JPS63206657A - Quantitative analysis of protein - Google Patents

Abstract

PURPOSE:To measure protein in a concn. region from low concn. to high concn., by using a bio-specimen as it is without dilution and using one or more kinds of a monoclonal antibody and a polyclonal antibody as the antibodies to protein to be measured in combination. CONSTITUTION:When one or more kinds of a monoclonal antibody and a polyclonal antibody are appropriately combined in use, it is unnecessary to dilute a specimen and proper turbidity is obtained from low concn. to high concn. without using a large amount of a reaction promoter. Therefore, the amount of the reaction promoter is sufficient within a range of 1-4% based on the amount thereof used in a usual dilution PCA method (measuring method for preliminarily diluting a specimen serum before measurement). Further, the use amount of the polyclonal antibody can be reduced to 1/5-1/10 as compared with a direct PCA method (method for measuring immunoglobulin using only the polyclonal antibody without preliminarily diluting a specimen) and turbidity is not also too intensive like the direct PCA method and can be properly adjusted.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention effectively measures proteins in biological samples, particularly human body fluids such as serum, plasma, and urine, by an immunoassay method that utilizes antigen-antibody reactions. Regarding how to.

[Prior Art] Measuring the content of various proteins in body fluids provides important clues in diagnosing divine diseases and investigating the course of treatment. There have been remarkable advances in methods for measuring proteins in body fluids in recent years, and many measurement methods have been developed, including the 5RID method, laser nephelometric method, turbidimetry, RIA method, EIA method, and latex agglutination method. and has been put into practical use.

Particularly recently, methods have been developed to perform this in a short time using laser nephelometers and automatic analyzers developed for multi-item biochemical measurements, and these methods are becoming popular.

However, since the antibodies used in these measurements are usually polyclonal antibodies obtained by immunizing animals, the degree of agglutination due to the antigen-antibody reaction is high and the degree of turbidity is too strong. It was necessary to dilute the sample by ~30 times, and in some cases by 100 to 300 times (hereinafter, this method will be abbreviated as the dilution PCA method).

).

An example of measuring immunoglobulin using only polyclonal antibodies without diluting the sample in advance (hereinafter, this method will be described directly)
It is abbreviated as the CA method. ), but in this case there are restrictions such as restrictions on the amount of sample sampled and limitations on the wavelength to be measured. For example, taking immunoglobulin G (IgG), the sample (serum) sampling volume is 3 to 54, and the turbidity after the reaction is strong, so measurements must be performed at a long wavelength of 600 nm or more. becomes. Furthermore, the polyclonal antibody Firefly used in the measurement requires about 10 times the amount of antibody per sample compared to the conventional diluted PCA method, which is about 1 mg per sample, which increases the cost per sample and poses an economic problem.

On the other hand, as an example of measuring immunoglobulin using only monoclonal antibodies, there is a well-known document Clinical Chemistry Vol. 27, 2044-2.
047 (1981) describes a method for quantifying immunoglobulins using monoclonal antibodies. However, in summary, what is stated here is that monoclonal antibodies do not cause turbidity due to antigen-antibody reactions when used alone, and can only be applied to measurements when multiple monoclonal antibodies are used in combination. Therefore, we are simply considering the use of monoclonal antibodies as a replacement for polyclonal antibodies.
It is not intended to bring out new effects by actively using this. Therefore, as a matter of course, the sample in this case is diluted as usual.

In contrast to these conventional techniques, some of the present inventors have developed a method for measuring immunoglobulin in serum using only monoclonal antibodies and without diluting the specimen in advance (hereinafter abbreviated as direct MCA method). They discovered it earlier and filed a patent application (Japanese Patent Application Laid-Open No. 60-237363). This method is
This is a method of measuring a sample (serum) without diluting it by using monoclonal antibodies, which react with antigens and produce turbidity even when used alone, or in combination of two or more, and is a method that can be used to measure samples (serum) without diluting them. This is an excellent method that solves the problems of the law all at once.

However, this method, which uses only monoclonal antibodies, requires the use of high concentrations of aggregation promoters (polyethylene glycol, polysaccharides, etc.) in order to agglutinate the antigen-antibody complex. There was also a risk that some of the components would cause non-specific aggregation due to the reaction accelerator, which would affect the measured values, so there was still room for improvement. In addition, this method can be used for short-time reactions (reaction time 2.5 to 10
When applied to an automatic biochemistry analyzer that is designed to perform measurements in minutes), the aggregation of antigen-antibody complexes at low concentrations does not result in light minutes within the reaction time, and accurate measurement values can be obtained. It has been newly revealed that there are cases where appropriate turbidity is not formed, causing problems with measurement accuracy in low concentration ranges.

