived mAbs.

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		<tr>
			<td>Complete Freund&#8217;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td>Animal immunization</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C1002</td>
			<td>Nuclear counterstain</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>Cell culture</td>
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		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>Cell culture</td>
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		<tr>
			<td>Dylight 549-conjugated goat anti-mouse IgG secondary antibody</td>
			<td>Abbkine</td>
			<td>A23310</td>
			<td>Indirect immunofluorescence analysis</td>
		</tr>
		<tr>
			<td>Enhanced Cell Counting Kit-8</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C0042</td>
			<td>Measurement&#160;of&#160;cell&#160;viability and vitality</td>
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		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10091</td>
			<td>Cell culture</td>
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		<tr>
			<td>Geneticin&#8482; Selective Antibiotic</td>
			<td>Gibco</td>
			<td>11811098</td>
			<td>Selective antibiotic</td>
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			<td>HAT Supplement (50X)</td>
			<td>Gibco</td>
			<td>21060017</td>
			<td>Cell selection</td>
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			<td>HT Supplement (100X)</td>
			<td>Gibco</td>
			<td>11067030</td>
			<td>Cell selection</td>
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			<td>Incomplete Freund&#8217;s adjuvant</td>
			<td>Sigma-Aldrich</td>
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			<td>isopropyl &#946;-d-1-thiogalactopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>ST102</td>
			<td>Bactericidal antibiotic</td>
		</tr>
		<tr>
			<td>Leica TCS SP8 STED confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>&#160;SP8 STED</td>
			<td>Fluorescence imaging</td>
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		<tr>
			<td>Lipofectamine&#174; 2000 Reagent</td>
			<td>Thermofisher</td>
			<td>11668019</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>LSRFortessa&#8482; fluorescence-activated cell sorting</td>
			<td>BD</td>
			<td>FACS LSRFortessa</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>BOX 998</td>
			<td>ELISA analysis</td>
		</tr>
		<tr>
			<td>Micro spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>Nano Drop one</td>
			<td>Nucleic acid concentration detection</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019308</td>
			<td>Culture broth</td>
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		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10002917</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>31985088</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 1500</td>
			<td>Roche Diagnostics</td>
			<td>10783641001</td>
			<td>Cell fusion</td>
		</tr>
		<tr>
			<td>PrimeScript&#8482; 1st strand cDNA Synthesis Kit</td>
			<td>Takara Bio</td>
			<td>RR047</td>
			<td>qPCR</td>
		</tr>
		<tr>
			<td>protein A agarose</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>P2006</td>
			<td>Antibody protein purification</td>
		</tr>
		<tr>
			<td>Protino&#174; Ni+-TED 2000 Packed Columns</td>
			<td>MACHEREY-NAGEL</td>
			<td>745120.5</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>SBA Clonotyping System-HRP</td>
			<td>Southern Biotech</td>
			<td>May-00</td>
			<td>Isotyping of mouse monoclonal antibodies</td>
		</tr>
		<tr>
			<td>Seamless Cloning Kit</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>D7010S</td>
			<td>Construction of plasmids</td>
		</tr>
		<tr>
			<td>Shake flasks</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>E3285</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Sodium carbonate-sodium bicarbonate buffer</td>
			<td>Beyotime&#160; Biotechnology</td>
			<td>C0221A</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Transfer Cell</td>
			<td>Bio-rad</td>
			<td>&#160;170-3940</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>Ultrasonic Homogenizer</td>
			<td>Ningbo Xinzhi Biotechnology</td>
			<td>JY92-IIN</td>
			<td>Sample homogenization</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid</td>
			<td>LP0021</td>
			<td>Culture broth</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
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production of monoclonal antibodies targeting aminopeptidase n in the porcine intestinal mucosal epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

In this study, the purified soluble APN protein (2.12 mg/mL) was used for mouse immunization. Mice immunized with the APN protein four times at 14-day intervals exhibited a higher antibody titer against APN in their sera. Although 14 hybridomas were obtained using the fusion experiments, only 9 hybridomas survived the three continuous freeze-thaw cycles, resulting in 9 stable clones that secreted antibodies against APN. All these cells are round, bright, and clear (Figure 1). The purified mA...

Watch this Scientific Journal Video about Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium at JoVE.com

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

production-monoclonal-antibodies-targeting-aminopeptidase-n-porcine

Research

JoVE Journal

Immunology and Infection

1.8K Views.  Yangzhou University. The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Video: Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...