Name: generation of monoclonal antibodies against natural products
Uploaded: 2019-04-06T12:30:00.000+00:00
Duration: 12 min 15 s
Description: Watch this Scientific Journal Video about Generation of Monoclonal Antibodies Against Natural Products at JoVE.com

This work was supported by the National Natural Science Foundation of China (grant numbers 81573573, 81473338, and 81503344) and the Classical Prescription Basic Research Team at the Beijing University of Chinese Medicine.

All of the animal procedures performed in this study have been approved by the Ethical Review Committee at the Beijing University of Chinese Medicine (approval number 2016BZYYL00109).
NOTE: Female BALB/c mice (8 weeks old) were immunized with hapten-carrier protein conjugates. When used alone, a small molecule (&#60;1,000 Da) cannot elicit an immune response. However, conjugating the small molecule to a carrier macromolecule results in antigen synthesis. In this context, the small molecule is labeled a hapten. Hapten conjugation is a necessary and effective strategy for mAb production. To avoid cross reactivity, two different protein carriers, such as bovine serum albumin (BSA) and ovalbumin (OVA) or keyhole limpet hemocyanin (KLH) and BSA, should be used as the immunogens (for animal immunization) and coating antigens (to coat the plate for anti-serum detection). BSA and OVA are used as an example in this protocol.
1. Preparation of the Immunogen and Coating Antigen
NOTE: For artificial antigen synthesis, use the appropriate functional group (e.g., hydroxyl, sulfhydryl carboxyl acid, or amino) as the side arm for covalent binding with the carrier protein. The conjugation methods include periodate oxidation, the carbodiimide method, a mixed anhydrides reaction, a glutaraldehyde reaction, and the succinate method. This protocol uses naringin (NAR), a well-known flavanone glycoside, as an example compound. NAR is a small compound (581 Da) present in citrus fruits as well as various traditional Chinese medicines.
<ol>
	<li>Use a periodate oxidation procedure to synthesize NAR-BSA and NAR-OVA conjugates.
	<ol>
		<li>Dissolve 50 mg of NAR in water at a final concentration of 1 mg/mL22.</li>
		<li>Add 100 &#181;L of freshly prepared sodium periodate solution (0.1 M) to 5 mL of the NAR solution. Stir the mixture at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>Dissolve 4 mg of BSA and OVA in 2.0 mL of 50 mM carbonate buffer (pH 9.6). Add this protein solution to the NAR/sodium periodate reaction mixture.</li>
	<li>Adjust the mixture to pH 9 with 1 M Na2CO3 solution and stir at room temperature for 6-8 h.</li>
	<li>Dialyze the reaction mixture in phosphate-buffered saline (PBS) using a dialysis membrane (MWCO 10 kDa) for 3 days to exchange the CBS.</li>
	<li>Analyze the hapten-carrier conjugate using matrix assisted laser desorption/ionization time of flight (MALDI-TOF)-MS.
	<ol>
		<li>Mix 1-10 pmol of the conjugate with a 103-fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid (TFA). Dry the mixture on a sample plate in air.</li>
		<li>Perform the analysis using a MALDI-TOF mass spectrometer equipped with a pulsed nitrogen laser operated at 337 nm. Acquire positive ion MALDI mass spectra in the linear mode within the mass range (m/z) of 15,000-100,000 as previously described22.</li>
	</ol>
	</li>
</ol>
2. Immunization
NOTE: A total of 5 BALB/c female mice (8 weeks old) were used: 4 for NAR conjugate immunization and 1 for control (PBS) immunization.
<ol>
	<li>For the first vaccination, mix 50 &#181;g of conjugate (diluted in 100 &#181;L of PBS) with an equal volume of Freund&#39;s complete adjuvant (CFA) and emulsify completely. Administer 100 &#181;L of this mixture to each mouse via dorsal subcutaneous injection.</li>
	<li>Two weeks later, deliver a booster vaccination (100 &#181;L) via subcutaneous injection with the same amount of conjugate mixed with incomplete adjuvant (IFA).</li>
	<li>Extract 200 &#181;L of blood from the tail vein of every mouse 7 days later to obtain serum.
	<ol>
		<li>Centrifuge the blood at 3,000 x g for 10 min. Transfer the serum supernatant to a fresh tube.</li>
