JPS62159047A - Reagent for quantitative determination of plasma protein - Google Patents

Abstract

PURPOSE:To make quantitative determination of plasma protein with high accuracy by a reagent which contains 2-7wt% polyethylene glycol, 0.01-0.4wt% phosphoric acid buffer soln. and is so adjusted as to have 11.0-13ms/cm specific conductivity. CONSTITUTION:The turbidity (absorbancy) of the antigen-antibody complex generated by reaction with a specific anti-serum by the protein component to be quantitatively determined exhibits an extremely exact linear relation over a wide range of the concn. thereof and passes the origin (the point where the concn. of the protein to be quantitatively determined and the absorbancy is zero) when the straight line is interpolated. A calibration curve having high accuracy is easily obtd. simply by measuring the turbidity with the only standard serum of the known concn. in the quantitative determination operation using the reagent which contains 2-7wt% polyethylene glycol, 0.01-0.4wt% polyoxyethylene nonionic surface active agent and 0.002-0.01M phosphoric acid buffer soln. and is so adjusted as to have 11.0-13.0ms/cm specific conductivity. The quantitative determination of the specimen of an unknown concn. is made possible in accordance with such calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to improvements in reagents used to quantify various proteins in plasma using immunoturbidimetric phenomena.

Conventional techniques and their problems To quantify proteins in plasma such as immunoglobulins, single immunodiffusion method (from SR) and laser nephelometric method have been used. Immunoturbidimetry is widely used because of its rapidity.

In immunoturbidimetry, immunoglobulin, etc. in the sample (serum) reacts with the corresponding specific antiserum, producing a turbidity of the antigen-antibody complex.This turbidity depends on the amount of antigen, so this is measured with a spectrophotometer. The immunoglobulin etc. are quantified by this method. In such measurements, a sample of unknown concentration is quantified based on a calibration curve prepared from standard serum of known concentration. Attempts are being made to increase the detection sensitivity of turbidity and improve the precision and applicable range of calibration curves.

For example, in Japanese Patent Publication No. 60-4938, polyethylene glycol/v (1% by weight) and nonionic surfactant (
Specifically, oxyethylene-oxypropylene copolymer or oxyethylene-oxypropylene copolymer added to ethylene diamine) was added at a relatively high concentration (
By using a reagent present at 3% by weight, ■g
Concentration of G antibody - cloudy! It is stated that the relationship between ll (expressed in % light scattering) exhibits linearity over a wide range. However, according to the specification, the concentration range of IgG that can be measured based on such linearity is at most several hundred mg/dJ, and the relationship between IgG and turbidity is not a strict linear relationship. Therefore, there is still room for improvement in the quantitative accuracy of the analyte. This special public service 1986-493
In the method described in No. 8, there is no particular mention of the buffer solution to be added to the reagent. - Furthermore, in JP-A-58-211659, polyethylene glycol and a nonionic surfactant (Tween 20
) and HEPES (N
A method for quantifying CRP (C-reactive protein) in serum by immunoturbidimetry using a reagent solution in which 2-hydroquinlethylhiberazine-N-2-ethanesulfonic acid is present is described. According to the description in this specification, the relationship between CRP concentration and turbidity (absorbance) shows relatively good linearity.

However, looking at the results obtained with this method,
The straight line connecting the absorbance values for each concentration does not pass through the origin, and in fact, this has been confirmed by additional trials of the method by the present inventor. This fact means that when creating a calibration curve, it is necessary to measure the turbidity of at least several standard sera with known concentrations, which increases the complexity of the operation.

OBJECTS, STRUCTURES, AND EFFECTS OF THE INVENTION The present invention aims to eliminate the drawbacks of conventional reagents for quantifying serum components by immunoturbidimetry, and to provide a simpler and more accurate reagent.

The present inventor has discovered that when measuring the turbidity of antigen-antibody complexes in order to quantify plasma protein components based on the principle described above, serum Concentration of protein components and related turbidity (
The inventors have found conditions under which the absorbance (absorbance) exhibits excellent linearity and such a straight line passes through the origin, leading to the present invention. That is, the present invention provides polyethylene glycol
/L/2 to 7% by weight, polyoxyethylene nonionic surfactant 0.01 to 0.4% by weight, and phosphate buffer 0.
Contains 002-0.0IM, specific conductivity 11.0-13
．． The present invention provides a reagent for quantifying plasma protein by immunoturbidimetry, which is prepared and used so that the reading time is 0.0 ms. Here, a particularly suitable surfactant for use in the present invention is polyoxyethylene sorbitan monoalkylate.

When the reagent of the present invention having the above configuration is used, the turbidity (absorbance) of the antigen-antibody complex generated when the protein component to be quantified reacts with the corresponding specific antiserum can be extremely accurately measured over a wide range of concentrations. shows a linear relationship, and when the resulting straight line is interpolated, it passes through the origin (ie, the point where the concentration and absorbance of the protein to be quantified are zero).

