a hybridoma technology for producing monoclonal antibodies against a metallopeptidase

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

To begin, intraperitoneally inject 100 micrograms of APM protein into the selected adult female mice for a final antigen boost. After 3 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 SP2/0 myeloma cells in a 25-square centimeter flask containing 5 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 5 to 6 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 aspirated SP2/0 cells with a pipette from 8 to 10 bottles and resuspend 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 SP2/0 cells at a ratio of 10-to-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 1 milliliter of polyethylene glycol 1,500 pre-warmed to 37 degrees Celsius drop-wise to the loosened cell pellet over 45 seconds while gently rotating the bottom of the tube.
Then, slowly add 1 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 degrees 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 5 days, add 100 microliters of fresh HAT medium to each well. And again, after 5 days of incubation, replace the medium with HT medium. Use a microtiter plate coated with 5 micrograms per milliliter APN protein diluted in 0.05 molar PBS to analyze monoclonal antibodies in the hybridoma supernatant using an ELISA assay.

a-hybridoma-technology-for-producing-monoclonal-antibodies-against

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

EoE Sub Articles

eoe sub articles

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal immunization
<ol>
	<li>Subcutaneous (s.c) inject female BALB/c mice, 6-8 weeks of age, with 50 &#181;g of APN protein or phosphate-buffered saline (PBS, negative control) mixed with adjuvants once every 2 weeks. Use complete Freund&#39;s adjuvant that contains the heat-killed Mycobacteria for initial immunization, and incomplete Freund&#39;s adjuvant for booster immunizations. Mix equal volumes of APN protein (or PBS) and Freund&#39;s adjuvant or incomplete Freund&#39;s adjuvant, respectively.</li>
	<li>Detect antibody titers against APN in the sera of these mice by indirect enzyme-linked immunosorbent assay (ELISA) using a microtiter plate coated with 5 &#181;g/mL APN protein diluted in 0.05 M PBS (pH 9.6).&#160;</li>
</ol>
2. Hybridoma technology to produce monoclonal antibodies against APN
<ol>
	<li>Intraperitoneally (i.p.) inject 100 &#181;g of APN protein into the selected mice for a final antigen boost.</li>
	<li>Three days later, euthanize the mice using pentobarbital sodium (50 mg/kg, v/v, intraperitoneal) and cervical dislocation.</li>
	<li>Collect spleens, and wash with DMEM twice to remove blood and fat cells. Filter the spleen-cell suspension using a 200-mesh copper grid to remove tissue debris, and harvest spleen cells using centrifugation (1500 &#215; g, 10 min) to remove the membrane of the spleen.</li>
	<li>Seed mouse myeloma SP2/0 cells in a 25 cm2 flask containing 5 mL of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 6% fetal bovine serum (FBS) and culture at 37 &#176;C, 6% CO2 atmosphere to maintain cell viability. After 5-6 days of culture, the cells reach 80%-90% confluence post-resuscitation and are in growth log phase. Under the microscope, the cells are round, bright, and clear.</li>
	<li>One day before hybridization, collect macrophages from peritoneal cavities of the mice according to a previously published method.</li>
	<li>Seed peritoneal macrophages at a density of 0.1-0.2 &#215; 105/mL in 96-well plates, each well containing 100 &#181;L of HAT medium (DMEM supplemented with 10% FBS and 1x HAT Supplement), and incubate at 37 &#176;C, 6 % CO2 humidified atmosphere overnight.</li>
	<li>For hybridization, gently aspirate SP2/0 cells with a pipette from 8-10 bottles, and suspend in 10 mL of serum-free DMEM medium. Wash cells with fresh DMEM, centrifuge (1500 &#215; g, 10 min) twice, and then re-suspend in 10 mL of DMEM.</li>
	<li>Mix the quantified spleen cells with SP2/0 cells at a ratio of 10:1 and transfer into 50 mL tubes. Centrifuge (1500 &#215; g, 10 min) and discard the supernatant. Collect the cell pellets at the bottom of the tubes and tap with palm to loosen the pellets prior to hybridization.</li>
	<li>Add 1 mL of polyethylene glycol 1500 (PEG 1500), pre-warmed to 37 &#176;C, dropwise using a dropper to the loosened cell pellet over the time period of 45 s while gently rotating the bottom of the tube.</li>
	<li>Slowly add 1 mL of DMEM pre-warmed to 37 &#176;C to the above mixture over the period of 90 s, followed by another 30 mL of fresh DMEM. Place the fusion tube into a 37 &#176;C water bath for 30 min.</li>
	<li>After incubation in the warm bath, harvest the cells and re-suspend in HAT medium. Then culture in a 96-well plate inoculated with peritoneal macrophages.</li>
	<li>Five days later, add 100 &#181;L of fresh HAT medium to each well, and incubate the plate for an additional 5 days, after which replace the medium with HT medium (DMEM supplemented with 10% FBS and 1x HT Supplement).</li>
	<li>Use a microtiter plate coated with 5 &#181;g/mL APN protein diluted in 0.05 M PBS (pH 9.6) to analyze monoclonal antibodies in the hybridoma supernatant using ELISA assay.
	<ol>
		<li>When the medium in the wells of the 96-well plate turns yellow (due to cell growth and metabolite release, pH in the medium decreases to 6.8, and phenol red turns from fuchsia to yellow) or cell clusters are observed, acquire 100 &#181;L supernatant from the selected wells and add to the wells of the coated ELISA plate. Use a microplate reader to measure the OD450 values.</li>
		<li>Use the polyclonal antibodies against APN and non-infected mouse serum as positive and negative control, respectively, and use PBS as blank control. In this study, OD450 ratio of sample to negative control (P/N) &#8805; 2.1 was recognized as positive selection standard.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Complete Freund&#39;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td>Animal immunization</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>Enhanced Cell Counting Kit-8</td>
			<td>Beyotime Biotechnology</td>
			<td>C0042</td>
			<td>Measurement of cell 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&#39;s adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>F5506</td>
			<td>Animal immunization</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>BOX 998</td>
			<td>ELISA analysis</td>
		</tr>
		<tr>
			<td>Polyethylene glycol 1500</td>
			<td>Roche Diagnostics</td>
			<td>10783641001</td>
			<td>Cell fusion</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning</td>
			<td>3599</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
</table>

