JP2001091516A - METHOD FOR DETERMINING QUANTITY OF HUMAN alpha-FETOPROTEIN AND MEASUREMENT KIT - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rapid, high-sensitivity determination method of the amount of AFP for measuring accurately even at a low-concentration region without any agglutination of nonspecific reaction. SOLUTION: In the method for determining the quantity of AFP for measuring the absorbance of a latex flocculation reaction, the Fc part of an anti-AFP antibody is eliminated, an insoluble carrier where the antibody with only F(ab')2 is sensitized is mixed with a sample, the amount of change in the absorbance of an antigen antibody flocculation reaction is measured, a latex particle is used as the insoluble carrier when determining the amount of AFP in the sample with un unknown concentration as the insoluble carrier, the anti-AFP antibody that is sensitized onto the carrier is used so that concentration in the flocculation reaction liquid ranges from 0.1 to 10 μg/ml, only an antibody where the anti-AFP antibody is set to F(ab')2 only is used so that the concentration in the flocculation reaction liquid becomes 0.01-2 wt.%, a specific macromolecule is added so that the concentration in the flocculation reaction liquid becomes 0.1-5 wt.%, the reaction between the insoluble carrier and the sample is made at a constant temperature for 5 seconds-15 minutes, and the concentration in the flocculation reaction liquid is set to 0.1-2 wt.% casamino acid and 0.05-2 M choline chloride for usage.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive and rapid method for quantifying human .alpha.-fetoprotein (hereinafter abbreviated as "AFP") in a sample, particularly to a method utilizing latex agglutination turbidimetry. The present invention relates to an immunological kit for measuring AFP using the method.

[0002]

2. Description of the Related Art AFP is a typical oncofetal protein having an average molecular weight of 64,000, which was discovered by Abelev in 1963, and appeared in blood from embryonic week 6 and showed the highest value at embryonic week 13; It drops at birth. AFP is scarcely found in the blood of healthy adults, but frequently appears in blood in liver diseases, and exhibits a high level particularly in hepatocellular carcinoma. AF
It has been recognized that the transition of P reflects the state of the tumor very well, and has been widely used as an important marker with extremely high specificity for hepatocellular carcinoma in the field of clinical examination (Kenji Koyama et al. Disease, Vol. 16, No. 6 (1986),
265-269).

Conventionally, AFP has been measured by a qualitative method, a semi-quantitative method, etc., and thereafter, a radioimmunoassay (RIA) and an enzyme immunoassay (EI) are used.
A) has become widespread, and numerous results have been reported. Recently, immunoassays based on latex agglutination, ferrite particle solid-phase assays, immunofluorescence assays, immunochemiluminescence, etc. have been developed with the aim of miniaturizing the reaction system, improving sensitivity, and shortening the reaction time. I have. In particular, for the immunoassay method using latex agglutination, various latex agglutination immunoreagents are commercially available (Hisao Okura et al., Clinical Chemistry, 24, 4 (1995), pp. 188-195).
However, in this method, aggregation due to a non-specific reaction is often observed, and there is a problem that the measurement cannot be performed accurately, particularly in a low concentration range, as compared with the radioimmunoassay method. Also,
In general, an immunoassay method uses an antigen-antibody reaction, so that the preferable measurement range is not wide.In the body such as an object to be measured of the present invention, those having a wide range of concentrations are often diluted. The concentration must be measured, and two or more operations are required at the time of inspection, which is inconvenient. For example, Japanese Unexamined Patent Publication
In JP-A-142360, spontaneous aggregation is extremely low and storage stability is attained by blending at least one compound selected from the group consisting of γ-collidine (specific imidazole or a derivative thereof) and a propane 1,3-diol derivative. A latex reagent of AFP has been disclosed which can obtain a reagent having excellent properties and can easily and accurately measure the blood AFP concentration. However, although the reagent of the present invention is excellent in storage stability, the sensitivity in a low concentration range is not sufficient, and there is a problem that some non-specific agglutination occurs depending on the sample. Furthermore, since the antigen-antibody reaction is used, there is a problem that aggregation occurs due to a reaction between a rheumatoid factor (hereinafter abbreviated as RF), which is a coexisting substance in the sample, and an antibody.

