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HB Pre-S1 Antigen Quantitative ELISA Kit,(Genotype D)

Product#: FNK-BCL-S1D-01

$1,011.42

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HB Pre-S1 Antigen Quantitative ELISA Kit,(Genotype D)

Cat. No.: FNK-BCL-S1D-01

Size: 1kit

Storage: 4℃ (stable for at least 6 months)

Background

Three types of hepatitis B virus surface antigen, L-, M-, and S-proteins are known.

-The L-protein is composed of S, Pre-S2, and Pre-S1 domains.
-The M-protein is composed of S and Pre-S2 domains.
-The S-protein is composed of S domains.

Pre-S1 domain plays an important role in HBV infection because it is the key molecule to recognize human hepatic cells. The “HBs Pre-S1 Antigen Quantitative ELISA Kit, Rapid” provide a good tool to explore the infection mechanism of HBV as well as to detect HBV escape mutants by determining Pre-S1 antigen.

The principle and outline of assay

The key components of the kit are two types of anti-Pre-S2 antibody and the standard Pre-S antigen. So-called sandwich system is employed in this kit, where one type of antibody captures the antigen on the microplate surface, and the captured antigen is detected by another antibody labeled with HRP.

Finally the amount of HRP attached was determined using chromogenic substrate. There are two methods to detect Pre-S2 antigen using the kit, and the approximate times required are as follows;

1-Step method: Total assay time = 90 min¹⁾
①reaction with capture and detection IgGs(60) → ②chromogenic reaction(30)

2-Step method²⁾: Total assay time = 150 min
①reaction with capture IgG(60) → ②reaction with detection IgG(60) → ③chromogenic reaction(30)

1): The indicated time does not include times for sample preparation, plate washing, abs measurement etc.
2): This method is recommended to use when one does not want to dilute samples such as due to low antigen concentration. Signal intensity and detection limit do not differ between the two methods.

The definition of Pre-S1 antigen activity

There is no established definition to express Pre-S1 antigen activity. In the kit we expressed the Pre-S1 antigen activity as follows; the Pre-S1 activity of 1 nUnit equals to that of 1 ng of standard antigen that is provided in the kit.

Storage condition and stability

All components can be stored at 4℃. At the condition, all reagents are stable for at least 6 months after shipment.

Materials and Reagents

Kit composition

The kit contains following materials. Please ensure that all materials are provided in the kit.

Anti Pre-S1 Detection IgG (HRP-labeled): 18 μL (1 tube)
Standard Pre-S antigen (lyophilized form, recombinant antigen): 10 μg (1 tube)
Buffer A, 20 x conc.: 20mL (1 bottle)
Reaction Buffer, 3 x conc.: 20 mL (1 bottle)
Dilution Solution For Detection IgG: 6 mL（1 bottle, red colored）
Chromogenic Reagent A: 6 mL (1 light shielded bottle)
Chromogenic Reagent B: 6 mL (1 bottle）
Stop Solution: 6.5 mL (1 bottle)
Assay Plate For Pre-S2 antigen: 1 plate (pre-coated with anti-Pre-S1 antigen antibody)
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 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 ready to use.
[note] Buffer A can be stored for a few months at 4℃.
Standard antigen: Pour 1 mL of 1 x Buffer A into the standard antigen tube and invert it several times for complete dissolving. This makes 100 nUnit/mL (100 ng/mL ) antigen solution.
[note] Prepared antigen solution can be stored for a few weeks at -20℃.
Detection Antibody Solution: Take 0.5 mL of red colored solution from the bottle of Dilution Solution for Detection IgG, and pour it into the tube of Detection IgG and invert it several times for complete antibody dilution. Return all the diluted antibody solution to the bottle and mix the content thoroughly. Repeat the same procedure two times more to achieve complete transfer of Detection IgG to the bottle. This makes 12 mL of Detection IgG solution.
[note] The prepared solution can be stored for a few days at 4℃.

[note] If necessary, prepared Buffer A, Reaction Buffer and dissolved antigen can be stored for a few weeks at 4℃.

1-Step Method

<①Preparation of standard solutions>

1. Put 1000 μL of Buffer A into the Standard Pre-S1 (D) antigen tube and mix well using a vortex mixer. This makes 100 nUnit/mL of standard solution.

2. Prepare the series of standard solutions according to the table below

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

Concentration after using antigen solution for dilution with reaction buffer.

As the dilution buffer, use Reaction Buffer for 1-Step Method and Buffer A for 2-Step Method.

<②Preparation of sample solutions>

1. Dilute the sample solution by Buffer A so that the expected activity is within the measurable range (0.01～20 nUnit/mL).

2. When human serum is assayed, serum sample should be diluted more than 100-fold by Buffer A to prevent the effect of serum components.

3. Sample can be directly applied without any dilution if the condition of sample is similar to PBS (PBS, pH 7.4).

[note] 1. Measurement of highly contaminated samples may not give accurate measurement. If you
measure such samples, it is recommended to ensure the accuracy by using samples which is
added by known amount of standard antigen.

2. Use PBS-T (PBS+0.05% Tween-20) when Buffer A is not enough for sample dilution.

<③Reaction 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 Buffer A (as the blank) to each well. Incubate at room temperature (around 25°C) for 3 hrs.

3. At the end of the reaction of sample with capture IgG, remove solution from the assay plate by decantation followed by gentle tapping on the paper towel.

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

5. Take adequate amount of Detection IgG solution (prepared at the start) into reservoir.

6. Add 100 μＬ of Detection IgG solution into each well by using 8-channel multi pipette.

7. Cover the plate by plate sealer, and incubate at room temperature (around 25°C) for 3 hrs.

<④Preparation of Chromogenic solution>

1. About 5 minutes before the end of reaction, prepare chromogenic reaction mixture as follows.

2. Transfer all the solutions from Chromogenic Regent A and B bottles into a reservoir, and mix well.

[note] 1.To get the best result, we recommend to pre-warm the Chromogenic Regent bottles to room
temperature before mixing.

2. The prepared chromogenic solution decreases the activity by oxidation within fairly short time. So please use the solution within 15 min after preparation.

<⑤Chromogenic reaction>

1. At the end of antibody reaction, remove the reaction solution from each well by decantation followed by gentle tapping on the paper towel.

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

3. Add 100 μL of the mixed chromogenic solution to each well by using an 8-channel pipette, and incubate for 30 min at room temperature in a light shielded container. To obtain reproducible result the incubation time with chromogenic solution should be accurately 30 min.

4. Stop the reaction by adding 50 μL/well of stop solution.

[note] The blue color generated by the chromogenic reaction changes rather quickly. Thus, to obtain accurate determinations, it is important to measure the absorbance soon after the addition of stop solution.

Example of standard curve

Below is an example of measurements of standard solutions and resulting standard curve.

An example of measurements of standard solutions and resulting standard curve.

[note] The above data is an example, and does not guarantee the performance of the kit. The results may differ due to the difference of the technique and other conditions.