Four months after GT, mice were intraperitoneally injected with 115 μg of Pneumovax vaccine (Sanofi Pasteur, Swiftwater, Pa). Serum samples were collected weekly for 3 weeks. For TNP-KLH challenge, mice were injected intravenously with 100 μg of TNP-KLH (Biosearch Technologies, Novato, Calif). Three weeks later, animals were injected intraperitoneally with 100 mg of TNP-KLH to boost the immune response. Antigen-specific IgM and IgG antibodies were evaluated in serum by means of ELISA, as previously described.32 (link)
Tnp klh
Manufactured by Biosearch Technologies
Sourced in United States
TNP-KLH is a conjugate of trinitrophenyl (TNP) and keyhole limpet hemocyanin (KLH). It is a widely used hapten-carrier protein complex employed in immunological research and applications.
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Lab products found in correlation
Imject alum, by Thermo Fisher Scientific (4 mentions)
Tnp lps, by Biosearch Technologies (2 mentions)
Sigma adjuvant system, by Merck Group (2 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
7 aad, by BD (1 mentions)
Eawgalanwavdsa, by GenScript (1 mentions)
Complete freund s adjuvant cfa, by Merck Group (1 mentions)
Sba clonotyping system hrp, by Southern Biotech (1 mentions)
Streptavidin conjugated to horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Pbs 0.05 tween 20, by Merck Group (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Alhydrogel, by InvivoGen (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Phycoerythrin pe, by Merck Group (1 mentions)
Tnp ficoll, by Biosearch Technologies (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Pnpp substrate, by Merck Group (1 mentions)
Tnp conjugated bsa, by Biosearch Technologies (1 mentions)
Ready set go, by Thermo Fisher Scientific (1 mentions)
16 protocols using tnp klh
1
Pneumovax and TNP-KLH Immunization in Mice
2
Investigating Antigen-Specific Immune Responses
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Mice were immunized with 20 µg trinitrophenyl-conjugated keyhole limpet hemocyanin KLH (TNP-KLH) (Biosearch Technologies, Novato, CA) emulsified in complete Freund adjuvant (CFA) through a subcutaneous injection. Immunized mice were sacrificed two weeks later. For recall response, 1×106 lymph node cells were stimulated with 20 mg/ml TNP-KLH for 3 days, supernatant was collected and cytokine production was examined by ELISA.
3
Immunization and Serum Collection Protocol
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Mice were immunized with synthetic TNP-KLH antigen 4 weeks before the end of the experiment. 100 μg of TNP-KLH (Biosearch Technologies) in 50% Imject alum (aluminum hydroxide) (Thermo Scientific) was injected intraperitoneally (i.p.). 3 weeks later, mice were boosted i.p. with 100 μg of TNP-KLH in PBS. Serum was collected before immunization and 1 week after the boost injection.
4
Rainbow Trout T Cell Proliferation Assay
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Gill CD8+ DCs were isolated by cell sorting as described earlier and cultured for 12 h in L-15 medium supplemented with 20% FCS in the presence of the T cell-dependent antigen TNP hapten conjugated to the keyhole limpet hemocyanin (TNP-KLH) (Biosearch Technologies). Because no antibodies are available against extracellular pan-T cell markers in rainbow trout, we used T cell-enriched cultures as responder cells. These T cell-enriched cultures were obtained from isogeneic splenocytes by depleting all IgD+, IgM+, and MHC II+ cells through cell sorting. The resulting negative population, representing approximately 10% of splenocytes, was then labeled with Cell Trace™ CFSE Cell Proliferation Kit (ThermoFisher Scientific). The enrichment in T cells was verified by stimulation with Concanavalin A (ConA, 4 µg/ml; Sigma), a typical T cell mitogen. To carry out the MLR, stimulated DCs were cocultured with isogeneic T cell-enriched splenocytes at a ratio of 1:30 (DCs:splenocytes). After 5 days of incubation at 20°C, cocultured samples were stained with 7-AAD (BD Biosciences) at 2.5 µg/ml to check cell viability and analyzed by flow cytometry to measure cell proliferation of the enriched T cell population through the degree of dilution of CFSE.
