50 μg RBD recombinant protein (Sinobiological: #40592-V05H) was dissolved in 100 μL PBS and then formulated in Freund’s complete adjuvant (Sigma: #9007-81-2) or Freund’s incomplete adjuvant (Sigma: #F5506-10ML) at a ratio of 1.2:1. Four subcutaneous immunizations were administered in conventional group (at Weeks 0, 2, 4, and 6). Or six subcutaneous immunizations were administered in extended group (at Weeks 0, 2, 4, 6, 9, and 12). On day 10 after each immunization, tail vein blood was collected and immediately used for antibody analysis.
F5506 10ml
Manufactured by Merck Group
Sourced in United States
The F5506-10ML is a laboratory product available from Merck Group. It is a 10 milliliter container of a specific chemical or substance. No further details about its core function or intended use can be provided in an unbiased and factual manner.
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5 protocols using f5506 10ml
1
SARS-CoV-2 RBD Protein Immunization
2
Nanobody Generation via Alpaca Immunization
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A healthy alpaca received immunizations at 2-week intervals, with 500 μg of FGFR4-HPF nanoparticles dissolved in PBS and mixed with freunds adjuvant (F5881-10ML, F5506-10ML, Sigma) administered near the cervical lymph node each time, for a total of four immunizations. The peripheral blood was collected both before and after immunization for peripheral blood mononuclear cells (PBMCs) isolation and serum ELISA, followed by total RNA extraction. Subsequently, cDNA was synthesized using the PrimeScript reverse transcription reagent kit (TaKaRa, Osaka, Japan).The sequences of Nbs were amplified and inserted into the pComb3XSS vector. Then recombinant plasmids were electroporated into E. coli SS320 electrocompetent cells. Subsequently, electro-transfected library was infected with M13K07 helper phages to generate Nb-displaying phages library.
3
Collagen-Induced Arthritis Mouse Model
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DBA/1J female mice, 8–12 weeks old, were supplied by Biogem SCARL (Ariano Irpino, Italy). CIA was elicited by intradermal immunization with 50 µL/mouse collagen type II (Sigma, C9301-5MG, Lot#016M4158V; St. Louis, MO, United States) emulsified in complete Freund’s adjuvant (Sigma, F5881-10ML, Lot#SLBQ1106V) on day 1 and in incomplete Freund’s adjuvant (Sigma, F5506-10ML, Lot#SLBL9742V) on day 31. Arthritis was induced in 10 mice (two groups of five mice each), while five mice composed the untreated negative control group. The injection has been performed at about 1.5 cm distal from the base of the tail, being careful to choose a tissue site and not a vessel. 50 μl of emulsion have been slowly injected intradermally into the tail while the mice were under gas anesthesia (2% isoflurane). Physical appearance, behavior and general and local clinical signs of the mice have been observed daily. To follow arthritis development, each paw has been evaluated and scored individually on a scale 0–4, with 0 indicating no inflammation and four indicating the most severe inflammation (Brand et al., 2007 (link)). Animal experiments were performed after approval by the ethical committee of Heath Ministery.
4
Collagen-Induced Arthritis Model in Mice
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DBA/1J female mice, 8–12 weeks old, were supplied by Biogem SCARL (Ariano Irpino, Italy). C57BL/6 female mice and additional female DBA/1J mice were purchased from Charles River (Wilmington, MA, United States) and housed in the animal facility of the University of Perugia. CIA was elicited by intradermal immunization with 50 μL/mouse collagen type II (Sigma, C9301-5MG, Lot#016M4158V; St. Louis, MO, United States) emulsified in complete Freund’s adjuvant (Sigma, F5881-10ML, Lot#SLBQ1106V) on day 1 and in incomplete Freund’s adjuvant (Sigma, F5506-10ML, Lot#SLBL9742V) on day 31. Arthritis was induced in 15 mice (3 groups of 5 mice each), while 3 mice composed the untreated negative control group. An additional 12 mice (4 mice/group) were immunized and utilized for gene expression profiling. Complete details of CIA mouse generation are provided in the Supplementary Material . Details of the CIA experimental procedure, with the arthritis score for each mouse, are given in Supplementary Material . Animal experiments were performed after approval by the ethical committee of the University of Perugia.
5
Alpaca Immunization for SARS-CoV-2 Spike Protein
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250 μg the extracellular domain of SARS-CoV-2 spike protein fused with His tag (S1+S2 ECD, S, cat.# 40589-V08B1, Sino Biological) was emulsified with 250 μL Freund’s complete adjuvant (F5881-10ML, Sigma) to immunize an alpaca (Female, 2-3 years old, Y-Clone, China). On day 14 and 28, the alpaca was boosted twice with 250 μg S protein in 250 μL Freund’s incomplete adjuvant (F5506-10ML, Sigma). One week following the 2nd immunization, we collected the blood samples to measure anti-serum titer. One week after the 3rd immunization, 100 mL of blood was collected to measure anti-serum titer and construct a phage library displaying Nb.
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