eoe sub articles

EoE Sub Articles

1. Production of mAbs
<ol>
	<li>The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% FBS per well in 96-well plates. Incubate overnight in a CO2 incubator (5% CO2) at 37 &#176;C.</li>
	<li>Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent. 
	NOTE: Perform the transfection in triplicates. The following protocol is for one well of a flat bottom 96-well plate.
	<ol>
		<li>Dilute 0.5 &#181;L of DNA transfection reagent into 10 &#181;L of 150 mM NaCl. Dilute 0.125 &#181;g of variable heavy chain (vH) and 0.125 &#181;g of variable light chain (vL) expressing vectors into 10 &#181;L of 150 mM NaCl. Vortex each dilution for 10 s.</li>
		<li>Add 10 &#181;L diluted DNA transfection reagent into the 10 &#181;L DNA solution. Vortex 15 s and incubate for 15 min at room temperature. Add the 20 &#181;L mix drop-wise on 293A cells, and mix by gently swirling the plate.</li>
	</ol>
	</li>
	<li>Replace the medium 16 h after transfection and culture cells for 5 days in serum-free medium. 
	NOTE: Use serum-free medium to avoid serum immunoglobulins (Igs) contaminating the produced antibodies.</li>
	<li>Eliminate cells and debris by centrifugation at 460 x g for 5 min.</li>
	<li>Harvest supernatants by aspiration with a multi-channel pipette and transfer them into a V-bottom 96-well plate.</li>
	<li>Coat relevant Ag overnight at 4 &#176;C in 100 &#181;L per well of reconstituted enzyme-linked immunosorbent assay (ELISA)/ enzyme-linked immunosorbent spot (ELISPOT) coating buffer 1x at a final concentration of 2 &#181;g/mL in 96-well ELISA plates.</li>
	<li>Block wells with 10% FBS DMEM medium for 2 h at 37 &#176;C.</li>
	<li>Add 50 &#181;L of supernatants of transfected 293A cells from step 1.5, and incubate for 2 h at RT.</li>
	<li>Add 100 &#181;L of an anti-human Immunoglobulin G antibody (IgG Ab) conjugated to horseradish peroxidase (HRP) enzyme at 1 &#181;g/mL and incubate for 1 h at room temperature. Add 50 &#181;L chromogenic substrate (3,3&#39;,5,5&#39; tetramethylbenzidine, TMB), and incubate for 20 min.</li>
	<li>Read optical densities at 450 nm on a spectrophotometer. 
	NOTE: The specific mAbs revealed by ELISA assay can be further produced at large scale and purified on protein A column presenting affinity for human IgG.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK 293A cell line</td>
			<td>Thermo Fisher scientific</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1X) Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco by life technologies</td>
			<td>21969-035</td>
			<td>(+) 4,5g/L D-Glucose 
			0,11g/L Sodium Pyruvate 
			(-) L-Glutmine</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>100X Conc</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum (FBS)</td>
			<td>Dominique Dutscher</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Jet PEI DNA transfection reagent</td>
			<td>PolyPlus</td>
			<td>101-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom96-well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>V-bottom 96-well plate</td>
			<td>Nunc/Thermofisher</td>
			<td>55142</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to generate monoclonal antibodies from antigen-specific b cells

Source: Devilder, M., et al. <a href="https://app.jove.com/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.

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

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Take eukaryotic expression vectors, encoding either an antibody heavy chain or an antibody light chain, cloned from antigen-specific B cells.
Introduce polyethylenimine, a cationic polymer, that electrostatically interacts with the negatively-charged vectors, forming polyplexes.
Incubate mammalian cells with the polyplexes, which adhere to the cells and are internalized via endocytosis. The resulting endosomes fuse with lysosomes.
The polymers in the polyplexes cause osmotic swelling of the vesicle, leading to its rupture and release of the vectors.&#160;
The released vectors enter the nucleus and are transcribed into heavy-chain and light-chain mRNA, which undergo translation via ribosomes on the endoplasmic reticulum.
The resulting peptide chains assemble into antibodies, which are transported to the Golgi and packaged into secretory vesicles.
Upon fusion of the secretory vesicles and the cell membrane, the antibodies are released extracellularly.
Harvest the antibody-containing supernatant for downstream assays.

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

125 Views. Source: Devilder, M., et al. Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood. 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 trans...

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

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 ...

JoVE Journal

Immunology and Infection

Generation of Monoclonal Antibodies Against Natural Products

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

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

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

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

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

Transcript

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

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Generation of Monoclonal Antibodies Against Natural Products

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations