JP4121432B2 - Casein for immunoassay reagents - Google Patents

Description

  The present invention relates to an immunoassay reagent. More specifically, the present invention relates to an immunoassay reagent that reduces non-specific reactions.

In order to prevent nonspecific reaction, immunoassay reagents contain various proteins, surfactants, and the like. In particular, casein has been reported to be useful as an inhibitor of nonspecific reactions (for example, Patent Document 1). Furthermore, heat-treated casein or casein treated with a degrading enzyme is also used (for example, Patent Documents 2 and 3).
JP-A-1-217266 JP-A-6-194366 JP-A-2-36353

  However, conventional casein-containing immunoassay reagents sometimes have a low effect of preventing nonspecific reactions of labeled ligands. Furthermore, the effect of preventing non-specific reactions is reduced over time, and the measurement sensitivity may be significantly reduced. That is, an object of the present invention is to provide a casein-containing immunoassay reagent excellent in the effect of preventing a nonspecific reaction of a labeled ligand.

As a result of intensive studies to solve the above problems, the present inventor has found that the above object can be achieved by using a specific casein, and has reached the present invention. That is, the casein (A) for immunoassay reagents of the present invention is characterized in that the content (% by weight) of casein (a), which is a fraction having a molecular weight larger than 900,000 daltons, has a molecular weight of 75,000 to 375,5. The gist is that it is 1 or less based on the weight of casein (A) which is 1,000 Daltons .
In addition, the method for producing the immunoassay reagent of the present invention is characterized in that the content (% by weight) of casein (a), which is a fraction having a molecular weight larger than 900,000 daltons, has a molecular weight of 75,000 to 375,000 daltons. a method of manufacturing a immunoassay reagent containing casein is 1 or less based on the weight of (a) (a) is, after the heating treatment at 20 to 60 ° C. a solution containing (a) , (A) is a point including the step of adsorbing and removing.

The casein (A) for immunoassay reagents of the present invention is extremely effective in preventing non-specific reactions of labeled ligands (particularly non-specific reactions of enzyme-labeled ligands). Moreover, it is difficult to reduce the effect of preventing non-specific reactions over time. Therefore, the immunoassay reagent containing the casein (A) for immunoassay reagent of the present invention enables a highly sensitive and highly stable clinical test.
Moreover, since the casein (a) precursor which can become casein (a) is removed from the immunoassay reagent produced by the method for producing an immunoassay reagent of the present invention, casein (a) is generated over time. There is nothing. Therefore, the production method of the present invention has an extremely high effect of preventing nonspecific reaction of a labeled ligand, and can easily produce an immunoassay reagent that is difficult to reduce the effect of preventing nonspecific reaction over time. Can do.

  The molecular weight of casein is in the range of about 75,000 to 375,000 daltons (ultracentrifugation) according to Chemistry Encyclopedia II, Kyoritsu Shuppan (1989), page 409, but it is actually an immunoassay using casein. When the reagent for preparation is prepared, there is casein which is a fraction having a molecular weight larger than 900,000 Dalton. This may be because part of casein contains high molecular weight casein, or part of casein has high molecular weight in an immunoassay reagent. Casein may be combined with other components in the immunoassay reagent to increase the molecular weight. These high molecular weight caseins correspond to casein (a) which is a fraction having a molecular weight larger than 900,000 daltons in the present invention. When the content (% by weight) of casein (a) is a certain amount or more, not only the effect of preventing the nonspecific reaction of the labeled ligand is reduced, but the nonspecific reaction may become remarkable. .

  That is, the content (% by weight) of casein (a) is preferably 1 or less, more preferably 0.5 or less, and particularly preferably 0.1 or less, based on the weight of casein (A). The content of casein (a) can be quantified by a conventionally known method. For example, when quantitative analysis is performed by gel filtration HPLC, casein (a) with respect to the peak area derived from casein (A) obtained by gel filtration HPLC (a ) Derived peak area ratio can be calculated. In this case, the molecular weights of casein (A) and casein (a) are appropriate molecular weight markers such as IgM (molecular weight 900,000 daltons), IgG (molecular weight 150,000 daltons), ferritin (molecular weight 450,000 daltons) and albumin (molecular weight 50,000). A calibration curve can be created and calculated by analyzing (Dalton) etc. under the same conditions. In particular, casein (a) is a fraction having a molecular weight larger than 900,000 daltons, and can be easily identified by comparing with IgM having a molecular weight of 900,000 daltons. Moreover, it can quantify similarly by electrophoresis etc. by SDS-PAGE.

