JPH0617910B2 - Antigen or antibody quantification method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying an antigen or an antibody.

(Prior Art) CRP is a protein that reacts with C polysaccharide to produce a precipitate, and is a non-specific protein component that appears in various inflammatory and tissue-disintegrating diseases. When the above-mentioned disease occurs in the living body, it has a characteristic that the dose is increased within 6 to 24 hours and the dose is reduced with the recovery. Therefore, the test is clinically indispensable. Various other tests are indispensable.

Capillary methods, latex agglutination methods, single immunodiffusion methods, etc. have been used as the conventional inspection methods, but these measurement methods cannot quantitatively grasp the fluctuation of the measured values over time. However, due to problems such as measurement sensitivity, accuracy, and operation time, it is difficult to understand fluctuations in measured values especially in the low range.

In addition, a laser nephelometry method was devised as a method for measuring the low value range by measuring the turbidity generated by the immunonephelometry using a laser nephelometer. This method
Not only is it highly sensitive but the equipment is extremely expensive,
There is a problem in that the pretreatment is complicated because there is a problem in the measurement accuracy and the measurement range and it is necessary to pay a special arbitrary degree to the clarity of the reagent or the sample serum due to its high sensitivity.

However, recently, a method of measuring the turbidity caused by the immunoturbidimetric method using an ordinary automatic biochemical analyzer has become widespread. In other words, it is a method of measuring the degree of turbidity by using transmitted light with a spectrophotometer. Using this method, the usual biochemical items such as GOT, G
Since the amount of various antigens or antibodies can be measured by an analyzer that measures PT, TG, CHO, etc., not only dozens of items can be measured simultaneously with one sample, but also completely automatic analysis is possible.

(Problems to be Solved by the Invention) However, since this method was applied to an analyzer made for biochemical examination, it was not possible to solve the phenomenon in which the calibration curve peculiar to the immune reaction draws a sigmoid curve. That is, since the biochemical automatic analyzer is a two-check amount mechanism that creates a calibration curve with two samples of known concentration, it cannot perform curve correction like an immuno-only device. In addition, since the reaction time is set to be short, the calibration curve draws a curve when the antigen-antibody reaction rate varies depending on the antigen-antibody ratio. In particular, when the concentration of the antigen or antibody is low, the antigen-antibody reaction is slow and the absorbance is measured before the reaction is completed, resulting in a low measured value. For example, in CRP, the reliability of measured values in the low value range is clinically important, but in this method, since the concentration in the low value range is low, there are large variations and a serious problem in diagnosis.

(Means for Solving the Problems) The present invention measures the absorbance (A ₁ ) by mixing a sample containing an antigen or antibody and a first reagent containing the same antigen or antibody as the antigen or antibody. Then, a second reagent containing an antibody or an antigen corresponding to the above-mentioned antigen or antibody is added to cause an antigen-antibody reaction to measure the absorbance (A ₂ ). From this measured value A ₂ to the measured value A ₁ and The present invention relates to a method for quantifying an antigen or antibody, which comprises quantifying the antigen or antibody in the sample from a value obtained by subtracting the absorbance due to a reagent (hereinafter, referred to as a calculated absorption value).

In the present invention, even a sample containing an antigen or antibody only at a concentration at which the antigen-antibody reaction does not proceed sufficiently,
By mixing with the first reagent containing the same antigen or antibody as the antigen or antibody, the concentration of the antigen or antibody as a whole can be increased to a concentration at which the antigen-antibody reaction sufficiently proceeds, and By calculating the calculated absorption value, it is possible to quantify with high sensitivity.

In the present invention, the sample and the first reagent are preferably mixed at 20 to 40 ° C. After the mixing, the absorbance (A ₁ ) may be measured at any time, but it is preferable to measure the absorbance within ₁ to 15 minutes after the mixing.
It may be measured immediately before the addition of the reagent. The concentration of the antigen or antibody component contained in the first reagent after mixing is preferably adjusted so that the antibody becomes excessive when the second reagent is added. The concentration after mixing varies depending on the antigen or antibody and cannot be generally stated. For example, for C-reactive protein (hereinafter abbreviated as CRP) that is an antigen, the concentration is about 0.
01 mg / d to about 20 mg / d is preferred, especially about 0.05 mg / d
~ About 4 mg / d is preferred.

