eoe sub articles

EoE Sub Articles

1. Preparation of porcine aminopeptidase N (APN) protein antigen
NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells pEGFP-C1-APN-IPEC-J2 were constructed in a previous study.
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	<li>Recover bacteria from a frozen glycerol stock and streak onto Luria-Bertani (LB) plates containing 50 &#181;g/mL kanamycin (Km+) for single colony isolation.</li>
	<li>Select a single colony from the freshly streaked plate, culture in 4 mL...

a method to produce recombinant antibodies against metallopeptidase

Source: Xia, P. et al., <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates a procedure for the production of recombinant antibodies against metallopeptidase. Recombinant plasmids carrying genes for the antibody, green fluorescent protein, and antibiotic resistance are transfected into epithelial cells with lipofectamine. The transfected cells are selected and cultivated to generate recombinant antibodies.

A Method to Produce Recombinant Antibodies Against Metallopeptidase

1. Preparation of porcine aminopeptidase N (APN) protein antigen
NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells ...

Begin with a tube containing a cationic lipid-based transfection agent and add recombinant expression plasmids.
The plasmid carries a gene encoding metallopeptidase-targeting recombinant antibodies and green fluorescent protein or GFP under one promoter, while the antibiotic resistance gene is under another promoter.
Positively charged lipids interact with a negatively charged plasmid, forming a complex.
Transfer the complexes into a multi-well plate containing mammalian cells, enabling the plasmid to enter the cytoplasm.
The plasmid reaches the nucleus and gets transcribed into mRNAs for antibiotic resistance proteins and bicistronic mRNAs encoding recombinant antibodies and GFPs.
These mRNAs lead to the production of recombinant antibodies, GFPs, and antibiotic resistance proteins.
Post-transfection, replace the medium with a serum-enriched medium.
Introduce an antibiotic, selectively eliminating non-transfected cells while the antibiotic-resistant transfected cells survive.
Using the fluorescence-activated cell-sorting technique, collect the green fluorescent cells expressing recombinant antibodies.
Seed the diluted transfected cells in a serum-free medium and incubate to produce recombinant antibodies against metallopeptidase.

a-method-to-produce-recombinant-antibodies-against-metallopeptidase

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

JoVE Journal

Bioengineering

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 ...

Immunology and Infection

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

A Method to Produce Recombinant Antibodies Against Metallopeptidase

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