JPH0727764A - Antibody for immunological measurement blocking fc part, reagent for immunological measurement containing said antibody, immunological measuring method using said reagent for immunological measurement and block reagent blocking fc part - Google Patents

Abstract

PURPOSE: To eliminate unspecific reaction caused by interference of rheumatoid factors(RF) in an immunological measuring method, by blocking an Fc part with a block reagent containing at least an antigen coupling part of an immune globulin which is specifically coupled with the Fc part of the antibody. CONSTITUTION: A block reagent eliminates interference by RF which blocks an FC part of an antibody used in an immunological measuring method, and contains at least an antigen coupling part of an immune globulin which is specifically coupled with the Fc part of the antibody. The antigen coupling part is a variable part which identifiers the antigen and can be coupled with the antigen. As an example of this block reagent, the entire immune globulin or a fragment thereof is exemplified. The immune globulin or the fragment thereof can prevent interference of the RF, and accordingly, it is preferable to have itself no part which is coupled with the RF. Although the origin and class of the immune globulin used in the block reagent are not specifically limited, the immune globulin such as a mouse or a rat can be used.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody for suppressing pseudoreaction caused by the binding of rheumatoid factor (hereinafter referred to as RF) to the Fc portion of the antibody in immunoassay. More specifically, an Fc region-blocking immunological measurement antibody that causes a non-specific reaction that causes a false reaction, an immunological measurement reagent containing the antibody, and an immunological measurement method using the reagent And to eliminate non-specific reactions,
It concerns a blocking reagent that blocks the Fc site.

[0002]

2. Description of the Related Art In vit utilizing the binding reaction between antigen and antibody
The immunoassay, which is a ro assay, is widely applied to clinical tests because it can detect a trace component specifically or accurately. Depending on the labeling method, there are SRID method, agglutination method (using blood cells, latex etc. as a carrier), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence. Immunological assay (FI
A), nephelometry, immunoturbidimetric method and the like.

However, in the application of immunoassays to clinical tests, nonspecific agglutination reaction due to RF and complement C1q present in biological fluids such as blood and urine collected from patients is false. There was the problem of producing positives.

Therefore, in order to suppress the non-specific reaction due to the influence of RF etc., the pretreatment of the sample with dithiothreitol, 2-mercaptoethanol or the pretreatment of the sample such as the addition of γ-globulin or its modified product, Improvements in immunoassays such as the use of Fab, F (ab ') ₂ etc. fragments of antibodies have been proposed.

For example, Journal of Clinical Microbiolo
gy, Feb. 1976, p.157-160 describes the pretreatment of specimens with dithiothreitol. Japanese Patent Publication 61-94
Japanese Unexamined Patent Publication No. 2 discloses a method for immunologically measuring an antigen or an antibody in a sample in the presence of γ-globulin of an unsensitized normal animal or its modified product.

Regarding the use of antibody fragments, for example, Japanese Examined Patent Publication (Kokoku) No. 63-38668 discloses that an F (ab ') ₂ fragment of immunoglobulin cleaved from the Fc portion is involved because it is involved in a non-specific reaction. An immunoassay for use as a measuring antibody is disclosed.

Particularly, in the agglutination method using latex, a derivative such as urea or formamide and di-lower alkyl sulfoxide or the like are blended to prevent nonspecific reaction due to RF or the like (Japanese Patent Publication No. 60-4941). , And avoiding RF interference by using latex particles sensitized with a mixture of F (ab ′) ₂ fragment of immunoglobulin and Fc fragment or Facb fragment and pFc ′ fragment (JP-B-63-
No. 63863 and Japanese Patent Publication No. 63-41426)
It has been known.

[0008]

However, the method of pretreatment of a sample has a problem that the procedure at the time of measurement increases and a problem that the sensitivity of the measurement decreases, and the antibody for immunological measurement is enzymatically hydrolyzed. Fab and F obtained by processing
The method using an antibody fragment such as (ab ′) ₂ has a problem such as influence on the antibody in the treatment.

The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, based on a previously unknown principle, a nonspecific reaction due to RF interference in an immunological assay method. I found a way to eliminate.

[0010]

The present invention provides an antibody for immunoassays in which the Fc portion is blocked by a blocking reagent containing at least an antigen-binding portion of an immunoglobulin that specifically binds to the Fc portion of the antibody. I will provide a. That is, the present invention provides an antibody in which the Fc portion is blocked based on an antigen-antibody reaction and which does not non-specifically react with substances such as RF.

The present invention also provides an immunological assay reagent containing the above antibody.

Furthermore, the present invention provides an immunological assay method using the above-mentioned immunological assay reagent. According to the immunological assay method of the present invention, nonspecific reaction with substances such as RF is eliminated.

In addition, the present invention is an immunoglobulin which specifically binds to the Fc part of an antibody for blocking the Fc part of an antibody used in an immunoassay and eliminating interference of substances such as RF. There is provided a blocking reagent containing at least the antigen-binding site.

In the present invention, the immunological measurement method is a method utilizing an immune reaction (antigen-antibody reaction), and includes both a qualitative method and a quantitative method. Examples of immunoassays include single radial immunodiffusion (SRID), agglutination (aggregation using a carrier such as latex or blood cells), radioimmunoassay (RIA), enzyme immunoassay. Law (E
IA), immunoturbidimetric assay (TIA), immunonephelometry (Immuno)
nephelometry), a fluorescent immunoassay, a luminescent immunoassay, a nephelometry based on an agglutination method, a nephelometry, a particle size distribution measurement method and the like.

The antibody of which the Fc portion is blocked according to the present invention is an antibody which is used in these immunological assay methods and which binds to a measurement substance in a sample. For example, a mouse,
Those derived from rabbits, guinea pigs, sheep, goats, etc., and those of the IgG, IgM and other classes are used.

Since RF easily binds to the Fc portion of this antibody, the original antigen-antibody reaction is interfered with by RF, resulting in an erroneous measurement of a substance in a biological fluid. It was prone to give false positives due to specific aggregation.

The present invention solves this problem in advance by blocking the Fc portion of the antibody to which RF specifically binds.

The blocking reagent of the present invention blocks the Fc portion of an antibody used in an immunoassay, eliminates RF interference, and specifically binds to the Fc portion of an antibody. It contains at least the antigen-binding site of immunoglobulin. The antigen-binding site recognizes the antigen,
It is a variable region ( _VL , _VH region) capable of binding to an antigen.

Examples of this blocking reagent include whole immunoglobulins or fragments thereof. Examples of fragments are Fv, Fab, Fab ', F (a
b ′) ₂ , Facb, Fab / c.

Since the immunoglobulin or a fragment thereof interferes with RF interference, it is preferable that the immunoglobulin itself does not have a site for binding to RF. For example, F
v, Fab, Fab ′, F (ab ′) ₂ fragments can be used. Preferably small molecules such as Fa
Fragments such as b, Fab ′, Fv are used. Further, in the agglutination method, in order to reduce the side effect of the blocking reagent, one having one antigen binding site per fragment, for example, one in which the S—S bond of the hinge region is cleaved is preferable. Particularly, fragments such as Fab and Fab ′ are typical. Further, these fragments may be modified with another compound or the like.

The origin and class of the immunoglobulin used in the blocking reagent are not particularly limited, but immunoglobulins of mouse, rabbit, rat, guinea pig, sheep, goat, chicken can be used, and IgG, IgM and other classes can be used. Things can be used. It is also possible to use chimeric immunoglobulins.

When chicken IgG that does not react with RF is used as the blocking reagent, the blocking reagent and the RF are used.
Since it does not have to take into account the interference of the whole, and its arbitrary fragments (eg Fv, Fab, Fab ', F
(Ab ′) ₂ , Facb, Fab / c) can be used.

