Anti-HB S Antibody Quantitative ELISA Kit (96assay)

Cat. No.: FNK-BCL-AKS-01

Size: 1kit

Storage: 4℃

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Introduction:

Three types of hepatitis B virus surface antigen (HBsAg), L-, M-, and S-proteins are known. The L-protein is composed of S, Pre-S2, and Pre-S1 domains, and the M-protein is composed of S, Pre-S2 domains, and the S-protein S domain alone. All three surface antigens have S antigen (S-protein) and detection of S antigen has been used for diagnosis of HBV infection because of its high content in serum of HBV-infected patient. The “HB-S Antigen Quantitative ELISA Kit, Rapid-II” can quantitatively detect the activity of S antigen.


The principle and outline of assay:

• Standard S Antigen (lyophilized form, recombinant antigen): 100 ng (1 tube)
• Buffer A, 20 x conc.: 20mL (1 bottle)
• Reaction buffer (3 x conc.): 20mL (1 bottle)
•  Anti S Detection IgG (HRP-labeled): 12µL (1 tube)
• Chromogenic Reagent A: 6mL（1 bottle）
• Chromogenic Reagent B: 6mL (1 bottle)
• Stop Solution: 6.5 mL (1 bottle)
• Assay Plate For S Antigen: 1 plate (pre-coated with anti-S antigen antibody, strip type)
• User’s manual 

Equipments required for the assay and Reagents not provided by the kit

• Microplate reader (equipped to measure absorbance at 450 nm)
• Micropipettes (for the handling of standard antigen and samples)
• Microtubes (for the handling of standard antigen and samples)
• Plate sealers (or plastic films)
• Multi (8-) channel pipette and its reservoir
• Distilled water or pure water 

Assay Procedure

<Preparation before starting>

• Buffer A: Warm up Buffer A (20 x conc.) bottle to room temperature for complete dissolution (chilled concentrated buffer often contains depositions of salts), and dilute the content to 20-fold with distilled or pure water. This makes 400 mL of Buffer A (1 x conc.) ready to use. Storage of diluted Buffer A should be at 4˚C.
• Reaction Buffer: Warm up Reaction Buffer (3 x conc.) bottle to room temperature for complete dissolution (chilled concentrated buffer often contains depositions of salts), and dilute the content to 3-fold with distilled or pure water. This makes 60 mL of Reaction Buffer (1 x conc.). Storage of diluted reaction buffer should be at 4˚C.
• Standard antigen: Pour 1 mL of Buffer A into the standard antigen tube and invert it several times for complete dissolving. This makes 100nUnit/mL (100ng/mL) antigen solution. 

1-Step Method


<①Preparation of standard solutions>

1. Prepare 7 new microtubes and print number from ① to ⑦. As indicated in the below table, transfer 900 μL of Reaction Buffer into No.1 microtube, add 100 μL of standard solution (100 nUnit/mL), and mix well. This makes 10 nUnit/mL of STD①. Likewise, prepare the series of standard solutions as indicated in below table.

2. To draw the standard curve, standard solution from STD① to STD⑦ are used.

3. Transfer 400 μL of Reaction buffer into a microtube as the blank.

STD ID	Reaction buffer (or Buffer A)*			Antigen solution for dilution					Final conc.
STD①	900	μL	+	100	nUnit/mL	100	μL	=	10	nUnit/mL
STD②	500	μL	+	10	nUnit/mL	500	μL	=	5	nUnit/mL
STD③	600	μL	+	5	nUnit/mL	400	μL	=	2	nUnit/mL
STD④	500	μL	+	2	nUnit/mL	500	μL	=	1	nUnit/mL
STD⑤	500	μL	+	1	nUnit/mL	500	μL	=	0.5	nUnit/mL
STD⑥	800	μL	+	0.5	nUnit/mL	200	μL	=	0.1	nUnit/mL
STD⑦	500	μL	+	0.1	nUnit/mL	500	μL	=	0.05	nUnit/mL

* Use Reaction buffer for 1-Step method and Buffer A for 2-Step method.


<②Preparation of sample solutions>

1. Dilute the sample solution by Reaction Buffer so that the expected activity is within the measurable range (0.05～10nUnit/mL). Sample should be diluted more than 10-fold by Reaction Buffer for accurate measurement.

2. When S antigen activity of a sample can not be estimated, please prepare multiple samples with different dilution factors.

3. Human serum sample should be diluted more than 100-fold by Reaction Buffer to prevent the effect of serum components.


<③One step reaction of antigen with capture IgG and detection IgG >

1. Take the assay plate out of the aluminum package.

2. In triplicate manner, add 100 μL of sample, standard and blank solutions to each well.

3. Dilute Anti S Detection IgG (HRP-labeled) to 500-fold with Reaction Buffer and transfer the solution to reservoir.

4. Add 50 μL of diluted Anti S Detection IgG into each well by using a 8-channel multi pipette.

5. Cover the plate by a plate sealer, and let it stay at room temperature (around 25°C) for 1 hr.

2-Step Method


<①Preparation of standard solutions>

Prepare standard solutions as indicated in “①Preparation of standard solutions“ for 1-Step Method. Use Buffer A for dilution instead of Reaction Buffer in 2-Step method.


<②Preparation of sample solutions>

Prepare sample solutions as indicated in “②Preparation of sample solutions“ for 1-Step Method. Use Buffer A for dilution instead of Reaction Buffer in 2-Step method.


<③ 2-Step reaction of antigen with capture IgG and detection IgG >

1. Take the assay plate out of the aluminum package.

2. In triplicate manner, add 100 μL of sample, standard solutions and blank to each well. Incubate at room temperature (around 25°C) for 1 hr.

3. Dilute Anti S Detection IgG (HRP-labeled) to 1000-fold with Reaction Buffer and transfer the solution to reservoir.

4. At the end of the reaction of sample with capture IgG, remove the reaction solution from each well by decantation followed by gentle tapping on the paper towel.

5. Wash each well with 300 μL/well of Buffer A three times and remove residual solution by gentle tapping on the paper towel.

6. Add 100μL of diluted Anti S Detection IgG (HRP-labeled) into each well by using 8-channel multi pipette, and incubate for 1 hr at room temperature (around 25°C).


<④Preparation of Chromogenic solution>

1. About 10 min before the end of reaction, prepare chromogenic reaction mixture as follows.

2. Transfer 6 mL of Substrate Solution A and 6 mL of Substrate Solution B into a reservoir for 8-channel pipette, and mix well.