[Means for Solving the Problems] In order to solve the problems, the present invention has the following configuration.

[In a method for quantifying proteins in a biological sample by irradiating light onto an antigen-antibody complex generated by an antigen-antibody reaction and measuring its optical change, the biological sample is used as it is without dilution, 1. A method for quantifying a protein in a biological sample, which is carried out using a combination of one or more monoclonal antibodies against the protein and a polyclonal antibody against the protein as antibodies against the protein to be measured. In other words, as a result of intensive research to solve the above problems, the present inventors unexpectedly found that by using monoclonal antibodies (MCA) and polyclonal antibodies (PCA) in appropriate combinations, it was possible to use only PCA or only MCA. Problems when assembling a measurement method using the direct PCA method: A large amount of antibody is required in the case of the direct PCA method, and the turbidity formed is also higher than necessary, making it necessary to minimize the amount of sample sampled during measurement (3-44). In addition, in the case of the direct MCA method, the purpose of promoting aggregation is that the degree of agglutination of the antigen-antibody complex is low in a short time. A large amount of reaction accelerator must be used in this method, and because a large amount of reaction accelerator is used, some blood components may cause non-specific aggregation, which may affect the measured value. Furthermore, when measuring directly using the MCA method, there are many problems that could not be solved with conventional techniques, such as the fact that the measured particle size in the low concentration range decreases due to insufficient turbidity formation at low concentrations. The inventors have discovered that these problems can be solved all at once, and have completed the present invention.

If one or more monoclonal antibodies and polyclonal antibodies are used in appropriate combination according to the method of the present invention, there is no need to dilute the sample, and there is no need to use a large amount of reaction accelerator. Appropriate turbidity can be obtained up to the concentration. Therefore, the amount of reaction accelerator is usually diluted with PCA.
The range of 1 to 4% used in the method is sufficient and direct M
The concentration can be significantly reduced compared to the 5 to 10% concentration used in the CA method. As a result, it was possible to avoid non-specific agglutination of some of the blood components and affect the measured values, making it possible to perform accurate measurements. Furthermore, the amount of polyclonal antibody used can be reduced to 175 to 1/10 of that in the case of the direct PCA method, improving economic efficiency compared to the direct PCA method, and the turbidity is also less strong than the direct PCA method. It is possible to adjust the turbidity to the appropriate level without overdoing it.
The measurement wavelength is 340, which is commonly used in automatic biochemical analyzers.
Any wavelength of ~700 nm can be selected, and the sample sampling amount is 5, which is commonly used in automatic biochemical analyzers.
~10IL! It is now possible to measure

As described above, the method of the present invention does not require pre-dilution of proteins in body fluids, nor is it subject to any specific restrictions on the amount of sample or measurement wavelength. It has become possible to quickly and accurately measure from low to high concentrations.

Specific measuring means for quantifying protein in body fluids by the method of the present invention include optical measuring methods such as nephelometry and nephelometric method. When measuring by turbidimetry, existing automatic analyzers and spectrophotometers can be used, and the wavelength is preferably in the range of 340 to 800 nm, but is not particularly limited. Furthermore, when measuring by the Hiroki method, one using a currently popular laser light source can be used, but the present invention is not particularly limited by the light source and detection method.

As for the buffer used in the measurement, all buffers that are normally used in measurement methods that utilize antigen-antibody reactions, such as Tris buffer, phosphate buffer, veronal buffer, and boric acid M buffer, can be used, and the pH is adjusted to match the antigen. Although it is not particularly limited as long as it does not inhibit the antibody reaction, a range of 6.0 to 9.0 is usually preferably used.

The monoclonal antibodies used in the present invention can be prepared using conventional methods, namely, Keller, Milstein (G., and C, M.
ilstein:Nature, 256.495(1
According to the cell fusion method established by (975)), cells from a mouse tumor line are fused with mouse splenocytes that have been previously immunized with the human protein to be measured to create a monoclonal hybridoma, and the hybridoma is then used to produce monoclonal hybridomas. The monoclonal antibody to be used may be collected by a conventional method.

