JP2003035712A - HBs ANTIBODY MEASURING REAGENT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring reagent which does not require a longer measuring time as compared with a conventional HBs antibody measuring reagent in which an HBs antigen is carried by a solid phase, which does not cause a nonspecific reaction, whose sensitivity is high and whose measuring range is wide. SOLUTION: In determining an HBs antibody qualitatively or quantitatively, use is made of a reagent in which a first aqueous solution containing a biotinized HBs antigen and albumin is combined with a second aqueous solution containing an enzyme-labeled HBs antigen, albumin and a bovine fetus serum, is used.

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] [0001] The present invention relates to an immunological assay reagent.
About. More specifically, hepatitis B virus surface antigen
Antibodies (hereinafter referred to as HBs antibodies)
) For qualitative or quantitative immunoassay reagents
Related. [0002] 2. Description of the Related Art In recent years, many trace components contained in body fluids
Is often measured by immunological methods for convenience.
It is getting worse. One of the items is hepatitis B virus
There is an inspection for Among them, measurement of HBs antibody is of type B
Monitoring of hepatitis and monitoring after vaccination
It is regarded as an important marker. Generally for measurement of HBs antibodies
Means aggregation method (hereinafter referred to as PA method), radioisotope labeling
Immunoassay (hereinafter referred to as RIA method)
Enzyme immunoassay using an iodine-labeled product (hereinafter referred to as EIA method)
U) etc. are used. PA method is simple and quick
Processing is possible, but on the other hand low sensitivity and narrow measurement range
And the like. The RIA method uses P in terms of detection sensitivity.
Although it is superior to method A, it uses
There is a disadvantage that handling is complicated. On the other hand, the EIA method
Is easy to handle because it does not use radioactive materials and has high sensitivity
In addition, due to the wide measurement range,
Is expected to increase. However, this EIA method
The time required for the measurement is long even if
In this case, there is a problem that the sample processing capacity is reduced.
ing. To solve this problem, recently,
HBs antigen is supported on a solid phase or magnetic particles
A device for B / F separation has been devised between
New non-specific reactions, reduced sensitivity, narrow measurement range
The problem such as becoming has come to be seen. [0003] An object of the present invention is to provide a solid phase
HBs antibody measurement reagent carrying HBs antigen
No non-specific reaction, no increase in measurement time,
Providing HBs antibody measurement reagent with high sensitivity and wide measurement range
To provide. Other objects and advantages of the present invention are as follows.
It will be clear from the description below. [0004] According to the present inventors, the present invention
The above objects and advantages of the present invention are as follows: (1) Biotinylated HBs anti-
Containing biotin and albumin and biotinylated HBs antigen concentration
A first aqueous solution having a degree of 1 to 100 μg / mL, and
(2) Enzyme-labeled HBs antigen, albumin and fetal calf blood
Containing enzyme and the concentration of enzyme-labeled HBs antigen is 1-10
A combination of a second aqueous solution of 0 μg / mL,
Qualitative or quantitative determination of the presence of HBs antibodies by the Dutch method
Attained by the combination reagents. [0005] BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
However, the substances, production methods and operation methods used in the present invention are limited to the following.
It is not specified. [0006]First aqueous solution The biotinylated HBs antigen in the present invention refers to HBs antigen.
Biotinylated, such as HBs antigen and biotin
By reacting the labeling agent with a known method.
Obtainable. Biotin labeling agents include, for example, Bi
otin-OSu, Biotin-AC_Five-OSu City
It can be obtained as a commercial product. [0007] Generally, HBs antigens are adw, adr, a
It is divided into four subtypes, yw and ayr,
The HBs antigen used in the present invention is any of the subtypes described above.
It may be. The first solution of the present invention comprises such H
Contains biotinylated HBs antigen obtained by biotinylation of Bs antigen
I do. At this time, the antigen concentration is 1 to 100 μg / mL.
You. If the concentration is lower than 1 μg / mL, the upper limit of the measurement range is low.
If the concentration is higher than 100 μg / mL, the
The degree will decrease. The first aqueous solution has at least the above concentration.
