USER GUIDE

Printed: 2022Feb-ver.1

HBsAgGi ELISA Kit (96 Test)
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@medglyco.com

CONTENTS
1.

PRODUCT DESCRIPTION ……………………………………………………. 2

2.

REAGENTS FOR HBsAgGi ELISA …………………………………………… 2

3.

SUMMARY ………………………………………………………………………. 2

4.

PRINCIPLE ………………………………………………………………………. 3

5.

PROCEDURE …………………………………………………………………… 3

6.

CALCULATION OF THE CONCENTRATION ………………………..………. 7

7.

PERFORMANCE CHARACTERISTICS ……..…………………………….…. 78.

PRECAUTIONS ……………………………………………………………….… 8

9.

STORAGE AND STABILITY ……………………..…………………….………. 9

10. REFERENCES ………………………………………………………………..… 9

1Product description
The HBsAgGi ELISA Kit is an enzyme-linked immunosorbent assay for thequantitative detection of HBV particles containing O-glycosylated M-HBsAg.
For Research Use Only. Not for use in diagnostic procedures.

2Reagents for HBsAgGi ELISA
2.1

2.2

3

Materials included in this kit
1)

HBsAgGi antibody coated plate：96 well (8-well strip x 12)

2)Standard M-HBsAg (800 ng/mL)：1 mL (2 mL tube)

3)

Dilution Buffer：24 mL (12 mL x 2 bottles)

4)

HRP-labeled HBsAgGi antibody：11 mL

5)

20X Wash Buffer：50 mL

6)

TMB Substrate：12 mL

7)Stop Solution (0.2 M Sulfuric acid)：12 mL

8)Plate seal：39)

User Guide

Materials and equipment required but not provided
1)Plate reader：450 nm

2)Plate washer

3)

Plate shaker (If available)

4)

Reagent reservoir

5)

Micropipette (20 µL, 200 µL, 1000 µL)

6)Multi-channel pipette

7)

Disposable microtubes and pipet tips

8)

Distilled water

Summary
The HBs antigen that constitutes the envelope of HBV particles consists ofthree glycoproteins of different sizes (S-HBs antigen, M-HBs antigen, and L-HBsantigen), and infectious HBV particles with HBV DNA or HBV RNA have all HBs
antigens. On the other hand, the blood of HBV-infected patients contains many
subviral particles that are not infectious, and the subviral particles mainly consistof S-HBs antigen. The anti-HBV surface antigen glycosylated isomeric antibody(HBsAgGi) used in this kit is specific for genotype C and binds mainly to infectious
HBV particles.

2

4Principle
This product is a kit for HBV measurement by sandwich ELISA method. An antiHBV surface antigen glycosylation isomer antibody (HBsAgGi) is adsorbed ontomicrowells. HBV particles and standards can be captured by HBsAgGi antibody(STEP 1). After washing, the HBV particles captured on the ELISA microwells will
be further reacted with the peroxidase conjugated antibody, HRP-labeled
HBsAgGi (STEP 2). After further washing, HRP substrate (TMB substrate) isadded into the well. After the incubation, the reaction is stopped by the addition ofStop solution and the absorbance at 450nm is measured by a plate reader (STEP3). The M-HBsAg protein concentration in the sample will be calculated by using acalibration curve prepared by measuring the standard material (M-HBsAg proteinin the kit) with different concentrations.

Figure 1 Procedures of HBsAgGi ELISA kit
5Procedure
5.1

Preparation
1)

Cell culture supernatant, serum, and plasma were tested with this assay.
Serum samples should be collected according to the usual method andassayed within 1 week at 4°C after collection. If stored longer, samples
should be aliquoted and must be stored frozen at -20°C or below and avoid
repeated freeze-thaw cycles. Prior to assay, the frozen sample should bebrought to room temperature. Samples containing a visible precipitate must3be clarified before use for the assay.
2)

Prepare 1X washing solution by mixing 50 mL of 20X Wash Buffer with 950
mL of purified water. After mix well, transfer to a clean bottle and store at 4–30°C. The 1X washing solution is stable for 30 days.