		<li>Analyze the serum using an ELISA as previously described21. Choose the mouse with the highest serum titer to use for cell fusion.</li>
	</ol>
	</li>
	<li>In preparation for cell fusion, administer a pulsed immunization to the chosen mouse via intraperitoneal injection of 50 &#181;g of the conjugate in 300 &#181;L of PBS without adjuvant 3 days before fusion. 
	NOTE: This step can be omitted if the titer is high enough.</li>
</ol>
3. Preparation for Cell Fusion
<ol>
	<li>Cell fusion medium preparation
	<ol>
		<li>For the hypoxanthine-aminopterin-thymidine (HAT) selective medium: Dissolve the 50x HAT media supplement in 10 mL of RPMI-1640 and add this mixture to 500 mL of RPMI-1640 containing 20% FBS. 
		NOTE: When 10 mL of the 50x concentrate are diluted in 500 mL of culture medium, the final concentrations of hypoxanthine, aminopterin, and thymidine are 100 &#181;M, 0.4 &#181;M, and 16 &#181;M, respectively.</li>
		<li>For the hypoxanthine-thymidine (HT) media: Dissolve the 50x HT media supplement in 10 mL of RPMI-1640 and add this mixture to 500 mL of RPMI-1640 containing 20% FBS. The final concentrations of hypoxanthine and thymidine are 100 &#181;M and 16 &#181;M, respectively.</li>
	</ol>
	</li>
	<li>Culture the Sp2/0-Ag14 cells.
	<ol>
		<li>At least 7 days before fusion, rapidly thaw a vial of liquid nitrogen-frozen SP2/0-Ag14 myeloma cells in warm water.</li>
		<li>Transfer the cell solution into 5 mL of fresh culture medium (RPMI-1640) and centrifuge for 10 min at 1000 x g.</li>
		<li>After centrifugation, remove the culture medium and resuspend the cells in fresh RPMI-1640 supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Transfer the cells and medium into a 25 cm2 flask and incubate at 37 &#176;C in an atmosphere of 5% CO2.</li>
		<li>Expand the cells to 3 or 4 75 cm2 flasks before fusion.</li>
	</ol>
	</li>
	<li>Preparation of abdominal feeder layer cells
	<ol>
		<li>Sacrifice a 12-week-old albino ICR mouse by cervical dislocation 1 day prior to cell fusion and submerge it in 75% ethanol.</li>
		<li>After removing the fur from the abdomen with hemostatic forceps, disinfect the area with 75% ethanol. Cut the outer skin to expose the abdominal cavity. Inject 3 mL of sterile RPMI-1640 medium into the abdomen and massage the abdomen to detach additional cells into the solution. Aspirate the feeder cell suspension into a 15 mL centrifuge tube.</li>
		<li>After centrifuging the cell suspension for 10 min at 1000 x g, discard the supernatant and resuspend the feeder cells in 20 mL of HAT selective medium.</li>
		<li>Transfer the cells into 96-well cell culture plates (100 &#181;L/well). Incubate the plates overnight at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
4. Cell Fusion
<ol>
	<li>After the chosen immunized mouse has been prepared for cell fusion (see step 2.4), administer an intraperitoneal injection of 10% chloral hydrate (anesthetic) and sacrifice the immunized mouse via cervical dislocation.</li>
	<li>Collect additional blood from the heart (1 mL), and prepare serum as described in step 2.3.1 to use as a positive control during hybridoma selection.</li>
	<li>Remove the skin and muscle tissue using scissors to expose the spleen.</li>
	<li>Isolate the spleen with tweezers carefully and wash the spleen with RPMI-1640. Cut the spleen into pieces and slowly pound the spleen to triturate. Prepare a spleen cell suspension by pressing the spleen tissue through an 800 mesh cell strainer into a 50 mL centrifuge tube.</li>
	<li>Wash the spleen cell suspension with RPMI-1640 medium. Harvest the spleen cells by centrifugation (1000 x g, 10 min, and 4 &#176;C), and then discard the supernatant. Repeat this washing step three times.</li>
	<li>Remove the cultivated and amplified myeloma cells from the incubator and gently tap/shake the flasks to obtain a cell suspension. Wash this suspension with RPMI-1640 medium twice to remove the FBS. 
	NOTE: This is an essential step as FBS may influence cell fusion.</li>
	<li>Count the cells and adjust the concentration before mixing with the spleen cells. The final ratio of spleen cells to myeloma cells should be between 1:5 and 1:10.</li>