Such an immunoglobulin can be obtained as a commercial product, or can be obtained from an animal using the Fc fragment as an antigen. For example, commercially available goat anti-mouse IgG Fc fragment (γ chain specifi
c) (Organon Technica), goat anti-rabbit IgG
There are Fc fragments (γ chain specific) (manufactured by Organon Technica) and the like, which are selected according to the antibody of interest. Further, methods for obtaining an immunoglobulin having a desired specificity from animals are known to those skilled in the art. The Fc fragment of an antibody that is an antigen to be given to an animal in order to obtain the blocking immunoglobulin may be one obtained from an animal of the same species as the immunological measurement antibody, and the measurement antibody is used as an antigen. No need.

Fragments of immunoglobulins can be obtained by digesting immunoglobulins with proteases. A method for producing an immunoglobulin fragment is known to those skilled in the art, and is described in "Sequel Biochemistry Experimental Course 5-Immunobiochemistry Research Method", Tokyo Kagaku Dojin, 1986, pp. 89-99, and
See RR Porter, Biochem. J., 73, 119 (1959).

Usually, the proteases used are papain, pepsin, plasmin, trypsin and the like. For example, a Fab fragment can be obtained by digestion with papain, and an F fragment can be obtained by digestion with pepsin.
(Ab ') ₂ fragments can be obtained.

Methods for cleaving the S--S bond in the hinge region of immunoglobulins or fragments thereof are known to those skilled in the art. For example, by treatment with a reducing agent such as dithiothreitol, 2-mercaptoethanol, followed by treatment with an SH reagent such as iodoacetamide, S-
S-bonds can be cleaved.

Separation and purification of the obtained fragment is not essential, but it is preferable to separate and purify the fragment by ion exchange chromatography, gel filtration chromatography or the like.

The blocked immunoassay antibody of the present invention can be obtained by binding a conventional immunoassay antibody with a blocking reagent and blocking the Fc portion of the antibody with the blocking reagent.

The antibody for immunological measurement and the blocking reagent of the present invention are usually bound so that the number of blocking reagents per molecule of the antibody for immunological measurement is 0.5 to 6 on average. Preferably, the number of blocking reagents per molecule of the antibody for immunological measurement is about 3 on average.

For example, the antibody and Fab or Fab adsorbed on the latex used in the latex agglutination method.
When binding a blocking reagent containing a b'fragment, the number of Fab or Fab 'fragments per antibody molecule is preferably 1.0 to 3.5 on average. In addition, the antibody solution used in the EIA method and Fab or Fa
When a blocking reagent containing a b'fragment is used, the number of Fab or Fab 'fragments per antibody molecule may be 5.0 to 6.0 on average.

The binding of the antibody for immunological measurement and the blocking reagent is usually carried out by mixing the two in a solution, and the mixing temperature is usually 2 to 50 ° C, preferably 4 to 37 ° C. . The mixing time is usually 1 minute to 2 minutes.
Is the day. Conditions such as mixing ratio, mixing temperature, mixing time, etc. differ depending on the affinity of the immunoglobulin that specifically binds to the Fc portion of the antibody, the antibody titer, the purity, the concentration or density of the antibody in the immunoassay reagent, etc. . For example, the difference in antibody titer for each lot of immunoglobulin raw material is well known to those skilled in the art, and those skilled in the art can easily determine the above-mentioned mixing temperature and mixing time, and The mixing ratio can be modified.

The immunological assay reagent of the present invention is the above-mentioned F
It includes an antibody in which the c site is blocked.

Examples of such immunological measurement reagents include those containing an antibody adsorbed on a carrier or a substrate. Examples of the carrier include latex, blood cells, colloidal gold, gelatin particles and the like, and examples of the substrate include glass, plastic, film and the like. Also, some include antibodies labeled with an enzyme or the like. Particularly, a latex reagent, an antibody adsorbed on a microplate, an enzyme-labeled antibody and the like can be mentioned.

The immunoassay reagent of the present invention is obtained by mixing a commonly used immunoassay reagent containing an antibody and a blocking reagent and binding the antibody and the blocking reagent as described above. be able to.

The immunological measurement reagent of the present invention may be subjected to a treatment which is usually carried out, such as the addition of a stabilizer such as bovine serum albumin.

The immunological assay method of the present invention is an assay method using the immunological assay reagent of the present invention as described above.

This immunological measurement method can be carried out by the same procedure as the measurement method using an ordinary immunological measurement reagent.

As an example, the latex agglutination method is
(1) One drop (eg, 20 μl) of a sample such as serum is dropped into the circle of the slide plate for judgment, and (2) One drop of a latex reagent containing an antibody blocking the Fc site is dropped into the circle of the slide plate for judgment. (3) Mix the sample and latex reagent with a stirring rod, gently rock the slide plate for judgment, and (4) visually judge the presence or absence of aggregation after shaking for several minutes (for example, 1 to 5 minutes). If agglutination is observed,
It can be performed by judging as positive.

Further, as an example of the EIA method, the microplate sandwich method is used. (1) A calibrator and a sample are incubated in each well of an antibody solid phase microplate containing an antibody having an Fc site blocked (eg, 37 ° C.). (15 minutes), (2) aspirate the solution in each well, add a washing solution, and (3) repeat the procedure of step (2) several times (for example, four times) to wash the well, and (4)
After removing water, an enzyme-labeled antibody containing an antibody that blocks the Fc portion is added to each well and incubated (eg, 37 ° C. for 15 minutes), and (5) the procedure (2) is repeated several times (eg, 4 times). , Wash wells, (6)
The enzyme reaction reagent is added to each well and incubated (for example, 37 ° C. for 10 minutes), (7) the enzyme reaction stop solution is added to stop the reaction, and (8) the absorbance of each well (for example, 450 nm) using the reagent blank as a control. In (9), a calibration curve is prepared from the measured values of the calibrator, and the substance in the sample is quantified. In the case of this example, a sufficient effect can be obtained if either the antibody solid phase microplate of step (1) or the antibody of the enzyme-labeled antibody solution of step (4) has its Fc site blocked. However, when only one of them is blocked, it is preferable that the antibody of the antibody solid-phase microplate has its Fc portion blocked.

As can be understood from the above, the immunoassay of the present invention can be carried out by replacing the conventional immunoassay with the immunoassay of the present invention. The procedure can be performed in the same manner as the method, and R
The interference of F can be suppressed.

In the immunological assay method in which the blocked antibody of the present invention is used, the sample is not particularly limited, and it can be applied to a target sample by a usual immunological assay method. Samples of human origin, especially human blood, urine,
Examples include feces. In particular, it is advantageous in the case of a specimen having a high RF content such as blood.

The substance to be measured in the immunological measuring method of the present invention is not particularly limited, and those measured by a usual immunological measuring method can be measured. Examples are CEA, AF
P, CA19-9, hCG, β _2m , tumor marker such as ferritin, protein C, protein S, ATIII, F
Coagulation and fibrinolysis system markers such as DP and D-dimer, CRP,
Infectious disease markers such as ASO and HBs antigens, hormones such as TSH, prolactin and insulin, IgE and Ig
Examples include immunoglobulins such as A, IgG, C3, and C4 and complement components, and tissue components such as myoglobin and myosin.

The present invention is a substance other than RF (F) which is contained in a sample and binds to the Fc portion of the antibody for immunological measurement, interferes with the immune reaction for measurement, and causes a non-specific reaction.
c-bonding substance). Examples of such Fc-binding substances include complement C1q and Fc receptor.

[0044]

The present invention will be described in more detail by the following examples, but the present invention is not limited to the following examples.