The type and number of monoclonal antibodies used in the quantitative method of the present invention are not limited, and one or more types may be appropriately selected and used depending on the required sensitivity. Further, the type (type of animal), origin, etc. of the polyclonal antibody used in combination with the monoclonal antibody are not particularly limited, and it is sufficient to use them.

The amount of monoclonal antibody used is not particularly limited, but 1 rn of antibody solution is usually used as a measurement reagent! It is used in a concentration range of 0.05 to 5 mg per serving.

The amount of polyclonal antibody used is not particularly limited, but usually 1 ml of antibody solution! Medium hit 0.01~
It is used in a concentration range of 0.8 mg, and the ratio of the combination of monoclonal antibody and polyclonal antibody is usually 1 monoclonal antibody to 0.02 to 0.02 mg of polyclonal antibody.
A ratio of 0.4 is used.

Examples of proteins in human body fluids that can be measured by the method of the present invention include immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), and complement C.
3, transferrin, haptoglobin, α1-antitrypsin, albumin, apolipoprotein AI, apolipoprotein AU, α2-macroglobulin, and the like.

The present invention will be explained in more detail with reference to Reference Examples and Examples below, but the present invention is not limited by these Reference Examples and Examples.

[Example] Reference example 1. Preparation of monoclonal anti-1gG antibody and hybridoma producing it +1) O in which immunized human IgG100p was dissolved,] 55M sodium chloride solution 01rnl and Freund's complete adjuvant 0.1rrI! were mixed to form an emulsion antigen solution, and 0.2rrLl of the emulsion was administered intraperitoneally to a BALB/c mouse (female, 6 weeks old). After 4 weeks, human IgG100p9 was added to 0. ]55M sodium chloride solution 0
．． 2rn and injected into the tail vein.

(2) Cell fusion Three days after the final immunization, the spleen of the immunized mouse was removed and
Loosen the tile lymphocytes in a plastic petri dish containing 1 m of RPMI-1640 medium, and centrifuge the tile lymphocytes (1000 rpm, 10 minutes) to turn them green.
Washed three times with 640 medium. Tile lymphocytes I x 10'
cells and mouse myeloma cells P3-NSI-11XIO' were mixed in a test tube and precipitated by centrifugation. After removing the supernatant by suction, the precipitate was slightly loosened. 50% polyethylene glycol (average molecular weight 6,000) I Fnl was added to the loosened precipitate, and a fusion reaction was performed at room temperature for 1 minute while rotating the test tube. RPMI-164 every 30 seconds thereafter
The reaction was stopped by adding 1 ml of 0 medium for 5 minutes.

Immediately centrifuge for 1 minute (1000 rpm, 5 minutes), remove the supernatant, and feeder cells at 1 x 1067 m! , 50rn RPMI-1640 medium containing 20% Fe2 (fetal bovine serum)
The cells were suspended in l. The cell suspension was dispensed into two 24-well plates at 1 ml per well and cultured in a CO2 incubator.

After 24 hours, HAT medium (hyboxanthin 1 x 10-'
M, aminopterin 4X10-'M, thymidine 1.6X
20% FC3 containing 10-5M RPM 1-16
40 medium) was added at 1 rnl per well.

On the second and third days, half of the medium was replaced with HAT medium every two days. On day 100, half of the medium was replaced with HT medium obtained by removing aminopterin from the above HAT medium. From the next day onwards, add normal medium, i.e. RPMI supplemented with 0% FC5, every 2 days.
-1640 medium was exchanged in half, and the culture supernatant on day 188 was subjected to assay for anti-TgG antibody production.

(3) Selection of hybridomas To select anti-1gG antibody-producing hybridomas, each cell culture supernatant from 48 wells was analyzed by ELISA.

First, add 10 py/g of IgG to a 96-well plate for ELISA.
The human IgG was dispensed in 0.1 ml portions at a concentration of 0.1 ml and left at 4° C. for 16 hours to immobilize human IgG on the plate. Tween2
0 (nonionic surfactant, Atlas Co., Ltd. brand name) to 0.
10mM phosphate buffer containing 0.05% pH 7.4 (washing solution)
After washing three times with
The mixture was divided into portions and allowed to stand at 37°C for 2 hours. Next, use cleaning solution for 3
After washing twice, dispense the cell culture supernatant into 0.1 m7 portions and store at 37°C.
It was left to stand for 2 hours. RP with 20% FC3 as a negative control
MI-1640 medium was dispensed at 0.1 rn1. After further washing three times with a washing solution, 0.1 ml of peroxidase-labeled anti-mouse immunoglobulin antibody solution was dispensed and left at 37° C. for 2 hours.