	<li>Mix the spleen cell suspension and myeloma cells. Centrifuge the cell mixture (1000 x g, 10 min, and 4 &#176;C), and remove the supernatant.</li>
	<li>Add 1 mL of 50% polyethylene glycol (PEG) solution to the cells and gently stir at 37 &#176;C for 1 min. Let stand for 30 s. 
	NOTE: The PEG solution used in this step should contain 50% (w/v) polyethylene glycol 1500 and 10% (v/v) dimethyl sulfoxide (DMSO) in PBS without calcium (Ca2+).</li>
	<li>Add 2 mL of RPMI-1640 medium for 2 min to terminate the reaction. Add an additional 2 mL of RPMI-1640 medium for another 1 min, and then add an additional 10 mL of RPMI-1640 medium.</li>
	<li>Centrifuge the cells at 800 x g for 10 min. After discarding the supernatant, add HAT solution and continue to cultivate the cells. Then add 100 &#181;L of the cell suspension to each well of the feeder layer plates and incubate the plates for 7 days at 37 &#176;C, 5% CO2. 
	NOTE: Under these conditions, the unfused myeloma cells will die while the hybridoma cells will continue to grow in the HAT medium. After 7 days, monoclonal hybridoma will form as a single cluster of cells in the well, while wells containing polyclonal hybridoma will have multiple clusters of cells.</li>
	<li>Aspirate the supernatant for analysis and replace the medium with HT medium. 
	NOTE: Be sure to substitute the HAT medium with HT medium first as switching directly to unsupplemented medium is detrimental to the hybridoma.</li>
</ol>
5. Indirect Competitive ELISA (icELISA)
<ol>
	<li>Coat a 96-well plate with NAR-OVA (1 &#181;g/mL, 100 &#181;L/well) and incubate for 1 h at 37 &#176;C or overnight at 4 &#176;C.</li>
	<li>Block the plate with 300 &#181;L of GPBS (PBS containing 1% m/v gelatin) for 1 h at 37 &#176;C to prevent non-specific adsorption.</li>
	<li>Wash the plate three times with TPBS (PBS containing 0.05% v/v Tween-20).</li>
	<li>Dilute unconjugated NAR into a series of concentrations with 10% methanol. Add 50 &#181;L of these dilutions to separate wells. In the other wells, add 50 &#181;L of anti-serum or hybridoma supernatant (containing mAbs). Incubate the plate for 1 h at 37 &#176;C.</li>
	<li>Add 100 &#956;L of peroxidase-labelled anti-mouse IgG into each well and incubate for an additional 1 h.</li>
	<li>After washing the plate three times with TPBS, add 100 &#181;L of substrate solution (0.1 M citrate buffer (pH 4) containing 0.015% v/v H2O2 and 2 mg/mL of 3, 3&#39;, 5, 5&#39;- tetramethyl benzidine (TMB)) to each well and incubate for 15 min.</li>
	<li>Stop the reaction by adding 50 &#181;L of 1 M H2SO4 to each well.</li>
	<li>Measure the absorbance using a microplate reader at 450 nm. Choose the hybridomas that secreted the highest concentrations of NAR antibodies into their supernatant for further preparation.</li>
</ol>
6. Preparation of Monoclonal Hybridomas
<ol>
	<li>Use the limiting dilution method to prepare the monoclonal hybridomas.
	<ol>
		<li>After removing the HT medium from the selected hybridomas (i.e., those positive for NAR antibody secretion), resuspend the cells in RPMI-1640 and count them.</li>
		<li>Dilute the cells to a concentration of 1 cell, 2 cells, and 4 cells per well in 100 &#181;L of RPMI-1640 in a 96-well plate. Incubate the plate at 37 &#176;C, 5% CO2.</li>
		<li>After 7-10 days, detect the NAR antibodies in the culture supernatant using the icELISA protocol (step 5). Transfer cells from the hybridomas that are positive for NAR antibodies to 24-well plates, 25 cm2 flasks, and 75 cm2 flasks for cultivation.</li>
	</ol>
	</li>
	<li>Cryopreserve the monoclonal hybridomas.
	<ol>
		<li>Harvest the cells and transfer them to centrifuge tubes.</li>
		<li>After centrifuging the cells (800 x g, 10 min), remove the supernatant and resuspend the cells in freezing medium (RPMI-1640, 20% fetal calf serum (FCS), and 10% DMSO). Transfer the cell suspensions to cryotubes.</li>
		<li>Store the cryotubes in a gradient cooling box at -80 &#176;C for 24 h. Then transfer the cryotubes to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
</ol>