Preparation Example 1 Papain elimination of goat IgG-anti-mouse IgG (Fc) antibody
Preparation of Fab blocking reagent by derivatization 50 mg of goat IgG-anti-mouse IgG (Fc) antibody (immunoglobulin) and 0.5 mg of papain were added to 10 mM cysteine-2 mM E
After dissolving in DTA-0.1M sodium phosphate buffer (pH 7.0) and digesting at 37 ° C for 75 minutes, 6.3 mg of iodoacetamide was added in the dark, and the mixture was incubated at 37 ° C for 15 minutes. Add 0.01 M sodium phosphate buffer (pH 8.0) to the reaction mixture.
), The internal solution was taken out, and the Fab fragment (blocking reagent) using a DEAE-Sepharose column.
Was separated and purified.

Preparative Example 2 Pepsin depletion of goat IgG-anti-rabbit IgG (Fc) antibody
Of Fab ′ blocking reagent by cyclization and cleavage of S—S bond
After manufacturing goat IgG- anti-rabbit IgG (Fc) antibodies (immunoglobulins) 50 mg pepsin 1.7mg were dissolved in 0.1M sodium acetate buffer (pH 4.0), and 18 hours digestion at 37 ° C., 10
It was dialyzed against mM Tris-hydrochloric acid buffer (pH 8.0). 2-mercaptoethanol was added to the inner solution at 10 mM and incubated at 37 ° C. for 75 minutes, then iodoacetamide was added at 11 mM in the dark, and the mixture was reacted at 4 ° C. for 40 minutes. The reaction solution
It was dialyzed against 50 mM glycine buffer (pH 8.6), the internal solution was taken out, and the Fab 'fragment (blocking reagent) was separated and purified by gel filtration.

Example 1 (1) Preparation of anti-D-Dimer antibody-sensitized latex reagent 0.75 mg of mouse IgG-anti-D-Dimer antibody was dissolved in 9.5 ml of sodium phosphate buffer to prepare 0.65 μm polystyrene latex (solid content). 10 wt% suspension) 0.5 ml,
After stirring at room temperature for 2 hours, the sensitized latex was centrifuged to collect the supernatant. From this supernatant, the amount of adsorbed IgG antibody was calculated by the following method.

(A) The supernatant is filtered with a 0.1 μm filter to remove a trace amount of latex which seems to be contaminated.

(B) Unadsorbed IgG remaining in the filtrate
The antibody concentration is calculated by measuring the absorbance at 280 nm.

(C) The amount of IgG antibody used was subtracted from the non-adsorbed amount calculated in (b) to obtain the Ig adsorbed on the latex.
Let G antibody amount.

On the other hand, the precipitate was obtained from the Fab obtained in Preparation Example 1.
The Fc portion of the mouse IgG antibody was blocked by suspending it in 10 ml of glycine buffer containing 2.25 mg of the fragment. Further, the mixture was centrifuged to collect the supernatant. From this supernatant, the amount of bound Fab fragment was calculated in the same manner as above.

The precipitate was dissolved in bovine serum albumin solution (0.1 M glycine, 0.05 M sodium chloride, 0.05% sodium azide,
0.2% BSA) to prepare a latex reagent sensitized with anti-D-Dimer antibody.

The molar ratio was calculated from the amount of IgG antibody adsorbed on the latex and the amount of Fab fragment bound to the IgG antibody calculated as described above, and the mouse I
Anti-mouse IgG bound per molecule of gG antibody
The number (average) of Fab fragments of the (Fc) antibody was used.

(2) Nonspecific reaction with RF RF positive human serum 653 IU / ml (International Immunology
Corporation) and anti-D-Dim prepared as described above.
er antibody-sensitized latex reagent mixed drop by drop,
The reaction plate was gently rocked and the aggregation image was observed for 10 minutes.
Fa that blocks the Fc portion of mouse IgG antibody
Anti-D-Dim with different numbers of b fragments (Preparation Example 1)
Table 1 shows the results of comparison using the er antibody-sensitized latex reagent.