The reaction was stopped by adding 0.05 mJ of cystic acid, and the OD490n
m was measured. Hybridomas growing in the culture supernatant showing an OD that was twice or more that of the negative control were selected as anti-IgG antibody-producing hybridomas. Anti-Jg in 3 out of 48 holes
G antibody production was observed.

(4) The thymus of a monocloned BALB/c mouse (female, 6 weeks old) was removed, and the thymus lymphocytes were loosened in a plastic petri dish lined with 10 m/ml of Rpyr-ts4o. The thymic lymphocytes were washed three times with RPMI-1640 medium by repeating centrifugation (too many rotations, 10 minutes). Thymus lymphocytes with 20% FC3 added, RPM l-1640 medium 50ml
I let J levitate. To this suspension, 0.1 rnl of a solution containing 7500 anti-1gG antibody producing hybrids/ml was added,
After mixing well, add 0.2 m per hole to a 96-well culture plate.
The mixture was dispensed into 1 portions and cultured in a CO2 incubator for 10 days. As a result of ELISA analysis of the culture supernatant in which cell proliferation was observed, 7 anti-1gG antibody-producing hybridoma clones were obtained. One of these clones was further subjected to the same procedure as above to obtain 6 anti-1gG antibody-producing hybridoma clones.
Perfected monocloning.

(5) Production of monoclonal antibody Clone G~2-32X1 obtained by the operations up to (4)
06 were suspended in 0.2 rnl of RPMI-1640 medium and administered intraperitoneally to BALB/c mice (male, 6 weeks old), and ascites was collected after a predetermined number of days. Ascites 4W
Saturated ammonium sulfate solution was gradually added to Lt to give a final ammonium sulfate saturation concentration of 50%, and after stirring at room temperature for 2 hours, centrifugation was performed (30%).
00 rpm for 10 minutes), collect the precipitate and add 5 rn of physiological saline.
1 was added and dissolved. The sample was serially diluted with physiological saline in 10-fold increments and analyzed by ELISA, and the dilution factor at which antibody activity was observed was determined to be 106. In addition, the mouse TgG content (a guideline for antibody titer)
When measured using an agarose plate containing rabbit serum (SRID method), it was 23n+g/ml.

(6) For clone G-3 obtained through the procedure up to (4), perform the same procedure as in (5) to obtain monoclonal antibody 5w.
I got Ll. In addition, when the antibody activity and mouse IgG content were determined using the same method as in (5), the antibody activity was 106,
Mouse IgG content was 22 [I1g/rnl.

Clone G-5 obtained in step (5) was subjected to the same procedure as in (5) to obtain 5 ml of monoclonal antibody. In addition, when the antibody activity and mouse IgG content were determined using the same method as in (5), the antibody activity was 105 and the mouse IgG content was 20 mg/Fn! Met.

Reference example 2. Preparation of monoclonal anti-IgA antibody and hybridoma producing it Mix 0.1 mJ of a 0.15M sodium chloride solution in which 200 pp of human IgA has been dissolved and 1 ml of Freund's complete adjuvant O to prepare an emulsion antigen solution, and 0.2 ml of this is mixed with B. It was administered intraperitoneally to ALB/CV mice (male, 5 weeks old). After 4 weeks, human I
100 pjl of gA was added to 0.15M sodium chloride solution.
2- and injected into the tail vein. Hereinafter, reference example 1 (2
) to &4) were performed to obtain 5 anti-IgA antibody-producing hybridoma clones. Furthermore, the operation (5) of Reference Example 1 was performed to prepare 5 types of anti-IgA monoclonal antibodies (A-2, A-
37°A-64, A-206, A-215) were obtained. The antibody activity and mouse IgG amount (a measure of antibody titer) of each clone were as shown in Table 1.