<ol>
	<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> Nature. 256, 495-497 (1975).</li><li>De Smet, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbal+remedies.">Herbal remedies.</a> The New England journal of medicine. 347, 2046-2056 (2002).</li><li>Rowinsky, E. K., Donehower, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+pharmacology+of+paclitaxel+(Taxol).">The clinical pharmacology of paclitaxel (Taxol).</a> Seminars in oncology. 20, 16-25 (1993).</li><li>Yan, X., Zhao, Y., Zhang, Y., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+Antibodies+and+Immunoassay+for+Medical+Plant-Derived+Natural+Products:+A+Review.">Monoclonal Antibodies and Immunoassay for Medical Plant-Derived Natural Products: A Review.</a> Molecules. 22, (2017).</li><li>Loungratana, P., Tanaka, H., Shoyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibody+against+ginkgolic+acids+in+Ginkgo+biloba+Linn.">Production of monoclonal antibody against ginkgolic acids in Ginkgo biloba Linn.</a> The American journal of Chinese medicine. 32, 33-48 (2004).</li><li>Fujii, S., Morinaga, O., Uto, T., Nomura, S., Shoyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+monoclonal+antibody-based+immunochemical+assay+for+liquiritin+and+its+application+to+the+quality+control+of+licorice+products.">Development of a monoclonal antibody-based immunochemical assay for liquiritin and its application to the quality control of licorice products.</a> Journal of agricultural and food chemistry. 62, 3377-3383 (2014).</li><li>Ishiyama, M., Shoyama, Y., Murakami, H., Shinohara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibodies+and+development+of+an+ELISA+for+solamargine.">Production of monoclonal antibodies and development of an ELISA for solamargine.</a> Cytotechnology. 18, 153-158 (1995).</li><li>Xuan, L., Tanaka, H., Xu, Y., Shoyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+monoclonal+antibody+against+crocin+and+its+characterization.">Preparation of monoclonal antibody against crocin and its characterization.</a> Cytotechnology. 29, 65-70 (1999).</li><li>Leu, J. G., Chen, B. X., Schiff, P. B., Erlanger, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+polyclonal+and+monoclonal+anti-taxol+antibodies+and+measurement+of+taxol+in+serum.">Characterization of polyclonal and monoclonal anti-taxol antibodies and measurement of taxol in serum.</a> Cancer research. 53, 1388-1391 (1993).</li><li>Zhu, S., Shimokawa, S., Shoyama, Y., Tanaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+analytical+ELISA-based+methodology+for+pharmacologically+active+saikosaponins.">A novel analytical ELISA-based methodology for pharmacologically active saikosaponins.</a> Fitoterapia. 77, 100-108 (2005).</li><li>Phrompittayarat, W., 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=Determination+of+pseudojujubogenin+glycosides+from+Brahmi+based+on+immunoassay+using+a+monoclonal+antibody+against+bacopaside+I.">Determination of pseudojujubogenin glycosides from Brahmi based on immunoassay using a monoclonal antibody against bacopaside I.</a> Phytochemical analysis. 18, 411-418 (2007).</li><li>Limsuwanchote, 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=Preparation+of+a+monoclonal+antibody+against+notoginsenoside+R1,+a+distinctive+saponin+from+Panax+notoginseng,+and+its+application+to+indirect+competitive+ELISA.">Preparation of a monoclonal antibody against notoginsenoside R1, a distinctive saponin from Panax notoginseng, and its application to indirect competitive ELISA.</a> Planta medica. 80, 337-342 (2014).</li><li>Tanaka, H., 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=Isolation+of+ginsenoside+Rb1+from+Kalopanax+pictus+by+eastern+blotting+using+anti-ginsenoside+Rb1+monoclonal+antibody.">Isolation of ginsenoside Rb1 from Kalopanax pictus by eastern blotting using anti-ginsenoside Rb1 monoclonal antibody.</a> Phytotherapy research. 19, 255-258 (2005).</li><li>Morinaga, O., Nakajima, S., Tanaka, H., Shoyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+monoclonal+antibodies+against+a+major+purgative+component,+sennoside+B,+their+characterization+and+use+in+ELISA.">Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA.</a> The Analyst. 126, 1372-1376 (2001).</li><li>Sakamoto, 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=Simultaneous+determination+of+soy+isoflavone+glycosides,+daidzin+and+genistin+by+monoclonal+antibody-based+highly+sensitive+indirect+competitive+enzyme-linked+immunosorbent+assay.">Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay.</a> Food chemistry. 169, 127-133 (2015).</li><li>Shan, W., 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=Development+of+a+Fluorescence-Linked+Immunosorbent+Assay+for+Baicalin.">Development of a Fluorescence-Linked Immunosorbent Assay for Baicalin.</a> Journal of fluorescence. 25, 1371-1376 (2015).</li><li>Qu, H., 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=Development+of+an+enzyme-linked+immunosorbent+assay+based+on+anti-puerarin+monoclonal+antibody+and+its+applications.">Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications.</a> Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 953-954, 120-125 (2014).</li><li>Zhang, 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=Development+of+an+enzyme-linked+immunosorbent+assay+and+immunoaffinity+chromatography+for+glycyrrhizic+acid+using+an+anti-glycyrrhizic+acid+monoclonal+antibody.">Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.</a> Journal of separation science. 38, 2363-2370 (2015).</li><li>Zhao, 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=Development+of+Fluorescence-Linked+Immunosorbent+Assay+for+Paeoniflorin.">Development of Fluorescence-Linked Immunosorbent Assay for Paeoniflorin.</a> Journal of fluorescence. 25, 885-890 (2015).</li><li>Qu, H., 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=Establishment+of+an+enzyme-linked+immunosorbent+assay+and+application+on+determination+of+ginsenoside+Re+in+human+saliva.">Establishment of an enzyme-linked immunosorbent assay and application on determination of ginsenoside Re in human saliva.</a> Planta medica. 80, 1143-1150 (2014).</li><li>Qu, H., 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=Development+of+ELISA+for+detection+of+Rh1+and+Rg2+and+potential+method+of+immunoaffinity+chromatography+for+separation+of+epimers.">Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers.</a> Journal of chromatography B, Analytical technologies in the biomedical and life sciences. 985, 197-205 (2015).</li><li>Qu, H., 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=Novel+immunoassay+and+rapid+immunoaffinity+chromatography+method+for+the+detection+and+selective+extraction+of+naringin+in+Citrus+aurantium.">Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium.</a> Journal of separation science. 39, 1389-1398 (2016).</li><li>Qu, H., 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=Rapid+lateral-flow+immunoassay+for+the+quantum+dot-based+detection+of+puerarin.">Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin.</a> Biosensors &amp; bioelectronics. 81, 358-362 (2016).</li></ol>