5.2

Preparation of standards
Prepare seven microcentrifuge tubes, labeled 400 ng/mL, 200 ng/mL, 100 ng/mL,50 ng/mL, 25 ng/mL, 12.5 ng/mL, and 6.25 ng/mL. Add 250 µL of Dilution Buffer toeach tube. Spin down the tube of Standard M-HBsAg (800 ng/mL) in the kit. Take
250 µL of the Standard M-HBsAg and add it to the 400 ng/ml tube and mix. Next,
take 250 µL from the 400 ng/ml tube and add it to the 200 ng/mL tube and mix.
Make a 2-fold serial dilution to 6.25 ng/mL in the same manner.

5.3

Sample dilution method
Serum samples should be diluted 100-fold with Dilution Buffer; for duplicate
assays (recommended), add 217.8 µL of Dilution Buffer to a dilution test tube, add
2.2 µL of sample, and mix.

5.4

Preparation of reagents1)

Antibody for detection: HRP-labeled HBsAgGi antibody should be usedwithout dilution. For the remainder of the HRP-labeled HBsAgGi antibody,close the tube tightly and keep storing at 2-8°C.

2)

TMB Substrate: Remove from refrigerator and allow to come to room
temperature. It can be used as is.

3)

Stop Solution: Remove from the refrigerator and let it come to room
temperature. It can be used as is.

5.5

Measuring protocol

41)

Determine the number of microwell strips required for measuring samples
and number of wells for standards and blanks. Each sample, standard, andblank should be assayed in duplicate (recommended). Remaining microwell
strips should be returned into the provided bag, which must be carefully
resealed to avoid moisture absorption and stored at 2-8°C

2)Standards: Add 100 µL of the Dilution Buffer (blank standards) in two wellsof the microwell strips as the blanks. In the same way, add 100 µL of eachstandard ranging from 6.25 ng/mL to 200 ng/mL to two wells (100 µL/well).

3)

Samples: Add 100 µL of samples 100-fold diluted with Dilution Buffer intotwo wells (100 µL/well).

4)First incubation: After all additions have been made, seal the microwell
strips and incubate for 2 hours at room temperature. If available, gently
shake the microwell strips (approximately 400 rpm/min).

5)

Wash: Remove the plate seal gently and empty wells. Wash microwell strips
5 times with Wash Buffer (350 µL/well).

6)

HRP-labeled HBsAgGi: Add 100 μL of HRP-labeled HBsAgGi to all wells,
including the blank wells.

7)

Second incubation: Seal the microwell strips and incubate for 1 hour atroom temperature. If available, gently shake the microwell strips(approximately 400 rpm/min).

8)

Wash: Remove the plate seal gently and empty wells. Wash microwell strips
5 times with Wash Buffer (350 µL/well).

9)

HRP reaction: Prepare TMB Substrate in a reservoir with enough amountfor the assay. By using a multi-channel pipette, add 100 µL of TMB
Substrate to all wells and incubate the strips at room temperature up to 30min. The color development of the well should be monitored and thereaction should be stopped at the appropriate timing.
The reaction will present blue coloration. Stop the reaction of the wells withblank being clear, around 25 ng/mL being light blue, and 200 ng/ml being
dark blue.

10)

Stop reaction: Add 100 µL of the Stop Solution to each well from thereservoir with enough amount for the assay by using a multi-channel
pipette. Mix the solution to stop the reaction by gently shaking the microwell
strips. The color of the wells will change from blue to yellow after theaddition of the stop solution. Note: The Stop Solution is 0.2 M H2SO4,
please use with caution.

511)

Measurement: After stopping the reaction, read the absorbance of eachmicrowell at 450 nm with a plate reader within 15 minutes. Optionally, readthe absorbance at 620 nm as the reference wave length. Determine theabsorbance of both samples and standards including blanks.

NOTE: In case of incubation using a shaker, the obtained values may be higher
than the value obtained without shaking.
6

Calculation of the concentration1)

Calculate the average absorbance of each concentration of the standard MHBsAg reactive with HBsAgGi antibody.

2)

Prepare a calibration curve by taking the concentration of the standard on
the horizontal axis and the absorbance of 450 nm on the vertical axis of thelogarithmic graph.

3)

Calculate the concentration of M-HBsAg from the absorbance readings ofeach sample according to the above calibration curve graph.

