To generate immune plasma, chickens were immunized with each of the rCP-PDs. rCP-PDs were mixed with Freund’s incomplete adjuvant (FUJIFILM Wako Pure Chemical Corporation). Four chickens per group were subcutaneously immunized with 20 μg of each rCP-PD generated, at 3 weeks of age. Three weeks after the first immunization, recombinant proteins with the same adjuvant were used to perform a second immunization. As a control, four chickens were immunized with PBS mixed with the same adjuvant. Chickens were euthanized by collecting heparinized whole blood from the hearts under deep general anesthesia by inhaling Isoflurane (Zoetis Japan, Tokyo, Japan) 3 weeks after the 2nd immunization, and immune plasma was isolated from the whole blood samples. Throughout the experimental period, we monitored the health status of chickens and observed no deaths, weight loss, or other clinical signs in all immunized groups.
Freund s incomplete adjuvant
Manufactured by Fujifilm
Sourced in Japan
Freund's incomplete adjuvant is a laboratory reagent used to enhance the immune response in immunological studies. It is composed of mineral oil and emulsifiers, and its core function is to create a depot effect, which slows the release of antigens and prolongs the immune response. This product is intended for research use only and should be handled with appropriate safety precautions.
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7 protocols using freund s incomplete adjuvant
1
Immune Plasma Generation from Chickens
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
Raising Antibodies against GST Proteins
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To produce specific antibodies against GSTs of PRMs, TFMs, and NFMs, four chickens per group were subcutaneously injected with 20 µg per 0.5 mL of each rGST emulsified with Freund’s incomplete adjuvant (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) at 3 and 7 weeks with 21-G needles. For the control group, we used phosphate buffer saline (PBS) with the same adjuvant. Three weeks after the second administration, chickens were euthanized by collecting heparinized whole blood from hearts of each immunized chicken separately, under deep general anesthesia with Isoflurane inhalation (Zoetis Japan, Tokyo, Japan), and approximately 25–30 mL of heparinized blood was obtained. The immune plasmas were isolated for further experimentation.
4
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.
5
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)].
6
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.
7
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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