Therefore, in quantitative operations using the reagent of the present invention, a highly accurate calibration curve can be easily obtained by simply measuring the turbidity of only one standard serum with a known concentration, and based on this calibration curve, a sample with an unknown concentration can be determined. Since quantitative measurements can be performed, the operation is extremely simple and the measurement time is shortened.

The reagent of the present invention is characterized in that it contains a significantly smaller amount of nonionic surfactant than the conventional reagent described in Japanese Patent Publication No. 60-4938 mentioned above. That is, according to the present invention, the amount of polyoxyethylene nonionic surfactant is adjusted to 0.01 to 0.4% by weight at the time of use.

The present inventors have discovered that among polyquinethylene nonionic surfactants, polyoxysorbitan monoalkylates such as polyoxysorbitan monooleate and polyoxysorbitan monolaurate are particularly suitable. These polyoxyethylene sorbitan monoalkylates can be sold under the trade names Tween (Katayama Chemical) and Rheodol TW (Kao Soap), for example. Thus, such nonionic surfactants can be combined with polyethylene glycol (preferably molecular weight i,
ooo ~20,000 polyethylene glycol: I-A
/) 2 to 7% by weight, an excellent linear relationship between plasma protein concentration and absorbance can be obtained.

Another feature of the present invention is that a phosphate buffer is used as the pH adjusting buffer. This phosphate buffer contains sodium,
Potassium phosphate contains gold, and a preferred example is hydrogen phosphate=sodium 14.5 per 10 yen.
g, phosphoric acid=potassium hydrogen 1g 1 potassium chloride Igt-containing. Such a phosphate buffer is used in a relatively small amount, that is, at a concentration of 0.002M to 0.01M, to 1) Ht-6 to 8 (preferably 7.
3 to 7.6). The present inventor discovered that when the concentration of the phosphate buffer exceeds the above range, the relationship between the concentration of the protein to be measured and its turbidity (absorbance) is no longer linear, and the line connecting the absorbance values for each concentration does not reach the origin. We have found that it will not pass.

Furthermore, the present inventor has discovered that in order for the relationship between the concentration and turbidity (absorbance) of the plasma protein to be quantified to exhibit linearity that passes through the origin, the electrical conductivity of the system is also involved; That is, it has also been found that it should be adjusted to 11.0 to 13.0 mS/crn. Such adjustment of the specific conductivity is achieved by adjusting the amount of the above-mentioned phosphate buffer added and also adjusting the amount of sodium chloride (NaCJ-). To make the specific conductivity within the above range,
Sodium chloride in relatively small amounts, typically 0.6
It is used at a concentration of ~0.9% by weight.

The detailed reason why the relationship between liquid quantitative plasma protein concentration and turbidity (absorbance) exhibits good linearity passing through the origin by using the reagent of the present invention is not understood. However, when measuring the turbidity of an antigen-antibody complex, the presence of a reagent component having the above-mentioned characteristics causes the complex to be formed uniformly and sequentially in proportion to the amount of antigen, thereby promoting turbidity. This is thought to be because there is no such thing as Thus, when using the reagent of the present invention, when the absorbance of a standard serum of known concentration (Cs) is measured against a blank, E (s
), then if the absorbance of a sample at an unknown concentration is E (A) with respect to the blank, then its concentration (C) can be easily determined from the following equation.

By using the reagent according to the present invention, various plasma proteins can be quantified over a wide range with high accuracy. The quantifiable range is 100 to 3.000 mg/dJL,
Engineering gA 50-500mg/dj! , Engineering gM20-30
0mg/di, CRP O, 5-2omg/df, aI
AG (a1 acidic mucopolysaccharide) 10-3o.

mg/if, Ca (third complement component) 10-300mg
/d1 Oyohi C4 (4th complement component) 5-1oomg/d
JL〇 Note that other substances known in the art can also be added to the reagent of the present invention as long as they do not impair the properties described above. For example, NaN3 can be added to preserve the reagents.

EXAMPLES Hereinafter, in order to further clarify the characteristics of the present invention, the present invention will be described with reference to Examples.

Example 1 A calibration curve was created to measure the concentration of engineered ggG antibody, IgA antibody, and engineered gM antibody in serum. 2.5 mf of diluted solution (physiological saline) was processed into 0.1 mJ' of serum of known concentration for those antibodies, diluted standard solution (26-fold diluted solution)
And so. Put 2.0ml of antiserum into a test tube, add the above diluted standard solution (■gG-containing standard solution is 0.01ml,
The standard solution contained is 0.05mJ! The standard solution containing -1 gM is o
, 1 mf) and immediately mix well. The antiserum was Na CJ! , NaNa, phosphoric acid '118 H(t
Hydrogen phosphate = sodium 14.5g per Okg, phosphoric acid =
Contains 1 g of potassium hydrogen and 1 g of potassium chloride), PEG (polyethylene+) with a molecular weight of 6000: 2-/L
/ ), Tween 80 (polyoxyethylene sorbicun monooleate manufactured by Katayama Chemical Co., Ltd.) was added to an aqueous solution containing anti-gG and anti-hyperglycium) ■gA, respectively.