[0004]

Conventionally, the normal value of AFP has been set at 20 ng / ml or less, so that the measurement range of most latex agglutinating immunoreagents is 20 to 1,000.
It was about ng / ml. However, in recent years, with the development and spread of diagnostic imaging, early hepatocellular carcinoma has been detected, and the serum AFP level at the time of detection has decreased.
AFP detection sensitivity up to about / ml has been required. Along with this, latex agglutination immunoreagents capable of measuring in the range of 0 to 250 ng / ml have been developed, but since these reagents achieve high sensitivity, it is said that specificity tends to decrease. . In particular, aggregation due to non-specific reactions is likely to occur, making accurate quantification in a low concentration range difficult. Further, since the conventional preparation is an immunological preparation, a specimen having RF reacts with the Fc portion of the antibody to cause non-specific aggregation, which is a cause of erroneous determination.

In view of the circumstances as described above, an object of the present invention is to provide a highly sensitive and rapid method for quantifying AFP, which is free from aggregation due to non-specific reactions and can be accurately measured even in a low concentration range. An object of the present invention is to provide an immunological kit using the method.

[0006]

In order to achieve the above object, according to the present invention, the Fc portion of the anti-human α-fetoprotein antibody is removed, and the F (ab ′) ₂ -only antibody is detected. The prepared insoluble carrier and the sample are mixed, an agglutination reaction is caused by an antigen-antibody reaction, the amount of change in absorbance of the reaction solution is measured, and human α-fetoprotein having a known concentration is measured in advance as a sample. Using lines,
In quantifying human α-fetoprotein in a sample of unknown concentration, (1) latex particles were used as an insoluble carrier, and (2) an anti-human α-fetoprotein antibody sensitized on the insoluble carrier caused an agglutination reaction. (3) use the reaction solution so as to have a concentration of 0.1 to 10 μg / ml.
The Fc portion of the anti-human α-fetoprotein antibody was removed, and the insoluble carrier sensitized with the F (ab ′) ₂ -only antibody was used at a concentration of 0.01% in a reaction solution that caused an agglutination reaction.
(2) average molecular weight of 3.0%
Polyethylene glycol of 00 to 1,000,000 and / or an average molecular weight of 300,000 to 1,000,000
Of polyvinylpyrrolidone is added so that the concentration in the reaction solution causing the agglutination reaction is 0.1 to 5% by weight. (5) The reaction between the insoluble carrier and the sample is carried out for 5 seconds to 15 minutes.
(6) using casamino acid such that the concentration in the reaction solution causing the agglutination reaction becomes 0.1 to 2% by weight, and (7) using choline chloride as the reaction solution causing the agglutination reaction (8) A method for quantifying human α-fetoprotein, characterized by measuring the increase in absorbance associated with the progress of latex agglutination reaction. Further, according to the present invention of claim 2, a kit for quantifying human α-fetoprotein using the method for quantifying human α-fetoprotein according to claim 1, comprising:
Latex particles sensitized with an anti-human α-fetoprotein antibody, and (2) an average molecular weight of 3,000 to 1,000,000
The present invention provides a kit for measuring human α-fetoprotein, comprising a polyethylene glycol having a molecular weight of 0 and / or a polyvinylpyrrolidone having an average molecular weight of 300,000 to 1,000,000, a diluent for diluting a sample containing casamino acid and choline chloride.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The components of the present invention will be described below in detail.

As the sample in the present invention, serum, plasma, etc., and various biological and non-biological samples are used.

Latex particles are used as the insoluble carrier in the present invention. As the particles, organic fine particles having a relatively constant particle size, a certain quality and performance, and which can be industrially mass-produced are preferable. For example, homopolymers and / or copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylates and methacrylates, styrene-butadiene copolymers, methyl methacrylate-butadiene copolymers and the like Butadiene-based copolymer. Polystyrene-based latex particles are particularly preferable because they have excellent antibody adsorption properties and can stably maintain biological activity for a long period of time.