Table 1 Reference example 3. Preparation of monoclonal anti-human C3 antibody and hybrid-7 that produces it Mix 0.1 ml of O, 15M sodium chloride solution in which human CC550p has been dissolved and 0.1 ml of Freund's combined Ashhant to prepare an emulsion antigen solution.
．． 2 mJ was administered intraperitoneally to BALB/c mice (#, 6 weeks old). After 3 weeks, human 03100p9
was dissolved in 0.2 ml of 0.15 M sodium chloride solution,
injected into the tail vein.

Thereafter, operations (2) to (4) of Reference Example 1 were performed to obtain anti-human C.
3 antibody-producing hybridoma clones were obtained.

Furthermore, the operation (5) of Reference Example 1 was carried out to obtain four types of anti-human C3 monoclonal antibodies (C3-1, C3-7, C3-9,
C5-10) was obtained. The antibody activity and mouse IgG amount (a measure of antibody titer) of each clone were as shown in Table 2.

Table 2. Antibody activity and mouse IgG amount reference example 4. Preparation of monoclonal anti-human transferrin antibody and pipulidonima used to produce it Human transferrin] 0.1 rnl of 0.15M sodium chloride solution in which OOPfl was dissolved and Freund's complete adjuvant 0. ] m/ was mixed to prepare a T emulsion antigen solution, and 0.2 ml of the solution was intraperitoneally administered to a BALB/c mouse (female, 6 weeks old). After 3 weeks, human transferrin [00 p] was dissolved in 0.2 rnl of 0.15 M sodium chloride solution and injected into the tail vein.

Thereafter, operations (2) to (4) of Reference Example 1 were performed to obtain four anti-human transferrin antibody-producing hybridoma clones. Furthermore, the procedure (5) of Reference Example 1 was carried out to prepare four kinds of anti-human transferrin monoclonal antibodies (TF-1, TF-
2, TF-3, TF-5) were obtained. Table 3 shows the antibody activity and mouse IgGffi (approximate antibody titer) of each clone.
It was as follows.

Table 3 Antibody activity and mouse IgG amount Example 1. Reagents for measuring human serum IgG using monoclonal and polyclonal antibodies: The following reagents were prepared.

■ Buffer (R1) Polyethylene glycol 8000 2.5gO,
+M Tris-HCl buffer, pH 7,4100ml Antibody solution (R2) Monoclonal antibody G-52mj polyclonal antibody obtained in Reference Example 1 (cow, 5mgAb/ml) 1rnl
The above buffer (R') 20rn
Sample: The following human sera were used.

IgG content 400.800.1200.1600.20
00 or 3000 mg/dl human serum.

Equipment used: Hitachi 705 automatic analyzer Parameter setting conditions 2 The conditions shown in Table 4 were used.

Table 4 RI, Buffer R2: Antibody solution Operation method: Measurement operations were carried out in a conventional manner by setting the parameters of a Hitachi 705 automatic analyzer under the above conditions.

Result: The obtained absorbance (OD) is plotted on the horizontal axis for each IgG i (
Fig. 1 shows a calibration curve obtained by plotting the vertical axis against (mg/dl).

Comparative Example 1 The results obtained by measuring under the same conditions as in Example 1 were obtained in the same manner as in Example 1, except that the antibody solution used in Example 1 except that only the polyclonal antibody was removed was used as the antibody solution. Shown in Figure 2.

As is clear from FIG. 1, the calibration curve obtained by the method of the present invention showed good linearity from low to high concentrations. On the other hand, in the case of Comparative Example 1 in which only a monoclonal antibody was used as the antibody, as is clear from Figure 2, almost no turbidity was obtained at low concentrations (1000 mg/dl or less), and at higher concentrations However, it was found that the calibration curve was not linear.

Example 2. Reagents for measuring 1 gA of human serum using monoclonal and polyclonal antibodies: The following reagents were prepared.

■ Buffer (R1) Polyethylene glycol 6000 4g Sodium chloride 0.8g 0.05M phosphate buffer, pH 7.2]00m/■ Antibody solution (R
2) Monoclonal antibody A-370 obtained in Reference Example 2, 4 ml
Monoclonal antibody A-640 obtained in Reference Example 2, 5 ml
Polyclonal antibody (sheep, 3mgAb/!nri 0.
5m! The above buffer (R')
1ornt■ Standard Serum A standard serum with an IgA content of 740 mg/df was used.

Samples: 15 human serum samples were used.

Equipment used: Hitachi 736 automatic analyzer Parameter setting conditions 2 The conditions shown in Table 5 were used.

