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.

<table><tbody><tr><td>Complete Freund&amp;rsquo;s adjuvant</td><td>Sigma-Aldrich</td><td>F5881</td><td>Animal immunization</td></tr><tr><td>DAPI</td><td>Beyotime&amp;nbsp; Biotechnology</td><td>C1002</td><td>Nuclear counterstain</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td>Cell culture</td></tr><tr><td>DMEM-F12</td><td>Gibco</td><td>12634010</td><td>Cell culture</td></tr><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&amp;nbsp; Biotechnology</td><td>C0042</td><td>Measurement&amp;nbsp;of&amp;nbsp;cell&amp;nbsp;viability and vitality</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10091</td><td>Cell culture</td></tr><tr><td>Geneticin Selective Antibiotic</td><td>Gibco</td><td>11811098</td><td>Selective antibiotic</td></tr><tr><td>GraphPad Prism 8.0 software</td><td>GraphPad</td><td>8.0</td><td>Scientific data analysis and graphing</td></tr><tr><td>HAT Supplement (50X)</td><td>Gibco</td><td>21060017</td><td>Cell selection</td></tr><tr><td>HT Supplement (100X)</td><td>Gibco</td><td>11067030</td><td>Cell selection</td></tr><tr><td>Incomplete Freund&amp;rsquo;s adjuvant</td><td>Sigma-Aldrich</td><td>F5506</td><td>Animal immunization</td></tr><tr><td>isopropyl &amp;beta;-d-1-thiogalactopyranoside</td><td>Sigma-Aldrich</td><td>I5502</td><td>Protein expression</td></tr><tr><td>kanamycin</td><td>Beyotime&amp;nbsp; Biotechnology</td><td>ST102</td><td>Bactericidal antibiotic</td></tr><tr><td>Leica TCS SP8 STED confocal microscope</td><td>Leica Microsystems</td><td>&amp;nbsp;SP8 STED</td><td>Fluorescence imaging</td></tr><tr><td>Lipofectamine 2000 Reagent</td><td>Thermofisher</td><td>11668019</td><td>Transfection</td></tr><tr><td>LSRFortessa 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></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</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 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&amp;nbsp; Biotechnology</td><td>P2006</td><td>Antibody protein purification</td></tr><tr><td>Protino 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&amp;nbsp; Biotechnology</td><td>D7010S</td><td>Construction of plasmids</td></tr><tr><td>Shake flasks</td><td>Beyotime&amp;nbsp; Biotechnology</td><td>E3285</td><td>Cell culture</td></tr><tr><td>Sodium carbonate-sodium bicarbonate buffer</td><td>Beyotime&amp;nbsp; 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>&amp;nbsp;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></table>

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

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

Biochemistry

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

Chemistry

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 ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

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 ...

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson&#8217;s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The ...

Biology

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Source: Xia, P., et al. <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.