[0010] The insoluble carrier is sensitized with the anti-AFP antibody by a method in which the antibody is physically or chemically adsorbed to the carrier.

The particle size of the particles as the insoluble carrier is as follows:
It is preferably from 0.02 to 0.5 μm, and particularly preferably from 0.05 to 0.2 μm. The particle size is 0.0
If it is less than 2 μm, it becomes difficult to disperse the particles when freeze-drying is performed. On the other hand, when the particle size exceeds 0.5 μm, self-aggregation proceeds, and dispersibility decreases.

The anti-AFP antibody of the present invention uses an enzyme or the like,
F (ab ') by removing the Fc portion from the immunoglobulin molecule
_It is necessary to use only ₂ The reason is that blood, which is a measurement sample, may contain RF, and since this RF is an antibody against the Fc portion of immunoglobulin, it also binds to the AFP antibody which is a component of the present invention. And increase agglomeration. Therefore, anti-AFP
An antibody is used as in the present invention, and an enzyme or the like is used to remove the Fc portion from the immunoglobulin molecule to make only F (ab ') ₂ , thereby preventing non-specific aggregation from occurring.

[0013] The anti-AFP antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody as long as the Fc portion is removed and only F (ab ') ₂ is obtained. For polyclonal antibodies, the animal species of origin (human, rabbit,
Goat and the like (purity of globulin fraction or affinity-purified fraction) are not particularly limited.

The enzyme used to remove the Fc portion from the immunoglobulin molecule of the present invention to obtain only F (ab ') ₂ is not particularly limited. For example, pepsin or the like can be suitably used.

AFP antibodies sensitized on an insoluble carrier are
The concentration in the reaction solution causing the agglutination reaction is 0.1 to 10
Use to make μg / ml. When the concentration of the antibody is 0.
If it is less than 1 μg / ml, the sensitivity is low and accurate measurement in a low concentration range cannot be performed. If it exceeds 10 μg / ml, agglutination due to non-specific reaction occurs, and accurate measurement cannot be performed.

The Fc portion of the anti-AFP antibody is removed, and F
The insoluble carrier sensitized with the antibody made only (ab ') ₂ is
It is used so that the concentration in the reaction solution that causes the agglutination reaction is 0.01 to 2% by weight. If the concentration is less than 0.01% by weight, the formation of aggregates is insufficient, and the required sensitivity cannot be obtained. On the other hand, if the concentration exceeds 2% by weight, the absorbance as the background is too high, and accurate quantification cannot be performed.

As the polyethylene glycol to be added to the reaction solution of the present invention, a polyethylene glycol having an average molecular weight of 3,000 to 1,000,000 is used for the purpose of improving sensitivity or accelerating the reaction. If the average molecular weight of the polyethylene glycol is less than 3,000, the effect of improving sensitivity or promoting the reaction is insufficient, and if the average molecular weight of the polyethylene glycol exceeds 1,000,000, the polyethylene glycol in the medium is The solubility is remarkably low, causing a problem in solubility.

The above average molecular weight is from 3,000 to 1,000.
The polyethylene glycol of 000 is used by adding it so that the concentration in the reaction solution that causes the agglutination reaction becomes 0.1 to 5% by weight. If the polyethylene glycol concentration is less than 0.1% by weight, the sensitivity is low, and accurate measurement in a low concentration range cannot be performed. On the other hand, if the polyethylene glycol concentration exceeds 5% by weight, aggregation due to non-specific reaction occurs, and accurate measurement cannot be performed.

Further, polyvinylpyrrolidone added to the reaction solution for the same purpose as polyethylene glycol includes:
Those having an average molecular weight of 300,000 to 1,000,000 are used. When the average molecular weight of polyvinylpyrrolidone is less than 300,000, the effect of improving sensitivity or accelerating the reaction is insufficient, and when the average molecular weight of polyvinylpyrrolidone exceeds 1,000,000, the amount of polyvinylpyrrolidone in the medium is reduced. The solubility is remarkably low, causing a problem in solubility.

Average molecular weight 300,000-1,000,
000 polyvinylpyrrolidone is added and used so that the concentration in the reaction solution that causes the agglutination reaction becomes 0.1 to 5% by weight. If the polyvinylpyrrolidone concentration is less than 0.1% by weight, the sensitivity is low, and accurate measurement in a low concentration range cannot be performed. Further, when the polyvinylpyrrolidone concentration is 5
If the content is more than 10% by weight, aggregation occurs due to non-specific reaction, and accurate measurement cannot be performed.

Average molecular weight 3,000 to 1,000,000
The polyethylene glycol having a molecular weight of 0 and / or polyvinylpyrrolidone having an average molecular weight of 300,000 to 1,000,000 is preferably used after being dispersed and dissolved in an appropriate aqueous medium. In this case, the insoluble carrier sensitized with the antibody and polyethylene glycol and / or polyvinylpyrrolidone may be dispersed or dissolved in the same medium to form a one-part latex reagent,
The insoluble carrier and polyethylene glycol and / or polyvinylpyrrolidone are separately dispersed or dissolved in an aqueous medium, and a latex reagent of the insoluble carrier and a solution of polyethylene glycol and / or polyvinylpyrrolidone (a diluent for diluting a sample) are used. It may be used as a liquid reagent. Preferably, a two-pack type reagent is used.

Here, the aqueous medium is preferably a phosphate buffer, a glycine buffer, a Tris-HCl buffer, a Good buffer, or the like. The pH of the aqueous medium is preferably 5.5
To 8.5, particularly preferably 6.5 to 8.0.

In the above-mentioned one-part latex reagent, bovine serum albumin, sucrose, sodium chloride for adjusting the salt concentration, and the like may be appropriately dissolved. Moreover, bovine serum albumin, sucrose, sodium chloride for adjusting the salt concentration, and the like may also be appropriately dissolved in a two-part reagent of a latex reagent and a reagent in the form of a solution (diluent for diluting a specimen).

In the present invention, the casamino acid in the reaction solution is
A hydrolyzate of casein with an acid, for example, DIF
CO products can be used. By containing casamino acid, aggregation of the latexes to which the antibody has been adsorbed can be suppressed as a protective substance against them. The casamino acid is used at a concentration of 0.1 to 2% by weight in a reaction solution that causes an agglutination reaction. If the concentration in the reaction solution is less than 0.1% by weight, it cannot work sufficiently as a protective substance against self-aggregation. , AFP cannot be measured sufficiently. If it exceeds 2% by weight, the sensitivity will be reduced because it interferes with the antigen-antibody reaction.

The reaction solution of the present invention contains choline chloride. By the presence of choline chloride at a high concentration, non-specific reactions, which are aggregations other than antigen-antibody reactions, can be suppressed. The concentration of choline chloride used is such that the concentration in the reaction solution that causes the agglutination reaction is 0.05 to 2M. If the concentration in the reaction solution is less than 0.05M, non-specific reaction cannot be suppressed, and if it exceeds 2M, even an agglutination reaction due to an antigen-antibody reaction is undesirably suppressed.

The reaction between the insoluble carrier and the sample in the present invention is an antigen-antibody reaction and a latex agglutination reaction associated therewith, and is carried out at a constant temperature for 5 seconds to 15 minutes. If the reaction time is less than 5 seconds, accurate measurement cannot be performed. If the reaction time exceeds 15 minutes, aggregation due to non-specific reaction occurs, and accurate measurement cannot be performed. The reaction is carried out,
It is preferably performed at 7 ° C.

In the measurement of absorbance, an increase in absorbance as the latex agglutination reaction proceeds is measured. The absorbance measurement is
Usually 500 to 1000 nm, preferably 500 to 800
nm, particularly preferably within the range of 550 to 650 nm.

The instrument used for the measurement of the present invention may be any instrument capable of measuring the absorbance of the reaction solution over time, but a general-purpose automatic biochemical analyzer is preferred. The measurement wavelength, sample amount, reagent amount, and the like can be appropriately selected according to the device.

The amount of AFP is determined by performing the above-described measurement on a sample having a known amount of AFP (for example, AFP standard serum and its dilution series), preparing a calibration curve from the measured value and the amount of AFP, and determining the AFP unknown. The amount of AFP can be determined by determining the corresponding AFP amount in the calibration curve from the measured value of the amount of the sample measured under the same conditions.

[0030]

According to the first aspect of the present invention, an anti-AFP antibody is obtained by removing an Fc portion from an immunoglobulin molecule using an enzyme or the like.
Since only (ab ') ₂ is used, it is possible to provide a highly sensitive and rapid AFP quantification method which does not cause aggregation due to non-specific reaction and enables accurate measurement even in a low concentration range. That is, blood which is a measurement sample may contain RF, and since RF is an antibody against the Fc portion of immunoglobulin, it also binds to AFP antibody which is a component of the present invention, and agglutinates. Increase. Therefore, anti-AF
As for the P antibody, an enzyme or the like is used as in the present invention, and the Fc portion is removed from the immunoglobulin molecule to make only F (ab ') ₂ , thereby preventing non-specific aggregation from occurring. Further, in the present invention, by adding casamino acid to the reaction solution, the self-aggregation of the latex to which the antibody has been adsorbed can be prevented, and by adding choline chloride, the non-specific reaction can be suppressed. Further, since the present invention has the above-mentioned constitution, a sample is mixed with an insoluble carrier sensitized with an anti-AFP antibody, an antigen-antibody reaction and a latex agglutination reaction accompanying the mixture are caused, and the change in absorbance of the reaction solution is caused. Is measured, and the amount of AFP in a sample of unknown concentration can be accurately measured using a calibration curve prepared in advance. Further, since the present invention of claim 2 has the above configuration, it is possible to provide an immunological kit for AFP measurement that accurately measures the amount of AFP in a sample of unknown concentration using the method for quantifying AFP. it can.

[0031]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which by no means limit the present invention.

(1) Reagents and Materials Reagents and materials used for quantification of AFP are as follows.

A) Latex Polystyrene latex having an average particle size of 0.403 μm (solid content: 10% (W / V), manufactured by Sekisui Chemical Co., Ltd.) was used.

B) Latex dilution buffer: 50 mM Na ₂ HPO ₄ and 50 mM NaH ₂ PO ₄
Was mixed so as to have a pH of 7.50, and this mixture was used as a latex dilution buffer.

C) Anti-AFP antibody Goat anti-AFP antibody (1 mg / ml, DAKO) purified from goat antiserum to immunoglobulin fractionation
The c-part was excised with an enzyme, and only F (ab ') ₂ was produced by affinity chromatography.

D) Antibody dilution buffer The latex dilution buffer was also used as an antibody dilution buffer.

E) Blocking buffer: 100 mM Na ₂ HPO ₄ and 100 mM NaH ₂ P
O ₄ was mixed to a pH of 7.40, and the mixture was added to bovine serum albumin (Bovine serum albumin Fraction).
V, Reagent Grade, manufactured by Miles Corp.) at 1% (W /
V) and 0.1% (W / V) of NaN ₃ (reagent grade, manufactured by Nacalai Tesque) was used as a blocking buffer.

F) Serum samples Serum from hepatocellular carcinoma patients and healthy subjects was used.

G) AFP standard product AFP obtained by adding AFP to human pool serum accurately to a concentration of 1000 ng is freeze-dried, and the AFP amount is measured using Elpier Ace AFP (manufactured by Diatron). 200, 50, 10 ng / m in saline
and used after dilution. A physiological saline only containing no AFP standard was used at 0 ng / ml.