R1, buffer solution R2, antibody solution operation method: Hitachi 736 automatic analyzer was set with parameters under the above conditions and measurement operations were carried out in a conventional manner.

Comparative Example 2 The same sample was measured under the same conditions as in Example 2, except that the antibody solution used in Example 2 except that only the polyclonal antibody was removed was used as the antibody solution.

The IgA concentration was measured using a method (manufactured by Kist Japan).

The results obtained in Example 2, Comparative Example 2, and Reference Example 5 are also shown in Table 6.

Table 6 y+=+, ooM+2.71, r=0.999Y2
= 1.04
It can be seen that there is a good correlation with that obtained by the method from low to high concentrations. In addition, in the case of Comparative Example 2 in which only a monoclonal antibody was used as an antibody, the amount of IgA was 100 mg.
Measured values for samples below /dZ are low. That is, it can be seen that the measured value is lower than that obtained by the 5RID method because the reaction has not progressed sufficiently and turbidity is insufficiently formed at a low concentration (100 mg/d1 or less).

Example 3. Reagents for measuring human serum C3 using monoclonal antibodies and polyclonal antibodies The following reagents were prepared.

■ Buffer (R') Polyethylene glycol 6000 3.5g Sodium chloride 0.9g 10mM Veronal buffer, pH 7,5]00rff■ Antibody solution (R2) Monoclonal antibody C3-10,2rn obtained in Reference Example 3
! Monoclonal antibody obtained in Reference Example 3 (:3-70
．． 2mZ polyclonal antibody (rabbit, 2mgAb/mt)
1ml of the above buffer (R')
Five oynt samples of each human serum were used.

C3 total amount 20,110,80,120,160,200
or 240 mg/dll human serum.

Procedure: Pour 20IL of sample directly into the test tube! Take this and add 2rn! of antibody solution to it! After adding and reacting at 37°C for 10 minutes, the layer length was 10 mm and the wavelength was 505 nm using a spectrophotometer.
The absorbance was measured at

Comparative Example 3 The same sample was measured under the same conditions as in Example 3, except that the antibody solution used in Example 3 except that only the polyclonal antibody was removed was used as the antibody solution.

Combining the results obtained in Example 3 and Comparative Example 3,
As shown in the figure. Here, -・- indicates the measurement results obtained in Example 3, -〇- indicates the measurement results obtained in Comparative Example 3, and the absorbances obtained for each amount of C3 (mg/d1) on the horizontal axis. It connects the points where (OD) is plotted along the vertical axis.

As is clear from Figure 3, the test using a combination of monoclonal and polyclonal antibodies was carried out using a combination of monoclonal and polyclonal antibodies. Either it shows good quantitative performance over a wide range of high concentrations, or in the case of Comparative Example 3 using only a monoclonal antibody, quantitative performance at low c33 concentration ranges is extremely poor, indicating that sufficient turbidity is not formed. I understand.

Example 4. Reagents for measuring human serum transferrin using monoclonal and polyclonal antibodies The following reagents were prepared.

■ Buffer solution (R1) Polyethylene glycol 6000 3g Sodium chloride 0.9g 0.05M boric acid M# solution, pH 7,4100ml ■ Antibody solution (R
2) Monoclonal antibody TF-21rnl obtained in Reference Example 4 Monoclonal antibody TF-5lIn! obtained in Reference Example 2) Polyclonal antibody (goat, 3.4mgAb/++Ll)
1 ml overlay and buffer* (R950m1 sample: The following human sera were used.

Trans 7 y-phosphorus content 50, 100, 200, 30
0,400,800 or 800 mg/d9 human serum.

Operation method: Place 10 pl of sample directly into a cuvette for laser nephelometer ZD-801 (manufactured by Wako Pure Chemical Industries, Ltd.)
After adding antibody solution 5004 to this and reacting at room temperature for 15 minutes, the laser nephelometer ZD-80
1, the light scattering intensity at a wavelength of 633 nm was measured.

Comparative Example 4 The same sample was measured under the same conditions as in Example 4, except that the antibody solution used in Example 4 from which only the polyclonal antibody was removed was used as the antibody solution.

Comparative Example 5 The same sample was measured under the same conditions as in Example 4, except that the antibody solution used in Example 4 with only the monoclonal antibody removed was used as the antibody solution.