[0055]

[Table 1]

The Fc portion of the mouse IgG antibody is anti-mouse I
Latex reagents prepared with antibodies blocked by the Fab fragment of the gG (Fc) antibody have R
Nonspecific aggregation of F No aggregation reaction was observed.

Example 2 (1) Preparation of anti-myoglobin antibody-sensitized latex reagent 1.5 mg of rabbit IgG-anti-myoglobin antibody was dissolved in 9.5 ml of glycine buffer to give 0.46 μm polystyrene latex (10% solids suspension). ) 0.5 ml was added and the mixture was stirred at room temperature for 2 hours, and then the sensitized latex was centrifuged to collect the supernatant. The precipitate was obtained using the Fa obtained in Preparation Example 2.
The Fc portion of the rabbit IgG antibody was blocked by suspending it in 10 ml of glycine buffer containing 1.5 mg of the b'fragment.
Further, the mixture was centrifuged to collect the supernatant. Precipitate the bovine serum albumin solution (0.1M glycine, 0.05M sodium chloride,
0.05% sodium azide, 0.2% BSA),
An anti-myoglobin antibody-sensitized latex reagent was prepared.

Further, as in Example 1, the amount of IgG antibody adsorbed on the latex and the Fa bound to the IgG antibody were
The amount of the b'fragment was calculated, the molar ratio was calculated, and the anti-rabbit Ig bound per molecule of the rabbit IgG antibody was calculated.
The number (average) of Fab ′ fragments of G (Fc) antibody was used.

(2) Nonspecific reaction with RF RF positive human serum 653 IU / ml (International Immunology
Corporation) and the anti-myoglobin antibody-sensitized latex reagent prepared as described above were mixed drop by drop, and the reaction plate was gently rocked to observe an agglutination image for 10 minutes. In addition,
Table 2 shows the results of comparison using anti-myoglobin antibody-sensitized latex reagents having different numbers of Fab ′ fragments (Preparation Example 2) blocking the Fc portion of rabbit IgG antibody.

[0060]

[Table 2]

The Fc portion of the rabbit IgG antibody is anti-rabbit I
Latex reagents prepared with antibodies blocked by the Fab 'fragment of the gG (Fc) antibody are:
RF non-specific aggregation No aggregation reaction was observed.

(3) Measurement of Myoglobin in Serum The serum of the subject and the latex reagent prepared in (1) were mixed drop by drop, and then the reaction plate was gently rocked to observe an agglutination image for 5 minutes. Agglomeration image measured in this way,
As a control, an agglutination image obtained by measuring in exactly the same manner using a sensitized latex reagent in which the Fc portion of the anti-myoglobin IgG antibody was not treated with a block, and 2 mg / gram of denatured γ-globulin in the serum of a subject. The results are compared with the agglutination image obtained by pretreatment of adding the solution to a concentration of ml and allowing it to stand for 1 hour, and the results of quantifying RF using RF-latex "Seiken" 3 shows.

[0063]

[Table 3]

The control method (no Fc site blocking) was:
In specimens Nos. 3 and 4 with high RF values, non-specific agglutination reaction due to RF is observed. It is also considered that sample Nos. 8 and 9 were also affected by the non-specific agglutination reaction. On the other hand, non-specific agglutination reaction of RF was not observed in the measurement after adding the denatured γ-globulin and pre-treating the sample, and the measurement by the reagent of the present invention showed that RF was not observed.
No non-specific agglutination reaction was observed.

Example 3 (1) Dab of goat IgG-anti-mouse IgG (Fc) antibody
Preparation of Fab blocking reagent by in-digestion Goat IgG-anti-mouse IgG (Fc) as in Preparation Example 1.
After papain digestion of the antibody, iodoacetamide was added to 10 mM in the dark and incubated at 37 ° C for 15 minutes.
The reaction solution was dialyzed against 100 mM glycine buffer (pH 8.6),
A coexisting substance of Fab fragment (blocking reagent) and Fc fragment was obtained.