4)

Quantification cannot be performed outside the range of the calibration
curve. If the measured value is near the upper limit of the calibration curve(O.D. 450 value is higher than 2.0), measure again after dilution to obtain
the amount of M-HBsAg.

5)

Example of standard curve of M-HBsAg is shown.
Concentration
O.D. 450of standard
HBsAgGi, standard curve(ng/mL)

7100.078

6.25

0.132

12.5

0.188

250.29450

0.482

1000.890

200

1.565

400

2.747

1A450 nm0 (blank)

0.1

0.01
1

10100

1000

M-HBsAg conc (ng/ml)

Performance characteristics
7.1. Sensitivity
The limit of detection of M-HBsAg was determined to be 0.89 ng/mL (mean plus
3 standard deviations obtained from 10 independent measurements).

6

7.2. Reproducibility (Intra-assay)
Intra-assay reproducibility was determined by assaying the serum samples at 6replicates. Coefficient of variation (C.V.) value on the same plate using a known
concentration of 3 samples is less than 10%.
SampleSerum 16110.4
1.82

Number of determinations
Mean (ng/mL)
C.V. (%)

8Serum 2
645.0
2.52

Serum 3689.2
2.65

Precautions
8.1

Precautions for handling (hazard prevention)
1)

Serum specimens and samples should be handled with the caution thatthey are infectious. In order to avoid the risk of infection, protective
measures such as safety cabinets, gloves, lab coats, goggles, and masks
should be taken during measurement.

2)This kit contains sodium azide as a preservative. If the reagent isaccidentally put into the eyes or mouth, or comes in contact with the skin,first aid measures such as rinsing thoroughly with water should be taken,
and medical attention should be sought as necessary.

3)

Since this kit contains sulfuric acid as a reaction stop solution, if it isaccidentally put into the eyes or mouth, or adheres to the skin, take first aid
measures such as rinsing thoroughly with water. If necessary, see a medical
doctor.

8.2

General precautions
1)

The designated reference material (recombinant Standard M-HBsAg) anddiluted standards should be used for calibration curve preparation.

2)

The specified sample diluent should be used for dilution of samples.

3)

A calibration curve should be prepared for each sample measurement.

4)

The reagent should be used immediately after opening the kit and stored at
2-8°C with the lid closed.

5)

The reagent should be stored under the specified conditions and should not
be used after the expiration date.

6)

This kit is for research use only and should not be used for any purpose
other than those specified.

78.3

Precautions for disposal
1)

Serum specimens and samples should be handled as potentially infectious,and precautions should be taken regarding the disposal of used tips, plates,and liquid waste.

2)

Chips used for specimens should be sterilized with sodium hypochlorite(soaked in 1% effective concentration for at least 1 hour). Chips, plates andwaste solution should be sterilized by autoclaving (121°C, 20 minutes orlonger).

3)

Waste solution should be disposed of in accordance with the Water
Pollution Control Law and other relevant regulations.

4)

Since the Dilution buffer contains sodium azide as a preservative, the liquid
waste should be wasted by a specialized waste disposal company

5)

Waste materials (used plastic products) should be disposed of as chemical
adherents by a specialized waste disposal company.

6)

Specimens should be handled in a safety cabinet, but if they are
accidentally scattered or contaminated, wipe them off with paper towels,
and then sterilize them by wiping with sodium hypochlorite (effectiveconcentration: 1%).

9Storage and stability
All kit components except Stop Solution must be stored at 2-8°C. All reagents arestable for 12 months after manufacturing when stored at the indicated conditions.

10 References
1.

Schmitt et al. Structure of pre-S2 N- and O-linked glycans in surface proteins fromdifferent genotypes of hepatitis B virus. (2004) J Gen Virol 85:2045-2053.

2.

Dobrica et al. N-Glycosylation and N-Glycan Processing in HBV Biology andPathogenesis. (2020) Cells 9:1404.

3.

Angata et al. O-glycosylated HBsAg peptide can induce specific antibody
neutralizing HBV infection. (2021) Biochim Biophys Acta Gen Subj. 1866:130020

Manufacturer
RCMG Inc.,
2-1-6 Sengen, Plaza Suite 106, Tsukuba,
Ibaraki 305-0047, Japan8

E-Mail: marketing@rcmg-glyco.com
Website: https://med-glycosci.com
Phone : +81-29-828-8010

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