Biotinylated HBs antigen and albumin within the range
It is rare. [0008] As albumin contained in the first aqueous solution
Is, for example, bovine serum albumin (hereinafter referred to as BSA),
Goat serum albumin, mouse serum albumin, donkey serum
Albumin, rabbit serum albumin and human serum albumin
Bumin is preferably used. Relatively inexpensive
For this reason, BSA is particularly preferably used. Albu
As the concentration of min, from the viewpoint of the sensitivity improving effect,
0.1 to 10% by weight is preferred. [0009] The first solution of the present invention may be, for example, NaC
1, KCl, Na_TwoHPO_Four, NaH _TwoPO_FourInorganic salts such as
Mouse IgG or mouse that does not react with HBs antigen
Organic substances such as IgG, NaN_Three, Microcide
Preservatives such as I and III can be contained. First water
The pH of the solution is desirably in the range of 5-9. this
Higher or lower than the protein contained in the aqueous solution
Are easily denatured and it is difficult to obtain a specific reaction. [0010]Second aqueous solution The enzyme-labeled HBs antigen in the present invention refers to HBs antigen.
Enzyme-labeled by a known method. Labeling enzyme
As for example, alkaline phosphatase, peroxy
Enzymes, β-glucosidase and the like. Easy to sign
Peru because of its excellent thermal stability
Oxidase is preferably used. H used in the present invention
The Bs antigen may be of any subtype. enzyme
The concentration of labeled HBs antigen is 1 to 100 μg / mL.
More preferably, it is 5 to 100 μg / mL. Lower darkness
Lower the upper limit of the measurement range, higher concentrations
Non-specific reactions have made accurate measurements impossible
I will. As the albumin contained in the second aqueous solution,
Are the same as those listed for the first aqueous solution.
However, because of the relatively low cost, BS
A is preferred. Although the concentration of albumin is particularly limited,
However, from the viewpoint of the sensitivity improvement effect,
It is preferred to use between 0% by weight. In the present invention, the calf contained in the second aqueous solution
Infant serum (hereinafter referred to as FCS) has a concentration of 1 to 50% by weight.
It is effective to use in the range. If the concentration is too low,
The effect of suppressing the reaction is reduced.
You. [0013] Combining the first aqueous solution and the second aqueous solution
To qualify or quantify the presence of HBs antibodies in the subject
At that time, a sandwich method is used. sandwich
The method may include (1) a first aqueous solution and an HBs antibody.
Reaction, and incubate under appropriate conditions.
(2) a compound having affinity for biotin
Contact with the supported solid phase and then do not bind to the solid phase
Of unreacted biotinylated HBs antigen and HBs antibody
(3) further contacting a second aqueous solution with the solid phase;
Removing unbound enzyme-labeled HBs antigen, (4)
The activity of the enzyme in the immune complex pair generated by this reaction was measured.
Qualitative or quantitative determination of HBs antibody
It is. Here, a compound having affinity for biotin
Examples include anti-biotin antibodies, avidin, etc.
You. In addition, a solid that carries a compound having an affinity for biotin
The shape and material of the phase are not particularly limited. Preferred
Suitable shapes include filters, plates, and tubes.
And spherical and spherical shapes. Preferred material
Are, for example, glass, inorganic substances such as magnetic substances, polystyrene,
Organic substances such as fats and celluloses are exemplified. The sandwich
In the batch method, the reaction temperature is preferably 15 to 50 ° C,
Below that, the reaction takes too long, and
Above is not preferable because the protein is modified. [0014] The present invention will be further described with reference to the following examples.
However, the present invention is not limited to these. Embodiment 1 (A) Preparation of reagent (1) Biotinylation of HBs antigen Biotinylation of HBs antigen is performed by Method Enzym.
ol. Vol. 184, 162
went. That is, 10 mM HEPES buffer (pH
8.5) 3 mg / mL HBs antigen (bath) was added to 440 μL.
50 µL and 20 mM biotin label
Agent Biotinamidocaprate-NHS
(Manufactured by Nacalai Tesque, Inc.)
