Measuring Immunoglobulin G Levels Against a Test Vaccine in a Mouse Serum

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

1. 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 IsdB and Hla genes, and ligate their products to the PGEX-6P-2 plasmid (obtained commercially; see Table of Materials). Screen with the vector Xl/blue s....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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