H) Diluent for sample dilution (R1) Polyethylene glycol 60 was added to the blocking buffer.
00 (average molecular weight 7,500, manufactured by Wako Pure Chemical Industries) 3%
(W / V), choline chloride (JIS special grade, Wako Pure Chemical Industries, Ltd.)
1M and casamino acid (manufactured by DIFCO) in a concentration of 0.5% by weight.
Used as 1).

I) Latex agglutination immunoreagent for AFP measurement Commercially available latex agglutination immunoreagent (Elpiace AF)
P, manufactured by Diatron).

(2) Preparation and Test Method (1) Preparation of Latex Reagent A polystyrene latex having an average particle size of 0.403 μm (1 volume of solid content 10% (W / V), 3 volumes of latex diluting buffer were added. Thus, a 2.5% latex solution was obtained.
The P antibody was diluted with an antibody diluting buffer so that the protein concentration became 66.7 μg / ml, and used as a sensitized antibody solution. 200 μl of the above 2.5% (W / V) latex solution was added at 25 ° C.
800 μl of the antibody sensitizing solution was quickly added while stirring with a magnetic stirrer in an incubator of
Stirred at C for 1 hour. Thereafter, 2.0 ml of a blocking buffer was added, and the mixture was continuously stirred at 25 ° C. for 2 hours. The mixture was further centrifuged at 15,000 rpm at 15 ° C. for 15 minutes. 4.0 ml of a blocking buffer was added to the obtained precipitate, and the precipitate was washed by centrifugation as described above. The washing operation was performed three times. After adding 2.0 ml of a blocking buffer solution to the precipitate and stirring well, a dispersion treatment was performed by an ultrasonic crusher to obtain a latex reagent having a solid content of 0.25% (W / V). The latex reagent thus prepared was stored at 4 ° C.

(2) Measurement of AFP Amount Using Latex Reagent The measurement of AFP amount using a latex reagent was performed using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.). The solid content obtained in the above operation (1) was 0.25% (W /
V) Latex reagent as it is in R2 solution (solid content 0.2
5% (W / V)). The measurement conditions are as follows.

Sample volume 20 μl Diluent for sample dilution (R1 solution) 210 μl Latex reagent (R2 solution) 70 μl Measurement wavelength 570 nm Measurement temperature 37 ° C.

The difference between the absorbance approximately 80 seconds after the addition of the reagent (R2 solution) and the absorbance approximately 320 seconds (ΔOD570)
Was measured, and the difference in absorbance multiplied by 10,000 was defined as the change in absorbance.

The same measurement as above was performed using an AFP standard product of known concentration in place of the sample, a calibration curve was prepared in advance, and the change in absorbance of the supernatant sample was extrapolated to the above calibration curve. The amount of AFP in the sample was calculated.

(3) Measurement of AFP Amount Using Commercially Available EIA Immunoreagent For all samples measured in the above operation (2), the amount of AFP in the sample was measured using a commercially available EIA immunoreagent. The measurement was performed using an automatic analyzer for biochemistry Model 7150 (manufactured by Hitachi, Ltd.) in accordance with the operation method attached to the kit.

3) Tests and Results (1) Examples 1 to 40 Using sera from patients with hepatocellular carcinoma and healthy subjects as samples, perform the procedures in 2) above, and AFP amount was measured. The operation (2) (3) "AFP using commercially available EIA immunoreagent"
The amount of AFP in the sample was measured in accordance with "Measurement of Amount". The results of these measurements are shown in Table 1. Further, FIG. 1 shows the correlation between the amount of AFP by the latex reagent shown in Table 1 and the amount of AFP by the commercially available EIA immunoreagent.

(2) Comparative Examples 1 to 40 Serums of the above hepatocellular carcinoma patients and healthy subjects were used as specimens.
Using a latex reagent using an antibody that did not remove the Fc portion of the AFP antibody and did not use only F (ab ') ₂ ,
The amount of AFP in the sample was measured. The results of these measurements are shown in Table 1. FIG. 2 shows the correlation between the amount of AFP by the latex reagent shown in Table 1 and the amount of AFP by the commercially available EIA immunoreagent.