  As a raw material of casein (A), a product obtained by separating and purifying casein contained in mammalian milk by a known method such as an acid addition method can be used. For example, casein separated and purified from milk is widely used in various food, medical and industrial applications and is also suitable in the present invention. Casein usually contains a plurality of fractions having different properties such as isoelectric point and solubility, for example, αs-casein, β-casein and κ-casein. In the present invention, these fractions are fractionated. A part of it may be used, or all fractions may be used. Further, sodium caseinate produced by adding sodium hydroxide to these caseins can also be used. Of these, sodium caseinate containing all fractions is preferable from the viewpoints of cost and solubility during preparation of the reagent.

When casein (a) is contained in excess of the above content, it is preferable to remove (a). As this removal method, a conventionally known method can be used. For example, after dissolving casein in an appropriate solvent, a method of removing (a) with a gel filtration column or the like can be applied. In addition, a method of adsorbing and removing (a) with an adsorbent (diatomaceous earth or the like) can also be applied. For example, 10 g to 200 g of diatomaceous earth (for example, trade name Celite No. 545, purchased from Wako Pure Chemical Industries, Ltd.) is added to 1 liter of a solution containing casein (a) (including a solution after adjusting an immunoassay reagent) After stirring at (10 to 25 ° C.) for 5 to 60 minutes, casein (a) is also adsorbed and removed by removing diatomaceous earth by filtration or centrifugation.
In this removal step, (a) can be removed before, during, or after the preparation of the immunoassay reagent, but the casein (a) -removed casein can be used as an immunoassay reagent. Since casein (a) may occur in the step of adjusting, it is preferable to perform the step of removing casein (a) in the final step of preparing the immunoassay reagent.

Further, even when the casein has a casein (a) content of not more than the above content, during the storage of the casein (A), during the preparation of the immunoassay reagent, or during the storage of the immunoassay reagent, Casein (a) precursor to be casein (a) is often included, and in this case, it is preferable to remove (a) after changing the casein (a) precursor to casein (a). .
As a method for changing the casein (a) precursor to casein (a), a casein-containing solution (including a solution after adjustment of an immunoassay reagent) is allowed to stand at a certain temperature for a certain period. As constant temperature (degreeC), 20-60 are preferable, More preferably, it is 30-50, Most preferably, it is 35-45. The standing period varies depending on the temperature, and is preferably 1 to 14 days, more preferably 2 to 10 days, and particularly preferably 3 to 7 days in the case of 35 to 45 ° C. It is preferable that the temperature is left longer as the temperature becomes lower.
Thus, the immunoassay reagent excluding the casein (a) precursor does not show the generation of casein (a) over time.

  The casein (A) of the present invention can be applied to immunoassay reagents for various uses. For example, reaction reagents such as immune reaction buffers, storage reagents containing antigens or antibodies, carrier blocking reagents, specimen dilutions It can be applied to a reagent for cleaning and a cleaning reagent used for B / F separation. These reagents can also serve multiple purposes. Among these uses, it is preferably applied to a reaction reagent and a storage reagent, and more preferably an immunoassay reagent that serves as both a reaction reagent and a storage reagent, that is, an immunoassay reagent that uses a storage reagent as it is as a reaction reagent. .

  The most important object of the present invention is to provide an immunoassay reagent that is highly effective in preventing non-specific reactions, in particular, non-specific reactions of labeled ligands. Casein (a) is non-specific reactive ( Adsorption is high, and nonspecific reaction of the labeled ligand is induced by nonspecifically binding (adsorbing) with the labeled ligand. Casein (A), excluding casein (a), does not bind (adsorb) non-specifically with the labeled ligand, but blocks the substances that react non-specifically with the labeled ligand. The effect of preventing non-specific reaction of the child occurs. Therefore, a particularly preferred application is an immunoassay reagent that uses a storage reagent containing a labeled ligand as it is as a reaction reagent.

  In the present invention, the labeled ligand is a conjugate of a ligand and a label. The ligand is a substance that selectively binds to the measurement object or a substance that shows the same immunoreactivity as the measurement object. As the substance that selectively binds to the measurement target, when the measurement target is an antigen, the ligand is an antibody against the antigen. When the measurement object is an antibody, the ligand is an antigen recognized by the antibody. When the measurement object is a sugar chain, the ligand is a lectin. When the measurement object is biotin, the ligand is avidin. When the measurement object is a gene, the ligand is a complementary gene. One of the measurement object and the ligand can be used as the ligand as in the case of the antigen and the antibody. In addition, a ligand that specifically binds to the measurement object and a second ligand that specifically binds to the ligand can be combined and used as a ligand.

  As the antibody, an antibody against the following antigen can be used, and examples of the antigen include drugs, low molecular hormones, cancer markers, viruses, high molecular hormones, cytokines, various growth factors, and appropriate DNAs and RNAs of the viruses. included. Examples of the drug include theophylline, phenytoin and valproic acid. Examples of low molecular hormones include thyroxine, estrogen, and estradiol. Examples of cancer markers include CEA, AFP, CA19-9 and the like. Examples of viruses include HIV, HCV and HBV. Examples of the high molecular hormone include thyroid stimulating hormone and insulin. Examples of cytokines include IL-1, IL-2 and IL-6. Examples of various growth factors include EGF and PDGF. These antibodies may be monoclonal antibodies or polyclonal antibodies, and may be F (ab ') 2, Fab', Fab which are degradation products of antibodies.

  As a lectin, concanavalin A (concanavalin A specifically binds to a sugar chain containing 2 or more residues of α-D-Man having an unsubstituted hydroxyl group at the C-3, C-4 or C-6 position). , Doricos bean lectin (Dorico bean lectin specifically binds to D-GalNAc), Datsura lectin (Datsura lectin specifically binds to chitin oligosaccharide and N-acetyllactosamine), Deigo bean lectin (Deigo bean lectin specifically binds to an asparagine complex-type sugar chain having an N-acetyllactosamine structure) and fucose binding protein (fucose bindin protein is α-linked to position 2 such as Gal. It binds specifically to L-Fuc).

  As the substance exhibiting the same immunoreactivity as the measurement object, the same substance as the measurement substance, a fragment of the measurement substance having the same immunoreactivity, an analog of the measurement object, and the like are used.

  As the labeling substance, known substances such as isotope [125I etc.], enzyme [peroxidase, alkaline phosphatase, β-galactosidase etc.], fluorescent substance [europium derivative etc.], and luminescent substance [acridium derivative etc.] can be used. Of these, enzymes and luminescent substances are preferred, and enzymes are more preferred.

  The binding between the ligand and the labeled substance is performed by a conventionally known method, for example, the method described in “Second Biochemistry Experiment Course 5 Immunobiochemistry Experiment Method” (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1986) p102-112. Can be implemented. When the label is an isotope, for example, radioactive iodine can be introduced into the tyrosine residue of the ligand using chloramine T as an oxidizing agent. When the label is a fluorescent substance, for example, when fluorescein isothiocyanate is reacted with a ligand in a buffer, it binds to a lysine residue of the ligand. When the label is an enzyme, for example, the amino group possessed by the enzyme and the thiol group possessed by the ligand can be bound with a bi-crosslinking reagent such as N-succinimidyl 6-maledohexanoate. When the label is a luminescent substance, for example, when acridinium derivative-I (manufactured by Dojindo Laboratories) is reacted with a ligand in a buffer solution, the acridinium derivative, which is a luminescent compound, can be bound to the amino group of the ligand. .

  The composition of the immune reaction reagent in the present invention can be freely set depending on the purpose of use. For example, in the case of a solution reagent, a solution obtained by adding casein (A) and other components to an appropriate buffer is usually used. In this case, the content of casein (A) (g / liter) is preferably 0.01 or more based on the volume of the immunoassay reagent at 25 ° C., more preferably 0.1 or more, and particularly preferably 0.00. 5 or more, preferably 30 or less, more preferably 20 or less, and particularly preferably 10 or less. Examples of the buffer include Tris / hydrochloric acid buffer, barbital buffer, and phosphate buffer, and can be appropriately selected depending on the purpose of use of the reagent. Usually, a buffer having a pH of 5 to 9 is used. The concentration of the labeled ligand can take various values, but is usually in the range of 0.01 to 100 mg per unit volume (L) of the reagent.

  As other components, proteins other than casein, salts, surfactants and the like are used. Examples of proteins other than casein include albumin (such as bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin, and lactalbumin), antibodies (such as normal rabbit IgG and normal mouse IgG), and gelatin. Examples of the salt include sodium chloride, potassium chloride and lithium bromide. Examples of the surfactant include nonionic surfactants such as sorbitan lauric acid monoester ethylene oxide adduct [eg, Tween 20 and Tween 40 (ICI America)].

  The immunoassay reagent of the present invention is not limited in reagent properties, shapes, etc., for example, solution reagents, dry reagents (granular and fine powders), reagents in which reaction tubes and insoluble carriers are coated with casein (A), etc. Is mentioned. Among these, a solution-like reagent is most preferable, and a composition in which casein (A) is dissolved in an appropriate buffer is used. When the immunoassay reagent in the present invention is a dry reagent, a solution obtained by drying the solution reagent is used. Examples of the drying method include freeze-drying. For lyophilization, a conventionally known method can be used. For example, after freezing a solution reagent at −20 ° C., the pressure is reduced to 0.5 Torr and drying is started. Dry to 0.1% or less by weight of reagent. In the case of a coated reagent, a solution obtained by contacting a reaction tube, an insoluble carrier or the like with the above solution reagent, and the like are used. For example, if the insoluble carrier is a test tube, dispense a solution reagent into the test tube, leave it at 2-10 ° C. for 24 hours, remove the solution with an aspirator, and air dry at room temperature (20-30 ° C.). Can be created. When coating, it is preferable to contain 0.006 to 0.03 mg of casein (A) per unit surface area (cm 2) of the insoluble carrier.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.
<Example 1>
In this example, when the casein (A) for immunoassay reagent of the present invention is applied to an enzyme-labeled antibody-containing immunoassay reagent, the nonspecific reaction is low.

