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/m...

<ol>
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A., Ortega, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopeptidase+N+(CD13)+is+involved+in+phagocytic+processes+in+human+dendritic+cells+and+macrophages.">Aminopeptidase N (CD13) is involved in phagocytic processes in human dendritic cells and macrophages.</a> BioMed Research International. 2013, 562984 (2013).</li><li>Melkebeek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+aminopeptidase+N,+a+newly+identified+receptor+for+F4ac+fimbriae,+enhances+the+intestinal+mucosal+immune+response.">Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.</a> Mucosal Immunology. 5 (6), 635-645 (2012).</li><li>Morgan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+aminopeptidase+N/CD13+produced+by+fibroblast-like+synoviocytes+in+rheumatoid+arthritis:+role+of+CD13+in+chemotaxis+of+cytokine-activated+T+cells+independent+of+enzymatic+activity.">Expression and function of aminopeptidase N/CD13 produced by fibroblast-like synoviocytes in rheumatoid arthritis: role of CD13 in chemotaxis of cytokine-activated T cells independent of enzymatic activity.</a> Arthritis &amp; Rheumatology. 67 (1), 74-85 (2015).</li><li>Du, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+and+arthritogenic+properties+of+the+soluble+form+of+CD13.">Angiogenic and arthritogenic properties of the soluble form of CD13.</a> The Journal of Immunology. 203 (2), 360-369 (2019).</li><li>Rasschaert, K., Devriendt, B., Favoreel, H., Goddeeris, B. M., Cox, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+and+transcytosis+of+enterotoxigenic+Escherichia+coli+F4+fimbriae+in+porcine+intestinal+epithelial+cells.">Clathrin-mediated endocytosis and transcytosis of enterotoxigenic Escherichia coli F4 fimbriae in porcine intestinal epithelial cells.</a> Veterinary Immunology and Immunopathology. 137 (3-4), 243-250 (2010).</li><li>Reguera, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bases+of+coronavirus+attachment+to+host+aminopeptidase+N+and+its+inhibition+by+neutralizing+antibodies.">Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.</a> PLoS Pathogens. 8 (8), 100859 (2012).</li><li>Delmas, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopeptidase+N+is+a+major+receptor+for+the+entero-pathogenic+coronavirus+TGEV.">Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV.</a> Nature. 357 (6377), (1992).</li><li>Xia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+aminopeptidase+N+binds+to+F4++enterotoxigenic+Escherichia+coli+fimbriae.">Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae.</a> Veterinary Research. 47 (1), 24 (2016).</li><li>Nakamura, R. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+better+monoclonal+antibody-based+therapeutics.">Building better monoclonal antibody-based therapeutics.</a> Nature Reviews Cancer. 15 (6), 361-370 (2015).</li><li>Bakshi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+single-domain+antibodies+as+carriers+for+targeted+vaccine+delivery+to+the+small+intestinal+epithelium.">Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium.</a> Journal of Controlled Release. 321, 416-429 (2020).</li><li>Kohler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> The Journal of Immunology. 174 (5), 2453-2455 (2005).</li><li>Chen, W., Liu, W. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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>
		</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&#160; Biotechnology</td>
			<td>C0042</td>
			<td>Measurement&#160;of&#160;cell&#160;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&#8482; Selective Antibiotic</td>
			<td>Gibco</td>
			<td>11811098</td>
			<td>Selective antibiotic</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&#8217;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td>Animal immunization</td>
		</tr>
		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<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>
</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 ...

