The peptide 4A124 (YHTTDKLFYMMRGGGS), peptide 4A125 (YSAYEFEYILSSGGGS), peptide 4A126 (KTMSAEEFDNWLGGGS), peptide 4A220 (LTSHTYRSQADTGGGS) and control peptide (MHSSFISPSALGGGS) were chemically synthesized (SBS, Beijing, China). Each peptide was then coupled through its C terminus to an immunogenic carrier, keyhole limpet hemocyanin (KLH, SIGMA, St. Louis, MO).
Keyhole limpet hemocyanin (klh)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Canada, France
KLH is a protein extracted from the hemolymph of the giant keyhole limpet (Megathura crenulata). It is commonly used as a carrier protein in immunological assays and as an antigen in the production of antibodies.
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Lab products found in correlation
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Ovalbumin (ova), by Merck Group (9 mentions)
Complete freund adjuvant (cfa), by Merck Group (8 mentions)
Human insulin, by Merck Group (6 mentions)
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Tween 20, by Merck Group (5 mentions)
Incomplete freund s adjuvant, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Chicken ovalbumin, by Merck Group (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (3 mentions)
Dsdna, by Merck Group (3 mentions)
Complete freund s adjuvant, by Merck Group (3 mentions)
Keyhole limpet hemocyanin (klh), by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase, by Merck Group (2 mentions)
Hen egg lysozyme, by Merck Group (2 mentions)
Human serum albumin (hsa), by Merck Group (2 mentions)
Ultra pure rabbit serum albumin, by Merck Group (2 mentions)
Pd 10 size exclusion column, by GE Healthcare (2 mentions)
110 protocols using keyhole limpet hemocyanin (klh)
1
Peptide Synthesis and KLH Conjugation
2
Immunization Using OCT4 Peptides
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Three OCT4 peptides were synthetized at ChinaPeptides Co., Ltd. (Shanghai, China). To elicit antibody production and immune responses against the hapten, KLH (H7017; Sigma-Aldrich, St. Louis, MO, USA) was chosen and 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (#22980; Thermo Fisher Scientific, Waltham, MA, USA) and N-hydroxysuccinimide (NHS) (#24500; Thermo Fisher Scientific, Waltham, MA, USA) were used to activate carboxylic acid as an active ester. The molar ratios of KLH, polypeptide, EDC, and NHS were 1:10:100:100, respectively, and this mixture was incubated at 4°C overnight. To remove excess KLH and EDC/NHS catalyst, the reaction mixture was washed with phosphate-buffered saline (PBS) three times on a 10,000 MWCO Microcon filtration device (Millipore, Billerica, MA, USA). The adjuvant, Toll-like receptor (TLR) 9 agonist, and Quick Antibody-Mouse 5W, was supplied by Biodragon-Immunotech Inc. (KX0210041; Beijing, China) and was mixed with different OCT4 peptides at a 1:1 volume.
3
MOG and KLH Proliferation Assay
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Twenty-one days after immunization with MOG peptide or 50 µg KLH (Sigma), inguinal lymph node (ILN) cells were isolated and cultured (2.0×105 cells/well) with MOG peptide (0, 1, 3.2 µM) or KLH protein (0, 0.1, 1, 10, 100 µg/mL) in 96-well flat-bottomed plates for three days. In some experiments, differentiated Th1-, Th17- or iTreg cells (2.5×104 cells) from OT-II TCR transgenic mice were co-cultured with X-ray irradiated (45 Gy) BM-DCs (1.0×104 cells) in the presence of OT-II peptide (2.5 µM) and 20 U/mL human IL-2 in 96-well plates for 3 days. ES-DCs were added at increasing doses. [3H]-thymidine (248 GBq/mM) was added to the culture (37 kBq/well) for the final 12 h. At the end of the culture, cells were harvested onto glass fiber filters (Wallac) and the incorporation of [3H]-thymidine was measured by scintillation counting.
4
Flounder CD28 Expression in Tissues
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A total of 18 flounder (50 ± 5.8 g) were used for detecting the expression of fCD28 in various tissues. Seven tissues including intestine, liver, PBLs, muscle, head kidney, spleen, and gills were sampled from nine healthy fish killed by overdose benzocaine (Sigma-Aldrich). To detect the variety of CD28 after being injected with KLH (Sigma, USA), a total of 180 flounder (50 ± 5.8 g) were intraperitoneally injected with 200 μg/fish KLH and the control group was injected with an equal amount of PBS. PBLs, head kidney, and spleen were randomly collected from nine individuals in each group at various periods. All the tissues had been flushed by PBS to remove blood. The expression levels were examined by real-time qPCR.
5
T Cell Proliferation Assay with KLH-Loaded DCs
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NOD mice were injected intraperitoneally with keyhole limpet hemocyanin (KLH; Sigma), 200 μg/mouse as a foreign antigen, and were emulsified in Alum (Pierce). Mice were killed 7 days after immunization, and CD4+ and CD8+ T cells were isolated from the splenocytes by magnetic bead–based negative selection. DCs were purified from IRAK-M−/− and WT NOD mice by a CD11c positive selection kit (Stemcell Technologies). DCs were or were not loaded with KLH (Sigma) 50 μg/mL for 5 h in complete medium at 37°C. Antigen-loaded DCs were then washed with PBS twice before coculturing with T cells. CD4+ and CD8+ T cells from immunized mice were cocultured with DCs from IRAK-M−/− NOD or WT NOD mice, with or without KLH, at a 2:1 ratio in complete medium. 3H-thymidine was added during the last 16 h of a 5-day culture, to determine antigen-specific T-cell proliferation responses by 3H-thymidine incorporation. In DC vaccine experiments, purified DCs from immunized mice were preloaded with KLH, as described above, and 106 cells were injected intravenously into WT NOD mice. The mice were killed 7 days after DC vaccination, and splenocytes were harvested and placed 105/well with KLH (1 μg/mL) in complete medium. Proliferative responses were determined by 3H-thymidine incorporation. 3H-thymidine was added during the last 16 h of a 5-day culture.
