For VLRA antibody production, four VLRA cDNAs isolated from lamprey lymphocytes were cloned in-frame with the constant region of human IgG1 (IgG1-Fc). The VLRA-IgG1-Fc fusion proteins were expressed in HEK-293T cells and purified from tissue culture supernatants by protein A (GE Healthcare) chromatography. Monoclonal anti-VLRA antibodies were produced by immunization of BALB/c mice with VLRA-IgG1-Fc proteins emulsified in TiterMax Gold adjuvant. Lymphocytes from the draining lymph nodes of immunized mice were fused with the Ag8.653 myeloma cell line using PEG-1500 (Roche). Three VLRA-specific hybridoma clones (9A6 (IgG1), 9B3 (IgG1) and 2A5 (IgG1)) were identified by ELISA with VLRA recombinant protein and immunoblotting of VLRA transfectants. Anti-VLRA polyclonal antisera were produced by immunization of rabbits with the VLRA-IgG1-Fc proteins (PickCell Laboratories BV, Netherlands).
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Most cited protocols related to «TiterMax»
For VLRA antibody production, four VLRA cDNAs isolated from lamprey lymphocytes were cloned in-frame with the constant region of human IgG1 (IgG1-Fc). The VLRA-IgG1-Fc fusion proteins were expressed in HEK-293T cells and purified from tissue culture supernatants by protein A (GE Healthcare) chromatography. Monoclonal anti-VLRA antibodies were produced by immunization of BALB/c mice with VLRA-IgG1-Fc proteins emulsified in TiterMax Gold adjuvant. Lymphocytes from the draining lymph nodes of immunized mice were fused with the Ag8.653 myeloma cell line using PEG-1500 (Roche). Three VLRA-specific hybridoma clones (9A6 (IgG1), 9B3 (IgG1) and 2A5 (IgG1)) were identified by ELISA with VLRA recombinant protein and immunoblotting of VLRA transfectants. Anti-VLRA polyclonal antisera were produced by immunization of rabbits with the VLRA-IgG1-Fc proteins (PickCell Laboratories BV, Netherlands).
To produce anti-human DO mAbs, mice were immunized with affinity-purified DM–DO complexes (10 (link)) emulsified in Titer Max (CytRx Corp.) and screened for an immune response by ELISA. Splenocytes were fused to SP2/O-Ag14 cells, and culture supernatants from the hybridoma cells were screened by immunofluorescence using DO positive and negative B cells. One hybridoma secreted an mAb that recognized DO. This clone (Mags.DO5; IgG1), recognizes free DO in addition to DM–DO complexes.
To generate MAbs, female BALB/c mice aged 6–8 weeks were each primed with 100 μg of RBD-mFc protein (Sino Biological) formulated with 0.5 mg of aluminum hydroxide adjuvant (Invivogen, USA) and 25 μg of CpG oligonucleotides (Sangon Biotech, China) via the i.p. route on day 0. The mice were boosted via the subcutaneous route on day 8 with RBD-mFc (50 μg/mouse) emulsified with Freund’s complete adjuvant (Sigma), and on day 13 with RBD-mFc (50 μg/mouse) emulsified with Titermax adjuvant (Sigma). On day 22, one mouse was injected with 75 μg of HEK 293F-expressed RBD protein in PBS in a tail vein. On day 26, splenocytes were isolated and fused with SP2/0 cells using polyethylene glycol 1450 (Sigma). Fused cells were then selected in a hypoxanthine, aminopterine, and thymidine (HAT; Sigma) medium. Eight days later, hybridoma supernatants were screened for their ability to bind to RBD protein and to block the ACE2-hFc/SARS-CoV-2 RBD binding by ELISA, as described below. ELISA-positive hybridoma cells were cloned by limiting dilution method and the resulting monoclonal cell lines were expanded. Purified MAbs were prepared from ascitic fluids using HiTrap™ Protein G HP column (GE Healthcare, USA).
Most recents protocols related to «TiterMax»
EXAMPLE 23
One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1224-PL, which is the ectodomain of human PD-L2 (rhPDL2-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-L2 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPDL2-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-L2 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000.
EXAMPLE 11
Two llamas (No. 146 and No. 147) were immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1086-PD, which is the ectodomain of human PD1 (rhPD1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPD1-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-1 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000 for both animals.
Example 2
Immunization with Recombinant Extracellular Domain of CD22.
Twelve UniRat animals (6 HC27, 6 HC28) were immunized with recombinant human CD22 protein. The animals were immunized according to standard protocol using a Titermax/Alhydrogel adjuvant. Recombinant extracellular domain of CD22 was purchased from R&D Systems and was diluted with sterile saline and combined with adjuvant. The immunogen was combined with Titermax and Alhydrogel adjuvants. The first immunization (priming) with immunogen in Titermax was administered in the left and right legs. Subsequent boosting immunizations were done in the presence of Alhydrogel and three days before harvest boosts were performed with immunogens in PBS. Serum was collected from rats at the final bleed to determine serum titers.
Serum Titer Results
Serum titer summary information is shown in
EXAMPLE 29
One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #165-B7, which is the ectodomain of human B7-H2 (rhB7-H2-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against B7-H2 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhB7-H2-Fc. After blocking and adding diluted sera samples, the presence of anti-B7-H2 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000.
EXAMPLE 17
One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #156-B7, which is the ectodomain of human B7-H1 (rh B7H1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against B7-H1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rh B7H1-Fc. After blocking and adding diluted sera samples, the presence of anti-B7-H1 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000.
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The platform allows users to easily locate the best procedures from scientific literature, preprints, and patents, and then utilize intelligent comparisons to identify the most accurate and reproducible methods.
By leveraging PubCompare.ai's leading solution for protocol optimization, researchers can boost the quality and confidence of their experiments.
TiterMax's AI-driven technology is a game-changer for researchers working with a variety of tools and techniques, including TiterMax Gold adjuvant, TiterMax Gold, Maxisorp plates, Titer-Max Classic adjuvant, ExpiCHO Expression System, PEG1500, and TiterMax adjuvant.
The TiterMax® Gold Adjuvant liquid is a particularly useful tool for enhancing immune responses in experiments.
With TiterMax, researchers can quickly and easily find the most effective protocols, reducing the time and effort required to achieve reliable and reproducible results.
The platform's intelligent comparison features allow users to identify the most accurate methods, boosting the quality and confidence of their research efforts.
Whether you're working with cell lines like ExpiCHO cells or exploring new experimental techniques, TiterMax's AI-driven platform can be a valuable asset in your research toolbox.
By leveraging the latest advancements in protocol optimization, you can take your experiments to the next level and drive your research forward with greater efficiency and confidence.