Hybridoma Screening by Antibody Capture: Dot Blot

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot103051
Author(s):  
Edward A. Greenfield
Keyword(s):  
Tissue Culture ◽  
Culture Supernatant ◽  
Screening Procedure ◽  
Dot Blot ◽  
Tissue Culture Supernatant ◽  
Antibody Capture

A dot blot is an appropriate hybridoma screening procedure when the antigen is a protein that is available in purified form. The antigen is bound directly to a nitrocellulose sheet and incubated with hybridoma tissue culture supernatant. A dot blot is widely used to determine the productivity of a given hybridoma, and this is described here. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones.

Hybridoma Screening by Antigen Capture: Reverse Dot Blot

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103135
Author(s):  
Edward A. Greenfield
Keyword(s):  
Tissue Culture ◽  
Monoclonal Antibodies ◽  
Culture Supernatant ◽  
Membrane Surface ◽  
Nitrocellulose Membrane ◽  
Dot Blot ◽  
Tissue Culture Supernatant ◽  
Antigen Capture ◽  
Mouse Immunoglobulin ◽  
Reverse Dot Blot

A dot blot is widely used to determine the productivity of a given hybridoma. This assay can also be used to screen a fusion or subclone plate for productive hybridoma clones. First, a nitrocellulose membrane is coated with an affinity-purified goat or rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are then “captured” on the coated nitrocellulose membrane surface and detected by screening with horseradish peroxidase (HRP).

scholarly journals Recombinant soluble human Fc gamma RII: production, characterization, and inhibition of the Arthus reaction.

1993 ◽  
Vol 178 (5) ◽  
pp. 1617-1628 ◽  
Author(s):  
F L Ierino ◽  
M S Powell ◽  
I F McKenzie ◽  
P M Hogarth
Keyword(s):  
Tissue Culture ◽  
Immune Complex ◽  
Culture Supernatant ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Form ◽  
Arthus Reaction ◽  
Tissue Culture Supernatant ◽  
Biodistribution Studies

A recombinant soluble form of human Fc gamma RII (rsFc gamma RII) was genetically engineered by the insertion of a termination codon 5' of sequences encoding the transmembrane domain of a human Fc gamma RII cDNA. Chinese hamster ovary cells were transfected with the modified cDNA and the secreted rsFc gamma RII purified from the tissue culture supernatant (to > 95%, assessed by SDS-PAGE) using heat aggregated human immunoglobulin G (IgG) immunoaffinity chromatography. The IgG-purified rsFc gamma RII was relatively homogeneous (approximately 31,000 M(r)) whereas the total unpurified rsFc gamma RII secreted into the tissue culture supernatant was heterogeneous relating to N-linked glycosylation differences. Functional in vitro activity of the rsFc gamma RII was demonstrated by: (a) ability to bind via the Fc portion of human IgG and mouse IgG (IgG2a > IgG1 > > IgG2b); (b) complete inhibition of binding of erythrocytes sensitized with rabbit IgG to membrane-bound Fc gamma RII on K562 cells; and (c) inhibition of the anti-Leu4-induced T cell proliferation assay. Blood clearance and biodistribution studies show the rsFc gamma RII was excreted predominantly through the kidney in a biphasic manner, with an alpha-phase (t1/2 approximately 25 min) and a beta-phase (t1/2 approximately 4.6 h); the kidneys were the only organs noted with tissue-specific accumulation. In vivo, the administration of rsFc gamma RII significantly inhibited the immune complex-mediated inflammatory response induced by the reversed passive Arthus reaction model in rats. There was a specific and dose-dependent relationship between the amount of rsFc gamma RII administered, and the reduction in the size and severity of the macroscopic inflammatory lesion. Histological analysis of the skin showed a diffuse neutrophil infiltrate in both control and rsFc gamma RII-treated rats, however the perivascular infiltrate and the red cell extravasation was less intense in the rsFc gamma RII-treated group. It is likely that complement activation leads to neutrophil chemotaxis, but neutrophil activation via Fc gamma RII, which results in inflammatory mediator release, is inhibited. The data indicate that rsFc gamma RII is a potential therapeutic agent for the treatment of antibody or immune complex-mediated tissue damage.

Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103127
Author(s):  
Edward A. Greenfield
Keyword(s):  
Culture Supernatant ◽  
Detection Method ◽  
Specific Activity ◽  
Binding Capacity ◽  
Sandwich Elisa ◽  
High Specific Activity ◽  
Tissue Culture Supernatant ◽  
Capture Assay ◽  
Antigen Capture ◽  
Elisa Plate

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

1990 ◽  
Vol 36 (9) ◽  
pp. 664-669 ◽  
Author(s):  
David R. Preston ◽  
G. Rasul Chaudhry ◽  
Samuel R. Farrah
Keyword(s):  
Tissue Culture ◽  
Nucleic Acid ◽  
Environmental Samples ◽  
Nucleic Acid Hybridization ◽  
Dot Blot ◽  
Polio Virus ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Hybridization Procedure ◽  
Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

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