As is clear from Table 1, FIGS. 1 and 2,
The amount of AFP measured by the latex reagent of the present invention was in good agreement with the amount of AFP by EIA both in the low concentration range and in the high concentration range, but the Fc portion of the AFP antibody was not removed, and only F (ab ') ₂ was removed. The amount of AFP in the reagent using the antibody that was not obtained was the amount of AFP by EIA,
In the low concentration range and the high concentration range, they did not always match. In addition, for those that did not match, the presence or absence of rheumatoid factor was further measured.
It contained more than 00 IU / ml of rheumatoid factor.

From the above results, the AFP quantification method according to the present invention has higher sensitivity than the conventional latex agglutination immunoreagent,
And it was confirmed that it was a quick and simple superior quantification method than EIA.

[0052]

[Table 1]

[0053]

The method for quantifying AFP of the present invention and the AFP
Since the configuration of the immunological kit using the quantification method is as described above, the non-specific reaction due to coexisting substances in the sample as seen with the conventional latex agglutination immunoreagent is suppressed,
AFP showing the same detection sensitivity as EIA even in low concentration range
Thus, an immunological kit for AFP measurement using the method for quantifying AFP can be provided. This kit can be used to quickly and easily
P can be accurately quantified, and is useful for finding a disease, grasping a disease state, determining a treatment method, and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the correlation between the amount of AFP by a latex reagent and the amount of AFP by a commercially available EIA immunoreagent in the specimens used in Examples 1 to 40. (The units of the vertical and horizontal axes are ng / ml.)

FIG. 2 shows AFP in a sample used in Comparative Examples 1 to 40.
FIG. 9 is a diagram showing a correlation between the amount of AFP by a latex reagent and the amount of AFP by a commercially available EIA immunoreagent using an antibody in which the Fc portion of the antibody was not removed and F (ab ′) ₂ was not used. (The units of the vertical and horizontal axes are ng / ml.)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/563 G01N 33/563

Claims (2)

[Claims] 1. Fc of anti-human α-fetoprotein antibody
The portion was removed, the sample was mixed with an insoluble carrier sensitized with the antibody only F (ab ') ₂ , and agglutination was caused by an antigen-antibody reaction. The change in absorbance of the reaction solution was measured. A human α-fetoprotein having a known concentration is measured as a sample, and the obtained calibration curve is used to quantify human α-fetoprotein in a sample of unknown concentration. (1) Using latex particles as an insoluble carrier, 2) using an anti-human α-fetoprotein antibody sensitized on an insoluble carrier so that the concentration in a reaction solution that causes an agglutination reaction is 0.1 to 10 μg / ml; The Fc portion of the fetoprotein antibody was removed, and the insoluble carrier sensitized with the F (ab ′) ₂ -only antibody was used at a concentration of 0.0% in a reaction solution that caused an agglutination reaction.
(4) polyethylene glycol having an average molecular weight of 3,000 to 1,000,000 and / or an average molecular weight of 300,0%.
00-1,000,000 polyvinylpyrrolidone,
(5) The reaction between the insoluble carrier and the sample is carried out at a constant temperature for 5 seconds to 15 minutes, and (6) casamino The acid is used so that the concentration in the reaction solution causing the agglutination reaction becomes 0.1 to 2% by weight, and (7) the concentration of choline chloride in the reaction solution causing the agglutination reaction is 0.05 to 2% by weight. (8) A method for quantifying human α-fetoprotein, which comprises measuring an increase in absorbance as the latex agglutination reaction proceeds. 2. A kit for quantifying human α-fetoprotein using the method for quantifying human α-fetoprotein according to claim 1, comprising: (1) latex particles sensitized with an anti-human α-fetoprotein antibody; (2) polyethylene glycol having an average molecular weight of 3,000 to 1,000,000 and / or an average molecular weight of 300,
000 to 1,000,000 polyvinylpyrrolidone,
A kit for measuring human α-fetoprotein, comprising a diluent for diluting a sample containing casamino acid and choline chloride.