Incubation for biotinylated HBs antigen
Was. (2) Purification of biotinylated HBs antigen To remove unreacted biotin labeling agent,
Biotinylated HBs antigen was replaced with PD-10 (Amercia)
m-Pharmacia). Above bio
0.15 M NaCl in 500 μL of tinned HBs antigen
PH 7.4 20 mM phosphate buffer (hereinafter PB)
S) and apply to PD-10.
did. Subsequently, 3.5 mL of PBS is allowed to flow, and the eluate is collected.
did. (3) Preparation of first aqueous solution The purified biotinylated HBs antigen was added to a final concentration of 40 μl.
g / mL to a final concentration of 20% goat serum.
So that the solution added to PBS becomes the first aqueous solution.
Was. (4) Preparation of second aqueous solution According to the method described in JP-A-11-23574.
Peroxidase (hereinafter referred to as HRP) labeled HB
s antigen so that the final concentration is 30 μg / mL.
S to a final concentration of 15% and BSA
Add the solution added to PBS to a final concentration of 1%.
Two aqueous solutions were obtained. (B) Measurement 50 µL of the test serum and 10 µl of the first aqueous solution prepared in (3)
And incubate at 40 ° C for 5 minutes.
50 μL of the anti-biotin antibody is supported on the solid phase
Into a cup (manufactured by Toyobo Co., Ltd.). Then 1
50 μL of PBS containing% BSA was added, and (4)
10 μL of the second aqueous solution prepared in the above was added. At 40 ° C
After incubation for 3 minutes, 0.05% Tween
Wash with 180 μL of PBS containing en20, followed by
3,5,3 ', 5'-tetramethylbenzidine was added to 30 µm.
L for 5.5 to 7.5 seconds and 40.5 to
650 nm laser reflection intensity for 42.5 seconds
Was measured. The above operation is performed using a commercially available analyzer ID-
1000 (manufactured by Toyobo Co., Ltd.). Displays measurement results
1 is shown. Comparative Example 1 200 μg of biotinylated HBs antigen concentration in the first aqueous solution
/ ML in the same manner as in Example 1.
Table 1 shows the measurement results. Comparative Example 2 0.5 μg of HRP-labeled HBs antigen concentration in the second aqueous solution
/ ML in the same manner as in Example 1.
Table 1 shows the measurement results. Comparative Example 3 Except that the first aqueous solution was adjusted without adding BSA,
Performed in the same manner as in Example 1. Table 1 shows the measurement results. Comparative Example 4 Except that the second aqueous solution was adjusted without adding BSA,
Performed in the same manner as in Example 1. Table 1 shows the measurement results. Comparative Example 5 Except that the adjustment was made without adding FCS to the second aqueous solution,
Performed in the same manner as in Example 1. Table 1 shows the measurement results. From the results in Table 1, it is found that biotin in the first aqueous solution
If the HBsAg antigen concentration is too high, the sensitivity decreases,
Measured when the concentration of HRP-labeled HBs antigen in aqueous solution is too low
There was a tendency for the range to be narrow. Also, BSA or F
In the absence of CS, there was also a tendency for specific responses to increase.
Was done. [0019] [Table 1] [0020] According to the combination reagent of the present invention, non-specific
No adverse reaction, no increase in measurement time, and
High sensitivity and wide range of measurement reagents can be supplied
Was.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Hisahiko Iwamoto             320-11 Hino, Hino-shi, Tokyo             And tea (72) Inventor Yoshinori Yoshimura             320-11 Hino, Hino-shi, Tokyo             And tea

Claims (1)

Claims: 1. Claims: (1) containing a biotinylated HBs antigen and albumin, and having a biotinylated HBs antigen concentration of 1 to 10
A sandwich comprising a combination of a first aqueous solution at 0 μg / mL and (2) a second aqueous solution containing enzyme-labeled HBs antigen, albumin and fetal calf serum and having a concentration of enzyme-labeled HBs antigen of 1-100 μg / mL. Combination reagent for qualitatively or quantitatively determining the presence of HBs antibody by the method.