The authors have nothing to disclose.

Here, we present a protocol for the successful production of mAbs against natural product-derived small molecules. The essential steps in the procedure have been outlined, and we have demonstrated the utility of this protocol using NAR as an example small molecule. The example spectra, reactivity analyses, and icELISA results all show representative experimental and control data that is obtained using this protocol. Example images of the hybridomas provide a visual representation of what the researcher should be looking for when differentiating between the monoclonal and polyclonal hybridomas. Taken together, we have demonstrated that the mAb production, characterization, and application strategy presented here results in the creation of an effective mAb against a small molecule as well as a novel ELISA based on the particular mAb that can be used to test the expression of the target molecule in other natural products.
Working with any type of antibody, the most common issue that may arise during this procedure is associated with the sensitivity of the mAb. Indeed, there is a high risk that the mAb will not work as expected due to low sensitivity, high cross-reactivity, or other factors. As the whole procedure takes at least 4 months to perform in full, it is important to take care during the initial screening of the mAbs secreted from the hybridomas. One essential aspect to avoid creating an ineffective mAb is to screen the anti-serum by icELISA before cell fusion to confirmed reactive with the hapten but not the carrier. This is performed by using two different protein carriers (in this case BSA and OVA) as the immunogen and coating antigen carriers, respectively. During immunization with the hapten-BSA conjugate, the animals will produce antibodies against both the hapten (e.g., NAR) and BSA. Thus, to avoid the false-positive detection of anti-BSA antibodies during screening, hapten-OVA should be used to detect the anti-hapten antibodies specifically. The creation of these two protein carriers as well as when to use them is explicitly highlighted in the protocol.
It is also important to note that during preparation of the monoclonal hybridomas, the limiting dilution method often needs to be repeated several times until all of the wells containing the monoclonal cells are positive. This repetition helps to confirm that every clone stably secrets the specific antibody.
The limitations of this method include the complicated process of hybridoma generation and the time needed for selection of the desired antibody-producing hybridoma. However, once the mAb is obtained and the icELISA is developed, the detection of the target compound in natural products can be performed quickly and efficiently. Notably, this protocol does avoid some of the costs associated with other analysis techniques and does not require the use of expensive instruments repeatedly for every natural product tested.
Once produced and screened, the hapten mAb can be widely used in a variety of analyses. In this protocol, we focused on the use of the mAb in an ELISA-based method that was used to study the biology of the small molecule as well as its pharmacokinetic interactions20,22. Other mAbs created with this protocol have also been used to establish an mAb-based immunoaffinity chromatography method for the separation of structural analogues18, including epimers21, as well as a lateral flow immunoassay23 for rapid and on-site detection of the target molecule. These studies highlight the broad application of mAbs produced using the protocol outlined here. Thus, this procedure, and the mAbs created, acts as a foundation for the development of various target mAb-based immunoassays that can be effectively utilized as analytical tools for the evaluation and quality control of nature products.

Monoclonal antibodies (mAbs), also known as mono-specific antibodies, are produced from a single B-lymphocyte clone and are composed of monovalent antibodies that all bind to the same epitope1. In recent years, many medicinal plant-derived natural products have been used in the treatment of various diseases2. Indeed, many small molecular compounds originally derived from natural products are now applied as first-line drugs, such as artemisinin for malaria and paciltaxel (taxol) for cancer2,3. The study of natural products has made rapid progress, largely due to the tremendous development and optimization of conventional analysis techniques, including high performance liquid chromatography (HPLC) and mass spectrometry (MS). However, there are still some limitations associated with these methods, such as their complex pretreatment protocols and associated costs with regards to time, labor/expertise, and required instruments4.
Recently, mAb-based enzyme-linked immunosorbent assays (ELISAs) have been applied to qualitatively and quantitatively analyze food and natural products. In fact, this method has been applied for both biological samples analysis and clinical testing and has been shown to be accurate, sensitive, and highly efficient while also avoiding the tedious pretreatment steps associated with other analyses5,6.
When using mAb-based ELISAs to study complex natural products, preparation of the monoclonal antibodies is one of the core steps. Unfortunately, the mAbs specific to the small bioactive components present in these types of substances6,7,8,9,10,11,12,13,14,15 are often limited compared to the protein antigens. To circumvent this issue, we have developed a protocol to specifically generate mAbs against small compounds. The protocol presented here includes artificial antigen synthesis, mouse immunization, cell fusion, indirect competitive ELISA, and monoclonal hybridoma preparation.
Notably, our research group has been studying the formation of mAbs against small bioactive compounds from traditional Chinese medicines and developing their applications for years. In our on-going studies, we have developed mAbs against baicalin16, puerarin17, glycyrrhizic acid18, paeoniflorin19, ginsenoside Re20, ginsenoside Rh121, and many other small molecules. Our ELISA protocols based on these mAbs have been used in a number of studies to evaluate the pharmacokinetics of these small molecules as well as their interactions with other bioactive compounds. Moreover, using these mAbs, we have also developed immunoaffinity chromatography methods for the separation of structural analogues, including epimers. Recently, we prepared a lateral flow immunoassay using our anti-puerarin mAb that was subsequently used for rapid, on-site detection of this compound. Our results indicate that our mAb-based assays are indispensable and convenient tools for studying the biology and quality of natural-product-derived compounds, particularly those used in traditional Chinese medicines.

<table><tbody><tr><td>800 mesh (40 &amp;mu;m nylon) filter&amp;nbsp;</td><td>FALCON</td><td>352340</td><td></td></tr><tr><td>24 well culture plate</td><td>NUNC</td><td>119567</td><td></td></tr><tr><td>25 cm2 Flask</td><td>Labserv</td><td>310109016</td><td></td></tr><tr><td>3,3&amp;rsquo;,5,5&amp;rsquo;-Tetramethylbenzidine(TMB)</td><td>Sigma Aldrich</td><td>860336 1G</td><td></td></tr><tr><td>75 cm2 Flask</td><td>Corning</td><td>430720</td><td></td></tr><tr><td>96 well culture plate</td><td>NUNC</td><td>117246</td><td></td></tr><tr><td>bovine serum albumin</td><td>AMRESCO</td><td>332</td><td></td></tr><tr><td>cell strainer</td><td>FALCON</td><td>352340</td><td></td></tr><tr><td>centrifuge tube 15 mL</td><td>Corning</td><td>430645</td><td></td></tr><tr><td>centrifuge tube 50 mL</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>cryotubes, 1 mL&amp;nbsp;</td><td>Sigma Aldrich</td><td>V7384-1CS</td><td></td></tr><tr><td>cultivator</td><td>DRP-9082&amp;nbsp;</td><td>Samsung</td><td></td></tr><tr><td>dialysis membrane (10kDa)</td><td>Heng Hui</td><td>45-10000D</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sinopharm Chemical</td><td>DH105-10</td><td></td></tr><tr><td>electronic balance&amp;nbsp;</td><td>BS124-S&amp;nbsp;</td><td>Sartorius</td><td></td></tr><tr><td>ELISA plates, 96 well</td><td>NUNC</td><td>655101</td><td></td></tr><tr><td>ethanol, 96%</td><td>Sinopharm Chemical</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270106</td><td></td></tr><tr><td>Freund&amp;acute;s adjuvant, complete</td><td>Sigma Aldrich</td><td>SLBM2183V</td><td></td></tr><tr><td>Freund&amp;acute;s adjuvant, incomplete</td><td>Sigma Aldrich</td><td>SLBL0210V</td><td></td></tr><tr><td>Gelatin</td><td>AMRESCO</td><td>9764-500g</td><td></td></tr><tr><td>Gradient cooler container</td><td>Nalgene</td><td>5100-0001</td><td></td></tr><tr><td>HAT media supplement</td><td>Sigma Aldrich</td><td>H0262-10VL</td><td></td></tr><tr><td>HRP-conjugated goat-anti-mouse IgG antibody</td><td>applygen</td><td>C1308</td><td></td></tr><tr><td>HT media supplement</td><td>Sigma Aldrich</td><td>H0137-10VL</td><td></td></tr><tr><td>Inverted Microscope</td><td>IX73</td><td>Olympus&amp;nbsp;</td><td></td></tr><tr><td>keyhole limpet hemocyanin</td><td>Sigma Aldrich</td><td>H8283</td><td></td></tr><tr><td>MALDI-TOF-MS&amp;nbsp;</td><td>Axima-CFR&amp;nbsp; plus&amp;nbsp;&amp;nbsp;</td><td>Axima&amp;nbsp;</td><td></td></tr><tr><td>Microplate Reader</td><td>BioTex</td><td>ELX-800&amp;nbsp;</td><td></td></tr><tr><td>mouse</td><td>Vital River&amp;nbsp;</td><td>BALB/c</td><td></td></tr><tr><td>ovalbumin</td><td>Beijing BIODEE</td><td>5008-25g</td><td></td></tr><tr><td>PEG</td><td>Sigma Aldrich</td><td>RNBC6325</td><td></td></tr><tr><td>Penicillin&amp;amp;Streptomycin solution</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Pipette 10 mL</td><td>COSTAR</td><td>4488</td><td></td></tr><tr><td>Pipette 25 mL</td><td>FALCON</td><td>357525</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CVR</td><td></td></tr><tr><td>skim milk</td><td>applygen</td><td>P1622</td><td></td></tr><tr><td>sodium periodate</td><td>Sinopharm Chemical</td><td>BW-G0008</td><td></td></tr><tr><td>Sulfo-GMBS</td><td>Perbio Science Germany</td><td>22324</td><td></td></tr><tr><td>TipOne Tips 1,000 &amp;micro;L</td><td>Starlab</td><td>S1111-2021</td><td></td></tr></tbody></table>

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.

Generation of monoclonal hybridomas
The molecular weight of the hapten-carrier conjugate was confirmed by MALDI-TOF-MS analysis. As the molecular weight of both BSA and the NAR are known, the number of small molecules conjugated with BSA could be calculated. Figure 1 shows representative spectral results for NAR-BSA22, which displays a broad peak at m/z 77,058. As the average molecular weight of BSA is 66,430, it appears that at least 18 NAR molecules (MW 581) were conjugated with the BSA (molar coupling ratio (NAR:BSA) = 18:1).
icELISAs were performed to determine the anti-serum titers following mouse immunization22. It appears that the serum antibody titers of the four mice immunized with the NAR-BSA conjugate were significantly higher than that observed for the control mouse (Figure 2). The mouse with the highest titer (over 1:5,000) was used for cell fusion.
After the spleen cells from this mouse were fused to the abdominal feeder layer cells, the hybridomas were grown for 7 days inselection medium. Using icELISAs, the cell culture supernatant was tested, andthehybridomas positive for NAR mAbs were recloned and expanded. The images in Figure 3 illustrate the conventional outcomes for stable monoclonal and polyclonal hybridoma cell lines.
Screening and application of the anti-NAR mAb
The critical point of this experiment is the screening of mAb specificity. The results in Table 1 demonstrate that the mAb in this experiment reacted with NAR but not the blocking buffer or carrier proteins22. Furthermore, the specificity of the mAb was further evaluated by testing its cross-reactivity with structurally related compounds. Table 2 shows the calculated cross-reactivity rates. As the cross-reactivity rate for the other flavonoids tested were all less than 2%, it is clear that this mAb is specific for NAR22.
An icELISA was developed using the anti-NAR mAb and highlights the application of this methodology. Using solutions containing known concentrations of NAR, a standard curve was plotted using the absorbances of these solutions and the linear range of the S model curve was calculated (shown in the upper-right figure inset). The linear regression equation (y=-0.176 ln(x) + 1.1243, R2 =0.9978) calculated for this curve can be applied to quantitative analysis of unknown samples.
Production and characterization of a mAb against glycyrrhizic acid
Using this splenocyte/myeloma cell fusion protocol, a monoclonal hybridoma secreting anti-glycyrrhizic acid mAb, named DF5, was also established18. The subtype of this DF5 mAb was identified as IgG1 with a kappa light chain (Table 3). Similar to the analysis above, the mAb was used for additional screening and applications, while the antigen-specific monoclonal hybridomas were expanded andcryopreserved in liquid nitrogen for long-term storage.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Coating substance</td>
			<td>A450 value</td>
			<td>Cross-reactivity (%)</td>
		</tr>
		<tr>
			<td>Nar-OVA</td>
			<td>0.97</td>
			<td>100</td>
		</tr>
		<tr>
			<td>OVA</td>
			<td>0.056</td>
			<td>&#60;0.01</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.068</td>
			<td>&#60;0.01</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>0.053</td>
			<td>&#60;0.01</td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>0.05</td>
			<td>&#60;0.01</td>
		</tr>
	</tbody>
</table>
Table 1. Reactivity of the anti-NAR mAb with NAR-OVA and carrier proteins.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Classification</td>
			<td>Compound</td>
			<td>Cross-reactivity (%)</td>
		</tr>
		<tr>
			<td>Flavonoids&#160;</td>
			<td>Naringin</td>
			<td>100%</td>
		</tr>
		<tr>
			<td></td>
			<td>Puerarin</td>
			<td>1.26%</td>
		</tr>
		<tr>
			<td></td>
			<td>Neohesperidin</td>
			<td>18.80%</td>
		</tr>
		<tr>
			<td></td>
			<td>Rutin</td>
			<td>1.95%</td>
		</tr>
		<tr>
			<td></td>
			<td>Baicalein</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Hyperoside</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td>Terpenes</td>
			<td>Ginsenoside Rg2</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Ginsenoside Rb1</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Ginsenoside Re</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Notoginsenoside</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Glycyrrhizic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Glycyrrhetic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Saikosaponin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Paeoniflorin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Gentiopicrin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td>Sterides&#160;</td>
