Novel antibody specific for HBV DNA virus
HB surface antigen glycan isomer (HBsAgGi)

RCMG Inc.
2-1-6 Sengen, Plaza Suite 106, Tsukuba, Ibaraki, 305-0047, Japan
Phone: +81-29-828-8010
Website: https://med-glycosci.com
Email: marketing@rcmg-glyco.com

A monoclonal antibody to O-glycosylated PreS2 on M-HBs, which is predominantly expressed by DNA-containing viral particle
HBsAg
gene

PreS1

PreS2

S

: N-glycan: O-glycan

L-HBsAgM-HBsAg

p39/gp42

gp33/gp36

S-HBsAg: HBsAgGiantibody

HBVSurface
gp27

p24

Figure 1: HBsAg and its glycan isomer (HBsAgGi)
HBsAgGi is a monoclonal antibody recognizing O-glycosylated PreS2
domain on M-HBs, which is predominantly expressed by DNAcontaining viral particle, but not on subviral and empty particles.

HBV envelope protein is composed of three types of surface
antigens, S-, M-, and L-HBs, which are produced from an HBsAg
gene containing PreS1, PreS2 and S-domains1. HBsAgs areheavily glycosylated with N-glycan and O-glycan2,3. Whole glycan
structural analyses revealed that PreS2 domain on M-HBs, but
not on L-HBs, contains highly conserved O-glycosylated site ingenotype C (gC)4. Compared to traditional HBsAg-testing which
recognizes all viral particles, HBsAgGi specifically recognizes
infectious HBV particles (DNA virion).
Features:
Binding to intact HBV particles (Immunoprecipitation)
to detect and enrich rare DNA virion from large pool of HBVparticle by simple and one-step procedure.
Measuring serum HBV particles (ELISA)
to detect and/or monitor infectious status in chronic hepatitisB patients including virally suppressed population.

Figure 2: Isolation of rare DNA virion by HBsAgGi
In sera from HBV infected patients, the majority (over 99.9%) of HBVparticle is sub viral particle (SVP), which mainly contains S-HBs. In
contrast, infectious particle (DNA virion) is less than 0.1% of the whole
HBV particles. HBsAgGi can differentiate glycopeptide structure (pink)
specific for rare DNA-containing particle from majority of HBV particle.

Figure 3: Correlation of liver cccDNA with HBsAgGi (Mouse)
In PhoenixBio (PXB) uPA/SCID chimeric mouse infected with human HBV,
serum levels of HBsAgGi were measured by ELISA (n=29). Correlation ofliver cccDNA with serum HBsAgGi was superior to that of qHBsAg or
HBcrAg in this mouse model (Unpublished data).
References:
1. Schadler and Hildt (2009) Viruses 1:185-209.
2. Schmitt et al. (2004) J Gen Virol 85:2045-2053.
3. Dobrica et al. (2020) Cells 9:1404.
4. Wagatsuma et al. (2018) Anal Chem 90:10196-10203.

1

RCMG Inc.
2-1-6 Sengen, Plaza Suite 106, Tsukuba, Ibaraki, 305-0047, Japan
Phone: +81-29-828-8010
Website: https://med-glycosci.com
Email: marketing@rcmg-glyco.com

Novel antibody specific for HBV DNA virus
HB surface antigen glycan isomer (HBsAgGi)
Applications:
1. Western
2. Immunoprecipitation/Separation of HBV particles
3. ELISA
4. HBV infection inhibition assay
5. Immunohistochemistry• Western blotting using PreS2 antibodies

1234kDa

75

75

50

50

37372525

1234

HBV DNA / 20 µL
60

Fold of originalkDa• Immunoprecipitation using HBsAgGi-gC50403020100HBsAgGi

PreS2

Left: HBsAgGi-gC
Right: anti-PreS2 antibody
Lane1: S-HBs (CHO) 50 ng
Lane2: M-HBs (HEK293) 50 ng
Lane3: L-HBs (yeast) 50 ng
Lane4: serum (HBV patient) 1 µg
Secondary antibody: HRP -labeled anti-mouse IgG

OrigIPSup

Original sample (Orig): HBV patient serum(4.1 logIU/20 µL)
IP condition: Biotinylated HBsAgGi-gC 2 µg,
Streptavidin-conjugated magnetic beads 10µL/1 mL TBS-T, 4℃, 16 hours
Quantification of HBV DNA: HBV DNA per 20µL from precipitated fraction (IP) andsupernatant fraction (Sup) was measured byreal-time PCR method.