derived monoclonal antibodies are primarily used in diagnostic therapeutic immune reagents highlighting the need to produce stable and reliable antibodies in limited production prepared. The methods described here proved that recombinant antibody production could increase monoclonal antibody production efficiency and minimize labor and time associated costs. The methods are used to develop aminopeptidase and antibody-based antibody drug conjugates and other therapeutic products, which aids in clarifying the role of APN in the prevention and treatment of bacterial and viral infections.Demonstrating the procedure will be Yan Li, a postgrad student from my laboratory. To begin, intraperitoneally inject 100 micrograms of APN protein into the selected adult female mice for a final antigen boost. After three days of injection, collect the spleens from mice 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 to remove the spleen membrane. Seed the mouse SP20 myeloma cells in a 25 square centimeter flask containing five milliliters of DMEM supplemented with 6%fetal bovine serum and culture the cells at 37 degrees Celsius and 6%carbon dioxide atmosphere to maintain cell viability. After five to six days of culture, the cells should reach 80 to 90%confluence post resuscitation and appear round, bright, and clear under the microscope.One day before hybridization, collect macrophages from the peritoneal cavities of the mice. Seed peritoneal macrophages at a density of 0.1 to 0.2 times 10 to the fifth per milliliter in 96-well plates, each containing 100 microliters of HAT medium and incubate them overnight. For hybridization, gently aspirate SP20 cells with a pipette from 8 to 10 bottles and suspend them in 10 milliliters of serum-free DMEM medium.Wash the cells with fresh DMEM and centrifuge them twice, then resuspend them in 10 milliliters of DMEM. Mix the quantified spleen cells with SB20 cells at a ratio of 10:1 and transfer them into 50 milliliter tubes. After centrifugation, discard the supernatant and collect the pellets at the bottom of the tubes.Tap the tube to loosen the pellets before hybridization. Using a dropper, add one milliliter of polyethylene glycol 1500 pre-warmed to 37 degrees Celsius dropwise to the loosened cell pellet over 45 seconds while gently rotating the bottom of the tube. Then slowly add one milliliter of DMEM pre-warmed to 37 degrees Celsius to the mixture for 90 seconds, followed by another 30 milliliters of fresh DMEM and place the fusion tube into a 37 degree Celsius water bath for 30 minutes.After incubation, harvest the cells. Resuspend the HAT medium and culture in a 96-well plate inoculated with peritoneal macrophages. After five days, add 100 microliters of fresh HAT medium to each well.And again after five days of incubation, replace the medium with HAT medium. Use a microtiter plate coated with five micrograms per milliliter APN protein diluted in 0.05 molar PBS to analyze monoclonal antibodies in the hybridomas supernatant using ELISA assay. Grow the pET28a plus recombinant antibodies aminopeptidase NBL21 transformed bacteria in the presence of 0.4 millimolar beta-d-1-thiogalactopyranoside in an orbital shaker at 37 degrees Celsius for 10 hours.Seed 100 microliters 0.5 times 10 to the fifth Chinese hamster ovary cells per well into a 96-well plate for incubation at 37 degrees Celsius and a 6%carbon dioxide atmosphere for 18 to 24 hours. When the cells reach 80 to 90%confluence, dilute the pIRES2-zs Green one recombinant antibodies aminopeptidase and plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-zs Green one recombinant antibodies aminopeptidases and plasmid with one microliter of Lipofectamine 2000 and 49 microliters of Opti-MEM.After 20 minutes of incubation, add 100 microliters of the mixture to each well of a 96-well plate containing CHO cells. At four to six hours post transfection, replace the medium with DMEM F12 supplemented with 10%FBS. After 48 hours of incubation, add 400 micrograms per microliter G418 to each well to select the stably transfected cells.After 10 days of selection using DMEM F12 medium supplemented with 10%FBS and 400 micrograms per microliter G418, sort the cells with fluorescence-activated cell sorting. Serially dilute the harvested positive cells. Seed them at an average of 0.5 to 2 cells per well in a 96-well plate and culture in 37 degrees Celsius, 6%carbon dioxide incubator.In representative microscopic analysis, all hybridomas cells appeared round, bright, and clear. The purified monoclonal antibodies possessing heavy 50 kilodalton and light 25 kilodalton chains were confirmed by SDS-PAGE and were found in the purified ascites. The titers of these anti-APN monoclonal antibodies in culture supernatants and ascites are shown here.Mouse monoclonal antibody isotyping revealed that antibodies derived from clones 5B31, 5B36, 3C48, 5C51, and 6C56 possessed immunoglobulin G2B subclasses while APN 2A20 was an immunoglobulin G2A kappa type antibody and monoclonal antibody APN 3FD9, 3F10, and 10F3 belonged to immunoglobulin M type and processed kappa light chains. Most of these monoclonal antibodies showed AV values of over 50%indicating that they targeted different epitopes in the APN while the APN 5C51 antibody recognized antigenic epitopes like those recognized by APN 3C48, 5B31, and 6C56 monoclonal antibodies. The amplified APN 5B36 VHVL gene was ligated into a pET28a positive or pIRES2-zs Green one vector to construct the recombinant expression plasmids pET28a positive RABS APN and pIRES2-zs Green one RABS APN respectively.The antibodies expressed by both pET28a plus recombinant antibodies aminopeptidase NBL21 and pIRES2-zs Green one recombinant antibodies aminopeptidase NCHO cells were purified and analyzed using ELISA and IFA assays. However, only the antibody expressed in the supernatant of pIRES2-zs Green one recombinant antibodies aminopeptidase NCHO cells recognize the APN protein. Binding of APN 5B36 monoclonal antibodies to APN proteins reached an equilibrium earlier than recombinant antibodies.Purification of the monoclonal antibodies in the supernatant using protein A agarose is better than using saturated ammonia sulfate precipitation.

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Research

JoVE Journal

Immunology and Infection

1.8K visualizações.  Yangzhou University. Os anticorpos monoclonais derivados são usados principalmente em reagentes imunes terapêuticos diagnósticos, destacando a necessidade de produzir anticorpos estáveis e confiáveis em produção limitada preparados. Os métodos aqui descritos provaram que a produção de anticorpos recombinantes pode aumentar a eficiência da produção de anticorpos monoclonais e minimizar os custos associados à mão de obra e ao tempo. Os métodos são usados para desenvolver conjugados de drogas à bas...