Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum
Published: April 30, 2024
Abstract
Source: Zeng, X. et al., Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection. J. Vis. Exp. (2023)
This video demonstrates a method for assessing the antibody immune response to a novel nanoemulsion adjuvant vaccine in pre-immunized mice. Immunization enhances antibody response as confirmed by determining the IgG antibody titers.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of the MRSA HI antigen protein Obtain Fe ion surface determining factor B (IsdB) and α-hemolysin mutant (Hla) clones from commercial sources (see Table of Materials), perform polymerase chain reaction (PCR) amplification to amplify Isd…
Divulgations
The authors have nothing to disclose.
Materials
| 5424-Small high speed centrifugeFA-45-24-11 | Eppendorf, Germany | 5424000495 | |
| 96-well plates | Corning Incorporated, USA | CLS3922 | |
| AFM Dimension FastScan | BRUKER, Germany | null | |
| Balb/c mice | Beijing HFK Bioscience Co. Ltd. | ||
| BCIP/NBT | Fuzhou Maixin Biotechnology Development Company,China | BCIP/NBT | |
| Bio-Rad 6.0 microplate reader | Bio-Rad Laboratories Incorporated Limited Co., CA, USA | null | |
| BL21 Competent Cell | Merck millipore,Germany | 70232-3CN | |
| BSA-100G | Sigma-Aldrich, USA | B2064-100G | |
| Centrifuge 5810 R | Eppendorf, Germany | 5811000398 | |
| Coomassie bright blue G-250 staining solution | MIKX,China | DB236 | |
| Decolorization solution | BOSTER,China | AR0163-2 | |
| Electro-heating standing-temperature cultivator HH-B11-420 | Shanghai Yuejin Medical Device Factory, China | null | |
| Electrophoresis apparatus | Beijing Liuyi Instrument Factory, China | DYCZ-25D | |
| Gel image | Tanon, USA | null | |
| Glutathione-Sepharose Resin GST | Mei5bio,China | affinity chromatography resin | |
| H2SO4 | Chengdu KESHI Chemical Co., LTD,China | 7664-93-9 | |
| HI recombinant protein | Third Military Medical University,China | 110-27-0 | |
| HRP -Goat Anti-Mouse IgG | Biodragon, China | BF03001 | |
| HRP- Goat anti-mouse IgG1 | Biodragon, China | BF03002R | |
| HRP- Goat anti-mouse IgG2a | Biodragon, China | BF03003R | |
| HRP- Goat anti-mouse IgG2b | Biodragon, China | BF03004R | |
| Inoculation loop | Haimen Feiyue Co.,LTD,China | YR-JZH-1UL | |
| IsdB and Hla clones | Shanghai Jereh Biotechnology Co,China | null | |
| Isopropyl nutmeg (pharmaceutic adjuvant) | SEPPIC, France | null | |
| isopropyl- β-D-1-mercaptogalactopyranoside | fdbio,China | FD3278-1 | |
| LB bouillon culture-medium | Beijing AOBOX Biotechnology Co., LTD,China | 02-136 | |
| Low temperature refrigerated centrifuge | Eppendorf, Germany | null | |
| Malvern NANO ZS | Malvern Instruments Ltd., UK | null | |
| Micro plate washing machine 405 LSRS | Bio Tek Instruments,Inc Highland Park,USA | null | |
| Mini-TBC Compact Film Transfer Instrument | BeiJingDongFangRuiLi Co.,LTD,China | 1658030 | |
| MMC packing | TOSOH(SHANGHAI)CO.,LTD | 22818 | |
| MRSA252 | USA, ATCC | null | |
| Nanodrop ultraviolet spectrophotometer | Thermo Scientific, USA | null | |
| New FlashTM Protein any KD PAGE Protein electrophoresis gel kit | DAKEWE, China | 8012011 | |
| PBS | biosharp, China | null | |
| PCR, Amplifier | Thermal Cycler, USA | null | |
| pGEX-target gene recombinant plasmid | Shanghai Jereh Biotechnology Co,China | B3528G | |
| Phosphotungstic acid | G-CLONE, China | CS1231-25g | |
| Pipette | Eppendorf, Germany | 3120000844 | |
| Polyoxyethylated castor oil (pharmaceutic adjuvant) | Aladdin, China | K400327-1kg | |
| Primary antibody | Laboratory homemade:from immunized mice with positive sera | null | See Reference 11 for details |
| Propylene glycol (pharmaceutic adjuvant) | Sigma-Aldrich, USA | P4347-500ML | |
| Protein Marker | Thermo Scientufuc, USA | 26616 | |
| PVDF Transfer Membrane | Invitrogen,USA | 88518 | |
| Scanning Electron Microscope | JEOL,Japan | JSM-IT800 | |
| Talos L120C TEM | Thermo Fisher, USA | null | |
| TMB color solution | TIAN GEN, China | PA107-01 | |
| Turtle kits | Xiamen Bioendo Technology Co.,LTD | ES80545 | |
| Tween-20 | Macklin, China | 9005-64-5 |
Tags
Citer Cet Article
Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum. J. Vis. Exp. (Pending Publication), e22215, doi: (2024).