6
FITC-KLH Conjugation and Purification
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Conjugation and purification of FITC-KLH was conducted as previously described (Pi et al., 2015[22 (link)]). To begin with, 1.5 mL of 10mg/mL KLH (Sigma-Aldrich, St. Louis, MO, USA) was dialyzed against 2 L of 100 mM NaCO3 buffer at pH 9.3, in dialysis tubing (MWCO: 3,500; VWR Scientific, Rancho Dominguez, CA, USA). The mixture was kept at 4 °C for 24 hours and the dialysis solution was changed once during this process. FITC (Sigma-Aldrich, St. Louis, MO, USA) and KLH were then combined at a 20 µg:1 mg ratio, and incubated for 1.5 hours at room temperature. Next, the FITC-KLH conjugate was dialyzed in 500 mL saline solution overnight at 4 °C. The buffer was changed, and the sample was dialyzed for 2 hrs at room temperature. The absorbance of purified FITC-KLH was read at 280 nm and 495 nm, and the labeling ratio (FITC:Protein) was determined based on the ratio of those absorbancies. Two batches, with labeling ratios 2.4 and 5.3, were made and pooled. Labeled FITC-KLH was stored at -80 °C, in a manner that ensured each aliquot was only thawed once before use.
7
Mycotoxin Detection Protocol
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T-2 toxin, HT-2 toxin, ochratoxin A, ochratoxin B, aflatoxin B1, deoxynivalenol, fumonisin B1, zearalenone, methanol, hexane, dichloromethane, phosphoric acid, sulphuric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium carbonate, sodium bicarbonate, sodium chloride, Tween 20, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), horseradish peroxidase (HRP), gelatine, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Dorset, UK and Zwijndrecht, The Netherlands). 3,3′,5,5′-Tetramethylbenzidine (TMB) was obtained from Neogen (Lansing, MI, USA).
A laboratory mill IKA A11 Basic was used for sample blending. Sigma 4K10 centrifuge was used for samples centrifugation and BioTek EL808 type ELISA plate reader for reading the microtiter plates.
A laboratory mill IKA A11 Basic was used for sample blending. Sigma 4K10 centrifuge was used for samples centrifugation and BioTek EL808 type ELISA plate reader for reading the microtiter plates.
8
Fluorescent Protein Conjugation Protocol
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Ultra-pure rabbit serum albumin, human serum albumin, chicken
ovalbumin, Hen egg lysozyme, and keyhole limpet hemocyanin were purchased
from Sigma (seeKEY RESOURCES TABLE ).
R-Phycoerythrin was purchased from ThermoFisher (seeKEY RESOURCES TABLE ). All carrier proteins were
resuspended in sterile PBS according manufacturers instructions.
Selected carriers were conjugated to fluorescein isothiocyanate
obtained from Sigma (seeKEY RESOURCES
TABLE ) through simple amine modification. Briefly, carrier
proteins were dissolved in 0.1M NaHCO3 and mixed with a
pre-determined amount of FITC dissolved in DMSO. Reaction tubes were rotated
for 2h at room temp. Total reaction volumes were loaded onto PD-10 size
exclusion columns obtained from GE healthcare, and unlabeled FITC was
removed as per manufacturers instructions. Labeled protein was assessed for
conjugation ratio through spectral analysis, and concentrated to desired
stock concentration.
ovalbumin, Hen egg lysozyme, and keyhole limpet hemocyanin were purchased
from Sigma (see
R-Phycoerythrin was purchased from ThermoFisher (see
resuspended in sterile PBS according manufacturers instructions.
Selected carriers were conjugated to fluorescein isothiocyanate
obtained from Sigma (see
TABLE
proteins were dissolved in 0.1M NaHCO3 and mixed with a
pre-determined amount of FITC dissolved in DMSO. Reaction tubes were rotated
for 2h at room temp. Total reaction volumes were loaded onto PD-10 size
exclusion columns obtained from GE healthcare, and unlabeled FITC was
removed as per manufacturers instructions. Labeled protein was assessed for
conjugation ratio through spectral analysis, and concentrated to desired
stock concentration.
9
Recombinant FVIII Production and Purification
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Recombinant human B domain deleted (BDD) FVIII (Xyntha) was from Pfizer (New York, NY). FVIII and FIX deficient plasma was from Haematologic Technologies (Essex Junction, VT). Anti-mCD20 IgG2a subtype (clone 18B12) was purified from transfected HEK293 cells (ATUM, Newark, CA). Rapamycin was purchased from LC laboratories (Woburn, MA). Keyhole limpet hemocyanin was purchased from Sigma Aldrich (St. Louis, MO).
10
Generating Antibody Against As3MT Protein
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As3MT polyclonal antibody was raised in rabbit using the following synthetic peptide (EpiCypher): [C]HGRIEKLAEAGIQSESYDIV. The amino acid in brackets was added to improve the solubility of the peptide and for coupling. Further, the peptide was conjugated to keyhole limpet hemocyanin (Sigma-Aldrich) and was sent to Pocono Rabbit Farm & Laboratory Inc. for antibody production. Rabbit serum was affinity-purified using an antigen column with a SulfoLink immobilization kit (Thermo Scientific). Commercial antibodies were used: rabbit anti-GAPDH (Life Science) and rabbit anti-Lamin A (Santa Cruz).
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