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

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

Take a mouse immunized with a metallopeptidase. Harvest the spleen and obtain a cell suspension.
Filter the suspension through a mesh and centrifuge, removing tissue debris.
The isolated cells include metallopeptidase-specific B cells, producing antibodies against metallopeptidase epitopes.
Add immortal myeloma cells lacking the HGPRT enzyme, preventing the salvaging of nucleobases for nucleotide synthesis. They survive via de novo nucleotide synthesis.
Centrifuge to obtain a pellet and discard the supernatant.
Add PEG and incubate, inducing myeloma-B cell fusion to form HGPRT-positive immortal hybridomas.
Add HAT medium comprising hypoxanthine, aminopterin, and thymidine. Transfer into wells containing a peritoneal macrophage feeder layer, promoting hybridoma growth.
Aminopterin blocks de novo nucleotide synthesis, causing unfused myeloma cells to die. Unfused B cells die due to their short lifespan.
The HGPRT-positive hybridomas salvage the nucleotide precursors hypoxanthine and thymidine, proliferate, and secrete metallopeptidase-specific monoclonal antibodies.
Harvest the supernatant to confirm antibody production via downstream assays.

72 Views. Una tecnologia di ibridomi per la produzione di anticorpi monoclonali contro una metallopeptidasi

Video: Una tecnologia di ibridomi per la produzione di anticorpi monoclonali contro una metallopeptidasi - Experiment

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.

Generazione di Murine anticorpi monoclonali da ibridomi Tecnologia

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

Ricerca

JoVE Journal

Immunologia e infezioni

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.

Generazione di anticorpi monoclonali contro prodotti naturali

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.

Metodo di screening a singola cellula per la selezione e il recupero di anticorpi con specifiche desiderate da popolazioni di cellule di memoria umana b arricchite

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

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Trascrizione

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase