Antibodies were produced from Japanese white rabbits. Rabbits were immunized with 5 ml AGEs-BSA emulsified with the same volume of Freund's complete adjuvant (Wako). The rabbits received first booster injection after three weeks, followed by two additional booster injections every week with emulsified Freund's incomplete adjuvant. After the whole blood was collected from the immunized rabbits under anesthesia, the antisera titers were determined by immunoblot analysis. Immunoglobulin fractions was purified using MEP HyperCel (Pall, Port Washington, NY, USA). Anti-AGE-specific antibodies were purified by using each of AGEs-BSA immobilized on Sepharose beads (GE Healthcare, Buckinghamshire, UK).
Freund s complete adjuvant
Manufactured by Fujifilm
Sourced in Japan
Freund's complete adjuvant is a laboratory reagent used to enhance the immune response in experimental settings. It is an emulsion of mineral oil, water, and heat-killed Mycobacterium tuberculosis cells. The adjuvant's primary function is to stimulate the recipient's immune system, leading to a more robust immune response when administered with an antigen of interest.
Automatically generated - may contain errors
Lab products found in correlation
Freund s incomplete adjuvant, by Fujifilm (5 mentions)
Sepharose beads, by GE Healthcare (1 mentions)
Hitrap nhs sepharose column, by GE Healthcare (1 mentions)
Ctla4 ig, by Bristol-Myers Squibb (1 mentions)
Human igg isotype control, by Thermo Fisher Scientific (1 mentions)
Pgex 4t expression vector, by GE Healthcare (1 mentions)
Formalin, by Fujifilm (1 mentions)
10 protocols using freund s complete adjuvant
1
Antibody Production Against Advanced Glycation End-products
2
Immunization of Mice with Protein Adjuvants
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Four‐week‐old female mice (outbred Jcl:ICR, CLEA, Tokyo, Japan) were used for the immunization experiments. For injections with adjuvant, the first dose was given subcutaneously with Freund's complete adjuvant (WAKO, Saitama, Japan), and doses 2–4 with Freund's incomplete adjuvant were provided intraperitoneally at weekly intervals. The quantity of injected protein was 30 µg and formulated in 100 µL of PBS or mixed with a 1 : 1 ratio of adjuvant when the latter was used. Injections without adjuvant were administrated subcutaneously with 30 µg of protein formulated in 100 µL of PBS, four times on a weekly base. Control mice were injected with PBS both in the presence and in the absence of adjuvant. Three days after inoculation, ~ 20 µL of tail‐bleed samples was collected and used for the weekly measurement of IgGs by ELISA. After the final dose, the mice were sacrificed, and heart‐bleed samples were collected and stored at −30 °C with 1 : 4 dilution in PBS supplemented with 20% glycerol. All of the animal experiments were performed in compliance with the Tokyo University of Agriculture and Technology (TUAT) review panel for animal experimentation and the Japanese governmental regulations.
3
Antigen Immunization and IgG Purification
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The NetB toxin was used for antigen immunization as described elsewhere [2 (link)]. Briefly, the toxin (200 µg/ml) was detoxified by
treatment with formalin at a final concentration of 0.4% (v/v) and kept at 37°C for 7
days. After intraperitoneal administration of 20 µg of toxoid to mice (ddY strain, male 4
weeks old; SLC Co., Ltd., Hamamatsu, Japan), the animals were kept under observation for 4
days to examine their survival status. For the first immunization, rabbits (Japanese
white, male, 14 weeks old, Oriental Yeast, Tokyo, Japan) were injected with 20 µg of
toxoid intradermally, emulsified in an equal volume of Freund’s complete adjuvant (Wako
Pure Chemical Co., Osaka, Japan). Subsequently, the animals were injected with the same
dose of toxoid emulsified in an equal volume of Freund’s incomplete adjuvant (Wako Pure
Chemical Co.) intradermally 3 times every 2 weeks. Two weeks after the fourth
immunization, 20 µg of NetB toxoid alone was inoculated subcutaneously as a booster. Two
weeks after the booster, whole blood was collected from the heart under anesthesia, and
serum was collected. The IgG fraction was isolated from the rabbit serum as described by
Harlow and Lane [18 (link)]. Thereafter, the IgG against
the toxin was purified with a HiTrap NHS Sepharose column (GE Healthcare) according to the
manufacturer’s instructions.
treatment with formalin at a final concentration of 0.4% (v/v) and kept at 37°C for 7
days. After intraperitoneal administration of 20 µg of toxoid to mice (ddY strain, male 4
weeks old; SLC Co., Ltd., Hamamatsu, Japan), the animals were kept under observation for 4
days to examine their survival status. For the first immunization, rabbits (Japanese
white, male, 14 weeks old, Oriental Yeast, Tokyo, Japan) were injected with 20 µg of
toxoid intradermally, emulsified in an equal volume of Freund’s complete adjuvant (Wako
Pure Chemical Co., Osaka, Japan). Subsequently, the animals were injected with the same
dose of toxoid emulsified in an equal volume of Freund’s incomplete adjuvant (Wako Pure
Chemical Co.) intradermally 3 times every 2 weeks. Two weeks after the fourth
immunization, 20 µg of NetB toxoid alone was inoculated subcutaneously as a booster. Two
weeks after the booster, whole blood was collected from the heart under anesthesia, and
serum was collected. The IgG fraction was isolated from the rabbit serum as described by
Harlow and Lane [18 (link)]. Thereafter, the IgG against
the toxin was purified with a HiTrap NHS Sepharose column (GE Healthcare) according to the
manufacturer’s instructions.
4
Murine Prorenin Tertiary Structure Visualization
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The tertiary structure of murine prorenin (PDB ID: 5MKT) was depicted by using graphical imaging software CueMol2 (Molecular Visualization Framework; http://www.cuemol.org/) (Fig 1A ). Prorenin consists of renin (white ribbons) and the prosegment (colored ribbon), which covers the enzymatic active site on renin [1 (link)]. From the 43 amino acids of prosegment, we selected three different amino acid sequences (Fig 1B ): E1 (L1PPTRTATFERIPLKKMP17P) and E2 (T7PFERIPLKKMP17P), both of which contains the HR I11PPLKK15P, and E3 (M16PPSVREILEER26P), which does not contain the HR. E1, E2, and E3 were conjugated to keyhole limpet hemocyanin (KLH) [11 (link)]. For each mouse, we prepared 100 μl of peptide vaccine solution, which contained 20 μg of peptide vaccine diluted in 50 μl of normal saline and 50 μl of adjuvant, as described below. We used Freund’s complete adjuvant (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the first vaccine and Freund’s incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) in the subsequent one [15 (link), 16 (link), 20 (link), 21 (link)].
5
Collagen-Induced Arthritis in DBA/1J Mice
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Arthritis was induced in DBA/1J mice between 8 and 10 weeks of age, as described previously33 (link). Chicken type II collagen (cCII; Sigma Chemical Co, St Louis, Missouri) was dissolved in 0.05 M acetic acid to a concentration of 4.0 mg/ml by overnight rotation at 4 °C and mixed with an equal volume of Freund's complete adjuvant (Wako Pure Chemical Industries, Ltd.). On day 0, DBA/1 J mice were immunized at the base of the tail with 100 μl of emulsion. The same injection was repeated on day 21. For CTLA-4 Ig treatment, mice were injected intravenously with 200 μg of CTLA-4 Ig (Bristol-Myers Squibb) labeled using a SAIVI Alexa Fluor 647 Antibody/Protein-Labeling Kit (S30044, Thermo Fisher Scientific), which can control the degree of labeling to prevent dispersion of the signal-to-background ratio, biodistribution, and clearance. We optimized the labeling conditions to achieve about four to five molecules of dye per antibody. Human IgG isotype control (02-7102, Thermo Fisher Scientific) was used for control experiments.
6
Recombinant Mouse Antibody Production
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For production of mouse antibodies, we designed two sets of primers. One pair amplified the Z-AR cDNA (AB372103) encoding polypeptide RVISCKRNNPASSSRRFFQL (AR20) corresponding to residues 695–714, and the other the CYP17A1 cDNA (AB284119) encoding polypeptide DEKEWVNPHLFNPDRFLDENGNRVYS (CYP1726), corresponding to residues 409–434. The DNA fragments were inserted into the BamHI/XhoI sites of the pGEX-4T expression vector (GE Healthcare). The GST-AR20 and GST-CYP1726 expression cassettes were transfected into E. coli BL21. The transfected cells were sonicated in ice-cold water using an Ultrasonic Processor (TAITEC, model VP-ST). We purified GST-AR20 and GST-CYP1726 proteins according to the manufacturer's protocol (GE Healthcare) and immunized 8-week-old BALB/c female mice three times with 50 µg of protein in complete Freund's adjuvant (Wako) at 2-week intervals. Sera were collected and tested 5 days after the last booster injection and used for immunohistochemistry.
7
Antibody Production and Characterization in Chickens
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Chickens were hatched from fertilized eggs (White Leghorn, Aoki Breeder Farm) and reared
exclusively in our laboratory. The viruses were inactivated with formalin (FUJIFILM Wako
Pure Chemical Corp.; final concentration, 0.1%) and purified by sucrose density-gradient
ultracentrifugation [17 (link)]. Chickens were immunized
at both 4 and 6 weeks old of age with the purified virus and Complete Freund’s Adjuvant
(FUJIFILM Wako Pure Chemical Corp.). Two weeks after the second immunization, whole blood
was sampled via cardiac puncture under isoflurane anesthesia. Serum samples were retrieved
in the same way as described above and stored as a polyclonal antibody for subsequent
antigenic analyses, the HI test, neuraminidase inhibition (NI) test [4 (link)], and neutralization assay. For the neutralization assay, polyclonal
chicken antiserum that was serially 2-fold diluted and 102.0 EID50of the virus were mixed and incubated for 1 hr at room temperature. This mixture was
inoculated into four 10-day-old embryonated eggs and cultured for 48 hr at 35°C. The HA
test was performed using the collected allantoic fluid, and the neutralizing titer was
expressed as the reciprocal of the highest dilution of the serum that showed 50% or more
inhibition of viral replication.
exclusively in our laboratory. The viruses were inactivated with formalin (FUJIFILM Wako
Pure Chemical Corp.; final concentration, 0.1%) and purified by sucrose density-gradient
ultracentrifugation [17 (link)]. Chickens were immunized
at both 4 and 6 weeks old of age with the purified virus and Complete Freund’s Adjuvant
(FUJIFILM Wako Pure Chemical Corp.). Two weeks after the second immunization, whole blood
was sampled via cardiac puncture under isoflurane anesthesia. Serum samples were retrieved
in the same way as described above and stored as a polyclonal antibody for subsequent
antigenic analyses, the HI test, neuraminidase inhibition (NI) test [4 (link)], and neutralization assay. For the neutralization assay, polyclonal
chicken antiserum that was serially 2-fold diluted and 102.0 EID50of the virus were mixed and incubated for 1 hr at room temperature. This mixture was
inoculated into four 10-day-old embryonated eggs and cultured for 48 hr at 35°C. The HA
test was performed using the collected allantoic fluid, and the neutralizing titer was
expressed as the reciprocal of the highest dilution of the serum that showed 50% or more
inhibition of viral replication.
8
Evaluation of CH401MAP Cancer Vaccine
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CH401 multiple antigen peptide (MAP), which contains the epitope sequence of the anti-HER2 mAb CH401, was used as the cancer antigen31 . The CH401MAP peptide was synthesized using a Rink amide resin (0.4‒0.7 mmol g−1), An ACT357 peptide synthesizer (Advanced Chemtech, Louisville, KY, USA). BAL6MAP, which contains a partial amino acid sequence of Pseudomonas aeruginosa BAM A (NYYAGGFNSVRGFKDSTLGP), was used as the control peptide59 (link). CH401MAP was emulsified with complete Freund’s Adjuvant (Wako Pure Chemical Industries, Ltd, Osaka, Japan) (50 μg/head, 100 μL 1:1 v/v) and administered to the hu-PBL hIL-4 NOG mice intraperitoneally. For the negative control, an equal volume of PBS was emulsified and injected into hu-PBL hIL-4 NOG mice. Boosters were administered using Freund’s incomplete adjuvant (Wako Pure Chemical Industries, Ltd) 2 weeks after the first immunization. Two weeks after the booster, the mice were sacrificed and used for subsequent analysis.
9
EAE Induction and HPV Vaccine Effects
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C57BL/6JJcl female mice were purchased from CLEA Japan. The mice were 8 weeks-old at the beginning of the study. On day − 1, sera were collected form mice, and EVs were collected from sera. MiRNA levels in serum EVs were then determined by RT-qPCR. EAE was induced on day 1 with a subcutaneous injection of 200 µg of myelin oligodendrocyte glycoprotein (MOG35–55) peptide (BEX, Japan) emulsified with complete Freund's adjuvant (WAKO, Japan) containing 500 µg of heat-killed Mycobacterium tuberculosis H37 Ra. This was followed by an intraperitoneal injection of 500 ng of pertussis toxin (CALBIOCHEM) immediately and 48 h later. HPV vaccine was provided by MSD. On day 0, mice were intramuscularly injected with 100 μl of PBS or HPV vaccine. After EAE induction, the mice were monitored daily using the following score: 0, no symptom; 0.5, tip of tail is limp; 1, limp tail; 1.5, limp tail and hind leg inhibition; 2, limp tail and weakness of hind legs; 2.5, limp tail and dragging of hind legs; 3, complete hind limb paralysis; and 3.5, in addition to the symptoms score 3, hind legs are together on one side of the body. Since EAE scores decreased gradually, we determined the accumulated EAE clinical score that is the sum of EAE scores every day during 30 days.
10
CH401 MAP Peptide Immunization in Humanized Mice
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CH401 multiple antigen peptide (MAP), which contains the epitope sequence of the anti-HER2 mAb CH401, was used as the cancer antigen 31 . The CH401MAP peptide was synthesized using a Rink amide resin (0.4-0.7 mmol/g), An ACT357 peptide synthesizer (Advanced Chemtech, Louisville, KY, USA). BAL6MAP, which contains a partial amino acid sequence of Pseudomonas aeruginosa BAM A (NYYAGGFNSVRGFKDSTLGP), was used as the control peptide 57 . CH401MAP was emulsi ed with complete Freund's Adjuvant (Wako Pure Chemical Industries, Ltd, Osaka, Japan) (50 μg/head, 100 μL 1:1 v/v) and administered to the hu-PBL hIL-4 NOG mice intraperitoneally. For the negative control, an equal volume of PBS was emulsi ed and injected into hu-PBL hIL-4 NOG mice. Boosters were administered using Freund's incomplete adjuvant (Wako Pure Chemical Industries, Ltd) 2 weeks after the rst immunization. Two weeks after the booster, the mice were sacri ced and used for subsequent analysis.
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