HBV particles were diluted into 1 mL (4.1 logIU/20 µL) and
precipitated by HBsAgGi-gC. HBsAgGi-gC could collect the HBVparticles containing HBV DNA in this condition.

HBsAgGi-gC (left) detects M-HBs but not L-HBs. On the other
hand, a commercially available PreS2 antibody (right) strongly
recognizes L-HBs but weakly M-HBs. In human serum of HBVpatients, HBsAgGi-gC recognizes M-HBs, while the PreS2
antibody recognizes M-HBs and L-HBs.

2Quantifying infectious HBV particles by HBsAgGi

RCMG Inc.
2-1-6 Sengen, Plaza Suite 106, Tsukuba, Ibaraki, 305-0047, Japan
Phone: +81-29-828-8010
Website: https://med-glycosci.com
Email: marketing@rcmg-glyco.com

An ELISA system using HBsAgGi antibody to detect HBV particles containing HBV DNA or HBV RNAFeatures:
Measuring serum HBV particles (ELISA)• to detect and/or monitor infectious status in chronic hepatitis
B patients including virally suppressed population.
• to detect HBV particles containing HBV RNA in patientstreated with NUC.
• to detect HBV which are not detected by conventional SHBsAg antibody caused by mutation(s) in S-domain.

➢ ELISA using HBsAgGi-gC coated plate and recombinant M-HBs
as a standard sample

Figure 4: HBsAgGi ELISA can measure HBV DNA particles
Sera of HBV patient mainly contains subviral particles over the HBVDNA particles. S-HBsAg antibody can capture all particles, whileHBsAgGi capture infectious HBV particles containing O-glycosylated
M-HBsAg. Measuring HBV by HBsAgGi will present pathologicalconditions not obtained by S-HBsAg measurement.

HBV virion is covered by envelope protein (HBsAg). As shown inFigure 1, PreS2 in M-HBsAg is uniquely modified with O-glycan
in subtype-specific manner1-3. Infectious virions contain all three
HBsAgs, but non-infectious particles mainly contain S-HBsAg.
For the current diagnosis or analysis of HBsAg, antibodies
against S-HBsAg are used to measure amount of HBsAg.
RCMG’s HBsAgGi (HBsAg glycan isomer) is a monoclonal
antibody recognizing O-glycosylated M-HBsAg that is included ininfectious HBV containing HBV DNA3. HBV particles captured
by HBsAgGi differ from those captured by S-HBsAg antibody.
The amount of infectious HBV particles is crucial to monitor
pathological condition of the patient. HBsAgGi ELISA is themost convenient method to measure HBV particles containing
HBV DNA or HBV RNA.

Standard material: M-HBs (3.7 – 900 ng/mL)
Tested samples: sera from HBV patients (2 µL)
Serum #1: HBsAg 6610 IU/mL, HBV DNA 3.1
Serum #2: HBsAg 8612 IU/mL, HBV DNA 0
Serum #3: HBsAg 36600 IU/mL, HBV DNA 8.3
Detection: Biotinylated HBsAgGi-gC and HRPlabeled streptavidin

ELISA plate was coated with HBsAgGi-gC and M-HBs expressedfrom HEK293 cells was used to obtain standard curve. Humansera of HBV patients (#1-3) were diluted (2 µL serum in 100 µL)
and measured on the same ELISA plate. Serum sample (HBVDNA = 0) after NUC treatment was also detected by HBsAgGigC.
References:
1. Schmitt et al. (2004) J Gen Virol 85:2045-2053.
2. Dobrica et al. (2020) Cells 9:1404.
3. Angata et al. (2021) Biochim Biophys Acta Gen Subj. 1866:130020

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