(2) Anti-D-Dimer Antibody Sensitized Latek
Preparation of reagent (A reagent) (control method) Mouse IgG-anti-D-Dimer antibody (1.25 mg ) was dissolved in sodium phosphate buffer (9.5 ml ) to prepare 0.65 μm polystyrene latex (solid content 10% by weight buffer) (0.5 ml). And add
After stirring at room temperature for 2 hours, the sensitized latex reagent was centrifuged and the supernatant was discarded. The precipitate was added to bovine serum albumin solution (0.1M glycine, 0.05M sodium chloride, 0.05% sodium azide, 0.2% BSA). Suspend and anti-D-Dim
er antibody-sensitized latex reagent was prepared.

(3) Adsorbed on latex in reagent A
Fc part of the mouse IgG-anti-D-Dimer antibody
Of the unpurified Fab fragment prepared in (1)
2.25 mg of the unpurified Fab fragment prepared in (1) was added to 0.5 ml of Block A reagent, incubated at 37 ° C for 30 minutes, centrifuged and the supernatant was discarded. The precipitate was suspended in bovine serum albumin solution. The reagents were prepared.

(4) Adsorbed on latex in reagent A
Fc part of the mouse IgG-anti-D-Dimer antibody
Of the purified Fab fragment obtained in Preparation Example 1
300 μg of the purified Fab fragment obtained in Preparation Example 1 was added to 0.5 ml of the Block A reagent, and the mixture was incubated at 37 ° C. for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was suspended in bovine serum albumin solution. The reagents were prepared.

(5) Non- Specific Reaction with RF The reactivity of 100 IU / ml RF positive control (Japan Roche Co., Ltd.) with the reagents A, B and C prepared as described above was compared. Table 4 shows the results of observing the agglutination image for 5 minutes by gently shaking the reaction plate after adding A reagent, B reagent and C reagent to each one drop of the RF positive control and mixing them. Show.

[0070]

[Table 4]

With the reagent A (control), nonspecific RF agglutination reaction is observed. On the other hand, non-specific agglutination reaction of RF was not observed in the reagent B and the reagent C in which the blocking reagent (Fab fragment of anti-mouse IgG (Fc) antibody) was added to the reagent A.

Example 4 (1) of solid phase antibody in EIA method (sandwich method)
100 μl of the blocking reagent obtained in Preparation Example 1 was dispensed into each well of a microplate coated with a blocking mouse monoclonal antibody (anti-FgDP / anti-FbDP antibody) at the Fc site, and the mixture was shaken at room temperature for 30 minutes, and then each well was shaken. The solution was aspirated and phosphate buffer was added for washing. This washing operation is repeated 3 times,
Wells (group B) in which the Fc portion of the solid phase antibody was blocked were obtained.

The number of molecules of the Fab fragment of the anti-mouse IgG (Fc) antibody in which the Fc portion was blocked was determined by the Protein Assay kit (BIORAD).
Was estimated using the solid phase antibody of group B (mouse Ig
About 1 molecule of anti-mouse IgG (F
c) The Fab fragment of the antibody is bound.

In addition, 100 μl of phosphate buffer was added instead of the above blocking reagent, and the wells treated in the same manner (A
Group) was obtained as a control.

(2) Nonspecific reaction with RF RF positive human serum 1421 IU / ml (Internati)
onal Immunology Corporation
on) and a phosphate buffer, an RF concentration of 1421 IU /
Samples of ml, 710 IU / ml, 89 IU / ml and 0 IU / ml were prepared.

100 μl of each sample was dispensed into the wells (Group A) and (Group B) obtained in (1), and the wells at 37 ° C.
Incubated for 5 minutes.

After the well washing operation was repeated 4 times, 100 μl of mouse monoclonal antibody labeled with peroxidase (anti-FbDP antibody-HRP) was dispensed into each well and incubated at 37 ° C. for 15 minutes.