It was prepared so that it had the following concentration and physical properties when mixed with the diluted standard solution by adding anti- and Toe gM'&:
NaCJ-0.65% by weight, phosphoric acid weak solution 0.005M,
PEG5.9% by weight, 'rweeno, 01% by weight
, NaN 30.1% by weight, pH 7.5, specific conductivity 12.
24m5/m.

After mixing, the mixture was heated at 37°C for 30 minutes, returned to room temperature, and within 2 hours, the absorbance at 340 nm (◯, D) was measured using a spectrophotometer using a blank as a control. The results are shown in FIG. As shown in Figure 1,
G, GA, and IgM all exhibit good linearity, and the straight line passes through the origin. Therefore, it is understood that when quantifying an actual specimen, a highly accurate calibration curve can be obtained simply by measuring the absorbance of only one standard serum with a known concentration.

Example 2 The same operation as in Example 1 was repeated using polyoxyethylene monolaurate as the nonionic surfactant,
The relationship between concentration and absorbance of IgG antibody was investigated. The result is shown in FIG. 2, and good linearity is also obtained in this case.

Example 3 Example 1 was carried out by changing the amount of nonionic surfactant polyoxyethylene monooleate (Tween 80).
The same operation as above was repeated to examine the relationship between the concentration and absorbance of the engineered gG antibody. The results are shown in FIG. As can be understood from the figure, when the amount of nonionic surfactant is small (0.005% by weight in the figure), the sensitivity (absorbance) gradually decreases, while on the other hand, when the amount of nonionic surfactant is large (0.005% by weight) In the figure, 0.
5% by weight), the sensitivity tends to rise and deviate from a straight line as the concentration increases. According to the present invention, when the amount of nonionic surfactant is within the appropriate range (0.01% and 0.3% by weight in the figure), the relationship between antibody concentration and sensitivity (0, D, ) is a good straight line. exhibiting gender.

Example 4 The same operation as in Example 1 was repeated while changing the amount of phosphate buffer, and the relationship between IgM antibody concentration and absorbance was investigated. The results are shown in FIG. .

As can be seen from the figure, if there is too much phosphate buffer,
The line (calibration curve) indicating the relationship between antibody concentration and absorbance tends to deviate in the + direction of the absorbance axis without passing through the origin.

Example 5 The same operation as in Example 1 was repeated to create a calibration curve for quantifying C4 (complement 4 component). However, the reagent used, when mixed with the diluted standard solution, was
a (JO, 65% by weight (excluding that in serum diluent),
NaNa: 0.1% by weight, molecular weight 600017)
PEG 4.5% by weight, nonionic surfactant (Tween
80) 0.25% by weight, pH 7.4, and specific conductivity 12.1 ms 7 cm. The results are shown in Figure 5.9 By using the reagent according to the present invention, the C4 concentration-absorbance exhibits linearity;
A good calibration curve can be obtained in which the straight line passes through the origin.

[Brief explanation of drawings]

FIG. 1 shows the results of engineering gG, I
It is a graph showing the relationship between concentration and spectroscopic intensity of gM meter and engineered gA antibody. FIG. 2 is a graph showing the relationship between the concentration of engineered GG antibody and the spectroscopic intensity when using the reagent according to the present invention. FIG. 3 is a graph showing the relationship between the concentration of engineered GG antibody and the spectroscopic intensity when the amount of nonionic surfactant is changed. FIG. 4 is a graph showing the relationship between the concentration of the engineered gM antibody and the spectroscopic intensity when the concentration of the buffer solution was completely changed. FIG. 5 is a graph showing the relationship between the concentration of the fourth complement component and the spectral intensity when using the reagent according to the present invention. Figure 1 0, D. mg/di Figure 2 Figure 3 rrK! /dI Figure 4 Figure 5 0, D.

Claims (2)

[Claims] (1) A reagent used to quantify plasma proteins by immunoturbidimetry, comprising 2 to 7% by weight of polyethylene glycol.
, polyoxyethylene nonionic surfactant 0.01~
0.4% by weight and phosphate buffer 0.002-0.01M
1. A reagent which is prepared and used so as to have a specific conductivity of 10.0 to 13 ms/cm. (2) Claim No. 1 in which the nonionic surfactant is polyoxyethylene sorbitan monoalkylate.
Reagents listed in section.