The results obtained in Example 4, Comparative Example 4, and Comparative Example 5 are also shown in FIG. Here, -・- indicates the measurement results obtained in Example 4, -〇- indicates the results obtained in Comparative Example 4, and -×- indicates the results obtained in Comparative Example 5. The light scattering intensity (m V ) obtained for each amount of transferrin (Il1 g/a ) is plotted along the vertical axis and the points are connected.

As is clear from FIG. 4, in the case of Example 4 in which a combination of monoclonal and polyclonal antibodies was used, the amount of transferrin and the light scattering intensity increased almost proportionally, but in the case of Comparative Example 4 in which only monoclonal antibodies were used. ,
Samples with low transferrin content (50, 100, 20 (
1 mg/dl), almost no turbidity formation occurred.

In addition, in the case of Comparative Example 5 using only a polyclonal antibody, it can be seen that turbidity formation due to excess antigen was suppressed and turbidity decreased when the amount of transferrin was 00 mg/dJ or more.

[Effects of the Invention] As described above, the present invention provides a method for effectively determining proteins in human body fluids such as serum, plasma, and urine by immunoassay using antigen-antibody reactions. According to the method of the present invention, (1) the sample can be directly used for measurement without being diluted, thereby eliminating the complexity and errors associated with the dilution operation.

(2) Since it is not necessary to use a large amount of an aggregation promoter such as polyethylene glycol, undesired non-specific agglutination reactions do not occur, and measurement values are not affected thereby.

(3) Measurements with high retardation can be performed in a wide concentration range from low to high concentrations.

(4) It is extremely easy to apply to automatic analyzers that measure multiple items on the same sample.

This invention has remarkable effects in these respects, and is a great invention in terms of its contribution to this industry.

[Brief explanation of the drawing]

Figure 1 shows the calibration curve obtained in Example 1, connecting the points where the absorbance (OD) obtained for each 1 gG amount (rng/dl> on the horizontal axis) was plotted along the vertical axis. Figure 2 shows the calibration curve obtained in Comparative Example 1, in which the absorbance (OD) obtained for each IgG amount (mg/df) on the horizontal axis is plotted along the vertical axis. Figure 3 shows the calibration curves obtained in Example 3 and Comparative Example 3, and the absorbance (OD) obtained for each C3M (mg/dl) on the horizontal axis is plotted vertically. It connects the points plotted along the axis. However, -.- indicates the results obtained in Example 3, and -0- indicates the results obtained in Comparative Example 3. FIG. 4 shows the calibration curves obtained in Example 4, Comparative Example 4, and Comparative Example 5, and the horizontal axis shows the amount of transferrin (mg
It connects the points where the light scattering intensity (mV) obtained for /dl) was plotted along the vertical axis. However, -・
- indicates the results obtained in Example 4, -〇- indicates the results obtained in Comparative Example 4, and -x- indicates the results obtained in Comparative Example 5. . Patent applicant: Wako Pure Chemical Industries, Ltd. Figure 2: IvG amount (S/d1) Figure 3: C3 amount (m/dQ)

Claims (6)

[Claims] (1) In a method for quantifying proteins in a biological sample by irradiating the antigen-antibody complex generated by an antigen-antibody reaction with light and measuring its optical change, the biological sample is used as it is without dilution. As an antibody against the protein to be measured,
1. A method for quantifying a protein in a biological sample, which is carried out using a combination of one or more monoclonal antibodies against the protein and a polyclonal antibody against the protein. (2) A patent in which the monoclonal antibody is a monoclonal anti-human protein antibody produced by a hybridoma formed by fusion of cells from a mouse tumor line and mouse splenocytes that have been previously immunized with the human protein to be measured. Quantification method according to claim 1. (3) The quantitative method according to claim 1 or 2, wherein the polyclonal antibody is a polyclonal antibody obtained from a rabbit, goat, sheep, horse, or cow by immunization with the protein to be measured. (4) The quantitative method according to any one of claims 1 to 3, wherein the amount of optical change is the amount of change in light scattering intensity. (5) The quantitative method according to any one of claims 1 to 3, wherein the amount of optical change is the amount of change in the amount of transmitted light. (6) The measurement target is immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM),
Complement C3, transferrin, haptoglobin, α_1
-antitrypsin, albumin, apolipoprotein A I
, apolipoprotein AII, or α_2-macroglobulin.