After measuring the absorbance (A ₁ ), the second reagent is added to cause an antigen-antibody reaction, and this reaction is preferably performed at 20 to 40 ° C. The absorbance (A ₂ ) is preferably measured 1 minute or more after the addition of the second reagent, and particularly preferably in the range of 3 to 15 minutes. When the second reagent is added, it is preferable that the amount of the antibody is adjusted to be excessive in the total volume. When the second reagent is added, the concentration of the antibody in the total volume varies depending on the antibody and cannot be generally stated. For example, in the case of an anti-CRP antibody, it is about 3 × 10 ⁻³ mg / d to 1 mg / d. It is preferably about 0.02 mg / d to 0.2 mg / d.

In the present invention, the absorbance due to the reagent means, for example,
In the same manner as measuring the absorbances A ₁ and A ₂ by using water, buffer solution or physiological saline instead of the above sample, the absorbance A ₁
And in response to A _2, respectively, the absorbance was measured A ₁ 'and A _2', which is a value obtained by subtracting the 'A _{1 from' A 2 (A 2 '-A} 1').

The calculated absorption value A is calculated from the absorbances A ₁ , A ₂ , A ₁ ′ and A ₂ ′ according to the formula A = A ₂ −A ₁ − (A ₂ ′ −A ₁ ′) (I).

On the other hand, as the above-mentioned sample, using a dilution series of a standard sample (sample containing a known amount of antigen or antibody), the calculated absorption value is obtained in the same manner as described above, and the calculated absorption value and the amount of the antigen or antibody (unit: For example, prepare a calibration curve of mg / d), obtain the calculated absorbance value as described above for a sample containing an unknown amount of antigen or antibody, and calculate the amount of the antigen or antibody corresponding to this value from the calibration curve. By determining, the amount of the antigen or antibody in the sample containing an unknown amount of the antigen or antibody can be quantified.

The amount of antigen or antibody in a sample containing an unknown amount of antigen or antibody is (In the formula, A is the calculated absorption value of a sample containing an unknown amount of antigen or antibody, and Cs and As are the amount and calculated absorption value of the antigen or antibody of a sample containing a known amount of each antigen or antibody. ), It is possible to quantify by determining C.

For A ₁ and A ₁ ′, the liquid volume is corrected as appropriate,
The sensitivity can be further increased. For example, for A ₁ , (total volume of sample and first reagent) / (total volume of sample, first reagent and second reagent), and for A ₁ ′, (water,
Buffer or physiological saline and total volume of first reagent) / (water, total volume of buffer or physiological saline, first reagent and second reagent)
Are summed up respectively.

In the present invention, examples of the sample include serum and urine, and examples of the antigen to be quantified include CRP and
There are transferrin, complement proteins C ₃ and C _{4, and the} like, and antibodies to be quantified include IgG, IgM, IgA, and the like. Further, in the above description, the wavelength for measuring the absorbance is arbitrary as long as it is not substantially impermeable to water, physiological saline, or the sample, but normally it may be selected from the range of 200 to 1,000 nm.

Next, the quantification reagent used in the quantification method of the present invention will be described. As the quantification reagent, a first reagent containing an antigen or an antibody and a second reagent containing an antibody corresponding to the antigen or the antibody or an antigen are used in combination.

The first reagent and the second reagent are usually used as a buffer solution. The buffer solution used at this time is phosphate (combination of monohydrogen phosphate and dihydrogen phosphate, and Na salt as the salt).
Salt, K salt, etc.), a combination of tris (hydroxymethyl) aminomethane and hydrochloric acid, an aqueous solution of a good buffer, etc., and the pH is preferably adjusted to 5-9.

The first reagent may contain a reaction accelerator, and polyethylene glycol having a molecular weight of 5,000 to 10,000 is particularly preferable. The reaction promoter is contained in the first reagent at a concentration of 0 to 10
The content is preferably in the range of wt%, particularly preferably 2 to 5 wt%. If the amount of the reaction accelerator is too much, nonspecific reaction is likely to proceed in addition to promotion of the antigen-antibody reaction. An appropriate amount of reaction promoter is selected depending on the type and properties of the antigen or antibody.