5
Immunization of Mice After Surgical Procedures
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On days 2 or 30 after sham or CLP surgery, B6 mice were immunized with the following reagents: (1) influenza A virus (A/PR/8; 105 PFU in 100 μl PBS i.p.; obtained from Dr. Ryan Langlois, University of Minnesota); (2) 2,4,6 trinitrophenyl-conjugated keyhole limpet hemocyanin [TNP-KLH; 50 μg i.p. (Biosearch Technologies, Novato, CA)] precipitated in alum (100 μg) or mixed with CpG containing oligonucleotide 1826 (10 μg; TCCATGACGTTCCTGACGTT), followed 3 weeks later by a second immunization; or (3) 2W1S:PE conjugates [i.p. injection of 0.6 μg 2W1S peptide (EAWGALANWAVDSA; GenScript, Piscataway, NJ) conjugated to 2.4 μg PE (ProZyme; Hayward, CA) emulsified in Complete Freund's Adjuvant (CFA; Sigma-Aldrich, St. Louis, MO)] (28 (link)). The 2W1S:PE conjugate was formed by combining biotinylated 2W1S peptide with streptavidin-PE at a 4:1 ratio.
6
Evaluating T cell-independent and T cell-dependent B cell responses
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To evaluate T cell–independent B cell responses, mice of the specified genotype (four per group) were immunized with 20 µg of TNP-LPS (Biosearch Technologies) in 100 µl of PBS. Mice were bled before and 7 d after immunization. To evaluate T cell–dependent B cell responses, mice of the specified genotype (four per group) were immunized intraperitoneally with 100 µg of TNP-KLH (Biosearch Technologies) emulsified in complete Freund adjuvant at day 0 and 14. Mice were bled before and 21 d after immunization. Serum antibodies specific for TNP were measured at the specified time points using plates coated with TNP-BSA (10 µg/ml). Plates incubated with serial sample dilutions were developed with isotype-specific antibodies (SBA Clonotyping System/HRP; Southern Biotech), and the concentration of bound antibodies was determined using IgM, IgG2a, IgG2b, and IgG1 isotype standards.
7
Immunization and Quantification of TNP-Specific IgG Antibodies
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Mice were immunized by intraperitoneal injection of 100 μg of trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH; Biosearch Technologies Inc., Novato, CA) in 50% Imject Alum (Thermo Scientific, Rockford, IL). The animals were boosted with 100 μg of TNP-KLH in PBS by intraperitoneal injection 3 weeks after the first injection. Serum was collected 1 week after the last injection. TNP-specific IgG antibodies were determined by sandwich enzyme-linked immunosorbent assay (ELISA). Plates coated with TNP-KLH were incubated for 3 h at room temperature with serial dilutions of the obtained sera. After washing with PBS/0.05% Tween 20 (Sigma Aldrich, St. Louis, MO), plates were incubated with goat anti-human IgG conjugated to biotin (Biosource, Life Technologies, Bleiswijk, The Netherlands, kindly provided by Dr. A. Mulder, Leiden University Medical Center, Leiden, The Netherlands) for 30 min at room temperature. After being washed, plates were incubated with streptavidin conjugated to horseradish peroxidase (Jackson ImmunoResearch, Newmarket, Suffolk, UK) for 30 min at room temperature. Azino-bis-ethylbenzthiazoline sulfonic acid (ABTS, Sigma Aldrich) was used as a substrate for detection using a Bio-Rad Model 680 microplate reader (Bio-Rad Laboratories B.V., Veenendaal, The Netherlands) at a wavelength of 415 nm.
8
Delayed-Type Hypersensitivity Response
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Mice (8-wk-old) were immunized subcutaneously with 100 µg of TNP-KLH (Biosearch Technologies) emulsified in CFA at the tail base. 6 d later, mice were challenged with 50 µg of TNP-KLH in the footpad of hind leg and PBS in the footpad of the contralateral hind leg as controls. ΔFootpad thickness was determined by measuring the footpad thickness before and after the induction of DTH using an electronic micrometer (Niigata Seiki) in a blinded fashion.
9
Immunization with TNP-KLH and SRBC
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Mice were immunized with 100 µg (100 µl in PBS) TNP-KLH (load 18, LGC Biosearch Technologies) in 100 µl alum (Imject Alum Adjuvants, ThermoFisher) intraperitoneally. On day 35, mice were boosted with 50 µg (50 µl) TNP-KLH in 50 µl alum intraperitoneally. Alternatively, mice were immunized with 2x109 sheep red blood cells (SRBC, Fiebig Nährstofftechnik) in 300 µl PBS intraperitoneally. Mice were 9-18 weeks old at the time of the first immunization.
10
Antigen-Specific Antibody Quantification
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Eight-to-ten-week-old animals were immunized intraperitoneally with 100 μg/mouse of trinitro-phenyl-conjugated keyhole limpet hemocyanin (TNP-KLH) (Biosearch Technologies) in Sigma Adjuvant System (Sigma-Aldrich). Serum was collected from tail vein at 14 days after immunization. An ELISA assay was applied for quantification of TNP-specific Igs in the sera as indicated in the supplementary data .
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