			<td>Cholic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Deoxycholic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td>Anthraquinones&#160;</td>
			<td>Rheumemodin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Rheinic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td>Other&#160;</td>
			<td>Salvianolic acid</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Curcumin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Gastrodin</td>
			<td>&#60;0.09%</td>
		</tr>
		<tr>
			<td></td>
			<td>Amygdalin</td>
			<td>&#60;0.09%</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Cross-reactivity (%) of the anti-NAR mAb against NAR and its related compounds.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="6">isotype of heavy chain</td>
			<td></td>
			<td colspan="2">isotype of light chain</td>
		</tr>
		<tr>
			<td></td>
			<td>IgG1</td>
			<td>IgG2a</td>
			<td>IgG2b</td>
			<td>IgG3</td>
			<td>IgA</td>
			<td>IgM</td>
			<td></td>
			<td>kappa</td>
			<td>lambda</td>
		</tr>
		<tr>
			<td>DF5</td>
			<td>&#9679;</td>
			<td>&#9675;</td>
			<td>&#9675;</td>
			<td>&#9675;</td>
			<td>&#9675;</td>
			<td>&#9675;</td>
			<td></td>
			<td>&#9679;</td>
			<td>&#9675;</td>
		</tr>
	</tbody>
</table>
Table 3. Isotype analysis of mAb DF5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57116/57116fig1v2.jpg" /> 
Figure 1:&#160;MALDI-TOF-MS analysis of the NAR-BSA conjugate. [M + H]+ and [M + 2H]2+ indicate the single- and double-protonated molecules of NAR-BSA, respectively. This figure has been modified from Qu et al., 201622&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57116/57116fig2.jpg" /> 
Figure 2:&#160;Analysis of anti-serum titer by icELISA. Mice 1- 4 were immunized with the NAR-BSA conjugate, while the control was immunized with vehicle (PBS). The data represent the mean &#177; standard deviation (SD) (n = 3). This figure has been modified from Qu et al., 201622. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57116/57116fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57116/57116fig3.jpg" /> 
Figure 3:&#160;Representative images of stable monoclonal (A) and polyclonal (B) hybridomas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57116/57116fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57116/57116fig4v2.jpg" /> 
Figure 4:&#160;Application of the anti-NAR mAb in an icELISA.Various concentrations of NAR were incubated with mAb in wells pre-coated with NAR-OVA (1 mg/mL). A is the absorbance in the presence of NAR, while A0 is the absorbance in the absence of NAR. The data represent the mean &#177; standard deviation (SD) (n = 3). This figure has been modified from Qu et al., 201622.&#160;

Watch this Scientific Journal Video about Generation of Monoclonal Antibodies Against Natural Products at JoVE.com

Generation of Monoclonal Antibodies Against 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 ...

generation-of-monoclonal-antibodies-against-natural-products

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.8K Views.  Beijing University of Chinese Medicine. Monoklonale antilichamen staan bekend als mono-specifieke antilichamen die worden geproduceerd uit een enkele B-lymfocyten kloon en de monovalente antilichamen die binden aan dezelfde epitoop. Onlangs is de monoklonale antilichaamgebaseerde ELISA uitgegroeid tot ons opkomende platform voor de kwalitatieve of kwantitatieve analyse van voedingsmiddelen of natuurlijke producten. Deze methode is een nauwkeurige, gevoelige en effectieve methode zonder vervelende voorbehandelingsstappen.Dat ...

Video: Generation of Monoclonal Antibodies Against Natural Products

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 ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

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, ...

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 ...

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 ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

Advanced Biology

Immunology

Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Source: Nogales-Gadea, G. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52830">Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Peripheral Blood ...

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Source: Devilder, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.

Generation of Monoclonal Antibodies Against Natural Products

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video