The first reagent may further contain a surfactant. Examples of the surfactant include nonionic surfactants such as polyoxyethylene nonylphenyl ether and polyoxyethylene sorbitan fatty acid ester, or polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl ether sulfuric acid and salts thereof. Of anionic surfactants are used. The surfactant is effective in removing the influence of turbidity of the sample and avoiding the influence of bubbles in the reagent and improving the accuracy of data. The concentration of the surfactant in the first reagent is in the range of 0 to 5% by weight, preferably 0.01 to 5%, particularly preferably 0.05 to 1.0%. When the amount of the surfactant is too large, the antigen-antibody reaction is hindered or the viscosity is too high and the analyzer is easily damaged.

The concentration of the antigen or the antibody in the first reagent is appropriately adjusted and used so that the antibody tends to become excessive at the start of the antigen-antibody reaction. For example, in the case of CRP, 0.01 to 20 mg / d is preferable, and 0.05 to 4 mg / d is particularly preferable. Generally, if the amount of antigen or antibody is too small, the calibration curve is unlikely to be linear in the low range, while if the amount of antigen is too large, the linear range of the calibration curve at high concentration tends to be narrow.

The antigen or antibody used here may be any one that substantially reacts with the corresponding antibody or antigen,
For example, in the case of CRP, human-derived ones are preferable, but animal-derived ones may be used as long as they react with an anti-CRP antibody. This also applies to the antibody or antigen contained in the second reagent. For example, as an anti-CRP antibody,
A γ-globulin fraction obtained from anti-human CRP goat serum, anti-human CRP rabbit serum, etc. can be used.

The concentration of the antibody or the antigen in the second reagent is appropriately determined at the time of starting the antigen-antibody reaction so that the antibody tends to be excessive.
For example, the anti-CRP antibody is preferably 0.02 to 5 mg / d,
0.1-1 mg / d is particularly preferred.

The first reagent and the second reagent may contain sodium chloride, potassium chloride or the like for adjusting the ionic strength. The concentration is preferably 0 to 1M, more preferably 0.01 to 1M, most preferably 0.05 to 0.2M. Since the reaction mode of the antigen-antibody reaction differs depending on the ionic strength, the ionic strength must not be too high or too low.

A preservative such as sodium azide may be added to the first reagent and the second reagent, and 0 to 1% is preferable in each reagent.

(Example) Next, the Example of this invention is shown.

The reagents and CRP standard serum used below are as follows.

(1) First reagent composition (pH 7.60) Sodium chloride 0.1M Polyethylene glycol 3.0% 6000 Polyoxyethylene octyl 0.3% Luphenyl ether Tris-HCl buffer 15 mM Sodium azide 0.1% CRP 1.0 mg / (2) Second Reagent composition (pH 7.60) Sodium chloride 0.1M Tris-HCl buffer 15 mM Sodium azide 0.1% Anti-human CRP antibody goat serum 0.5 mgAg / m (anti-human CRP antibody content) (2) CRP standard serum Human serum-derived CRP concentration 5.0 mg / d Example 1 The CRP standard serum was diluted with physiological saline to prepare CR.
A 10-step dilution series was prepared from P concentration of 0.5 mg / d to 5.0 mg / d at 0.5 mg / d intervals and used as a sample. Each sample was quantified as follows and a calibration curve was created.

The analysis was performed using a Hitachi 705 type automatic analyzer under the following analysis conditions. That is, 10 μm of the sample is added (injected) to 500 μm of the first reagent, and pre-incubated at 37 ° C. for 4 minutes and 20 seconds to 4 minutes and 40 seconds, and the main wavelength is 340n.
Absorbance (Ax ₁ ) was measured at m (sub wavelength 700 nm). After 5 minutes, 100 µ of the second reagent was added (injected) to start the reaction. After 10 minutes, the absorbance (Ax ₂ ; turbidity) was measured at the same wavelength as above. Using physiological saline instead of the above sample under the same analytical conditions, the absorbances Ax ₁ and A
Ab ₁ and Ab ₂ were measured corresponding to x ₂ . The calculated absorption value was calculated from Ax ₁ , Ax ₂ , Ab ₁ and Ab ₂ by the formula A = Ax ₂ −Ax ₁ − (Ab ₂ −Ab ₁ ), and a calibration curve was prepared. The result is shown as graph 1 in FIG.