Thereafter, the well was washed four times, 100 μl of a tetramethylbenzidine solution was dispensed, and the well was washed at 37 ° C.
And incubated for 10 minutes.

Next, the enzyme reaction was stopped by adding 100 μl of 1M sulfuric acid to each well. After stopping the reaction,
Microtiter plate reader (Reader 5
10 Organon Technica Co., Ltd.) was used to measure the absorbance at 450 nm.

Table 5 shows the measurement results.

[0081]

[Table 5]

From the results in Table 5 above, in the EIA method,
It can be seen that the group B in which the blocking reagent was added to the solid phase antibody suppressed the non-specific reaction of RF as compared to the group A (control) which was not treated.

[0083]

EFFECTS OF THE INVENTION According to the present invention, since non-specific reactions due to substances such as RF can be eliminated, false positives can be reduced and specificity can be improved, and more reliable clinical tests can be performed.

Further, according to the present invention, the pretreatment of the sample itself is not required, rapid examination is possible, and the problem of the decrease in measurement sensitivity is solved. Further, since the measuring antibody itself which directly reacts with the measuring component in the sample does not need to be enzymatically hydrolyzed in order to avoid the influence of substances such as RF, the measuring antibody is enzymatically hydrolyzed. It is possible to avoid the effects (decrease in measurement sensitivity, decrease in antibody stability) associated with the above.

Claims (18)

[Claims] 1. An antibody for immunoassay, wherein the Fc portion is blocked by a blocking reagent containing at least an antigen-binding portion of an immunoglobulin that specifically binds to the Fc portion of the antibody. 2. The blocking reagent is Fv, Fab or F.
The antibody according to claim 1, which is a reagent containing an ab ′, F (ab ′) ₂ , Facb, or Fab / c fragment. 3. The antibody according to claim 1, wherein the blocking reagent is a fragment of immunoglobulin containing one antigen-binding site per fragment. 4. The blocking reagent is Fv, Fab or F.
The antibody according to claim 3, which is an ab 'fragment. 5. The antibody of claim 4, wherein the Fc portion of the antibody is blocked with an average of 0.5-6 Fab or Fab 'fragments per site. 6. A reagent for immunological measurement, which comprises the antibody according to any one of claims 1 to 5. 7. The immunological measurement reagent according to claim 6, wherein the antibody is adsorbed on a carrier. 8. The immunological assay reagent according to claim 7, wherein the carrier is latex, glass or plastic. 9. The carrier is a latex.
The reagent for immunological measurement according to 1. 10. The immunological measurement reagent according to claim 6, wherein the antibody is adsorbed on a substrate. 11. The immunological measurement reagent according to claim 6, wherein the antibody is labeled. 12. An anti-IgG comprising a latex carrier sensitized with an IgG antibody, wherein the Fc portion of the IgG antibody is heterogeneous.
The immunological assay reagent according to claim 6, which is a latex reagent blocked with a Fab or Fab ′ fragment of the (Fc) antibody. 13. An immunological assay method using the immunological assay reagent according to any one of claims 6 to 12. 14. A single radial immunodiffusion method (SRID)
Method), agglutination method, radioimmunoassay (RIA), enzyme immunoassay (EIA), immunoturbidimetric assay (TIA), immunonephelometry (Immunonephelometry), fluorescent immunoassay, luminescent immunoassay, or A turbidimetric method, a brazing method or a particle size distribution measuring method based on the agglutination method.
The immunological assay method according to item 3. 15. A blocking reagent for blocking the Fc portion of an antibody for immunoassay, which comprises at least an antigen binding site of an immunoglobulin that specifically binds to the Fc portion of the antibody. 16. Fv, Fab, Fab ′, F (a
The blocking reagent according to claim 15, comprising b ′) ₂ , Facb or Fab / c fragment. 17. An immunoglobulin fragment containing one antigen-binding site per fragment.
The blocking reagent according to item 5. 18. The blocking reagent according to claim 17, which is an Fv, Fab or Fab ′ fragment.