Comparative Example 1 A calibration curve was prepared in the same manner as in Example 1 except that CRP was omitted from the first reagent in Example 1. This is the first
Shown as graph 2 in the figure.

Example 2 The CRP standard serum was diluted with physiological saline to prepare CR.
P concentration is 2mg / d or 10mg at 2mg / d intervals up to 20mg / d
A serial dilution series was prepared and used as a sample. A calibration curve was prepared in the same manner as in Example 1 except that these samples were used. The result is shown as graph 3 in FIG.

Comparative Example 2 A calibration curve was prepared in the same manner as in Comparative Example 1 except that the dilution series of Example 2 was used as the dilution series. The result is shown as graph 4 in FIG.

Example 3 Using 95 human serum samples as samples, the correlation between the present invention and the SRID method (using My Plate CRP manufactured by Eiken Chemical Co., Ltd.) was determined.

Under the same conditions as in Example 1, physiological saline was used instead of the sample, and CRP standard serum (CRP concentration C _s : 50 mg / d) was used as the sample.
The calculated absorption values A _B and A _S were obtained using

Further, the calculated absorption value A _x was obtained in the same manner as in Example 1 using each of the above-mentioned samples as a sample.

From A _x , A _B and A _S , CRP concentration of each sample C _x (mg / d)
Is the expression Decided by.

FIG. 3 shows the correlation between this result and the result of the SRID method for the same specimen.

Comparative Example 3 In the same manner as in Example 3, except that 64 samples of human serum were used as samples and CRP in the first reagent was removed, S
The correlation with the result of the RID method was obtained, and the result is shown in FIG.

As is apparent from FIGS. 1 and 2, according to the present invention,
The calibration curve shows excellent linearity even in the low concentration range, including the low concentration range. Therefore, if a calculated absorption value is obtained for one sample containing a known amount of antigen or antibody when creating a calibration curve,
It can be seen that a calibration curve can be created.

Further, by comparing FIG. 3 and FIG. 4, it can be seen that according to the present invention, the sample marked with (−) and the sample marked with (±) by the SRID method can be clearly distinguished.

(Effect of the Invention) According to the method of the present invention, the antigen or antibody can be quantified accurately and easily, including in the low concentration range.

[Brief description of drawings]

FIG. 1 shows the calibration curves obtained in Example 1 and Comparative Example 1,
The figure shows the calibration curves obtained in Example 2 and Comparative Example 2, FIG. 3 shows the correlation between the present invention and the SRID method in Example 3, and FIG. 4 shows the correlation between the comparative method and SRID method in Comparative Example 3. Show the relationship. DESCRIPTION OF SYMBOLS 1 ... Calibration curve obtained in Example 2 ... Comparative Example 1 〃 3… Example 2 〃 4… Comparative Example 2 〃

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Masaaki Mukai, 4-13-1, Higashi-cho, Hitachi, Ibaraki Prefecture, Hitachi, Ltd., Yamazaki Plant, Hitachi Chemical Co., Ltd. (72) Hiromi Iijima 4-13, Higashi-machi, Hitachi, Ibaraki No. 1 in Ibaraki Laboratory, Hitachi Chemical Co., Ltd. (56) Reference JP-A-60-100764 (JP, A)

Claims (1)

[Claims] 1. A sample containing an antigen or an antibody and a first reagent containing the same antigen or antibody as the antigen or the antibody are mixed to measure the absorbance (A ₁ ), and then the above-mentioned antigen or antibody is prepared. antibody or by adding a second reagent containing an antigen antigen - measured absorbance by antibodies reacting (a _2), this measured value a ₂
A method for quantifying an antigen or antibody, which comprises quantifying the antigen or antibody in the sample from a value obtained by subtracting the measured value A ₁ and the absorbance due to the reagent from