1. Fractionation of casein 5 g of casein sodium salt (manufactured by Sigma-Aldrich, containing αs-, β- and κ-) was added to 100 mL of a phosphate buffer (0.02M, pH 7.2), and heated to 95 ° C with stirring. After dissolution, it was filtered through a membrane filter having a pore diameter of 0.45 μm to obtain casein solution A. Next, 20 mL of this casein solution A was fractionated with a gel filtration column (Pharmacia, Superdex 200 prep grade 60/600) at a flow rate of 1 mL / min [using phosphate buffer (0.02 M, pH 7.2)]. And from the result of fractionating mouse IgM under the same conditions, a fraction having a molecular weight equal to or less than mouse IgM was fractionated as casein solution B. Further, a fraction having a molecular weight larger than that of mouse IgM was collected as casein solution C. In addition, casein solution B was again separated with a gel filtration column in the same manner, and a fraction having a molecular weight of mouse IgM or less was fractionated as casein solution D.
As a result of analyzing the casein solutions A to D by gel filtration HPLC (column: Shodex GS-620HQ manufactured by Showa Denko), the ratio (% by weight) of casein (a) larger than 900,000 daltons in the casein solution to the total casein is as follows. It was street.
Casein solution A: 3.2%
Casein solution B: 0.3%
Casein solution C: 92.4%
Casein solution D: 0.1%

2. Preparation of Reagent 1) Preparation of Anti-β2-Microglobulin Polyclonal Antibody-Binding Beads Anti-β2-microglobulin polyclonal antibody (manufactured by Dako Japan) was dissolved in 0.1 M carbonate buffer at pH 9 at a concentration of 20 μg / ml. 1000 polystyrene beads (manufactured by Immunochemical Co.) having a diameter of 3.2 mm were added to 20 ml of this solution, and allowed to stand at 2 to 10 ° C. for 48 hours to physically adsorb the anti-β2-microglobulin polyclonal antibody to the polystyrene beads. Thereafter, the solution was removed by suction with an aspirator, and the beads were washed twice with 20 mL of 0.1 wt% bovine serum albumin-containing phosphate buffer (pH 7.2) to prepare anti-β2-microglobulin polyclonal antibody-bound beads. . The anti-β2-microglobulin polyclonal antibody-bound beads were immersed again in 50 mL of a phosphate buffer solution containing 0.1% by weight bovine serum albumin and stored refrigerated (2-10 ° C.).

2) Preparation of peroxidase-labeled anti-β2-microglobulin polyclonal antibody Using anti-β2-microglobulin polyclonal antibody (manufactured by Dako Japan) and horseradish-derived peroxidase (manufactured by Toyobo Co., Ltd.), literature [S Yoshitake, M・ Imagawa, Yi Ishikawa, Etol; Biochem, Vol. 92 (1982) 1413-1424], a peroxidase-labeled anti-β2-microglobulin polyclonal antibody was prepared and stored frozen (−30 ° C.).

3) Preparation of buffer for immunoassay (reaction) 1 g of bovine serum albumin (manufactured by Miles), 0.85 g of sodium chloride and 0.05 g of sodium azide in 100 mL of phosphate buffer (0.02 M, pH 7.2) Dissolved and used as a buffer for immunoassay. Stored refrigerated until use.

4) Preparation of enzyme-labeled antibody solution Using the casein solutions A to D and peroxidase-labeled anti-β2-microglobulin polyclonal antibody prepared above, an enzyme-labeled antibody solution was prepared as follows. That is, casein solutions A to D were added to 10 mL of a phosphate buffer (1M, pH 6.0) so that the weight of casein was 0.1 g, and 100 μg of peroxidase-labeled anti-β2-microglobulin polyclonal antibody was added in a protein amount. Then, 0.05 g of sodium azide and 0.05 g of 4- (methylthio) phenol were added and dissolved by stirring, and the resulting solution was made up to 100 mL with deionized water, and this was used as an enzyme-labeled antibody solution. At this time, by using various combinations of casein solutions A to D, a labeled antibody solution is prepared so that the ratio (weight%) of casein (a) having a molecular weight of 900,000 daltons (Da) is as shown in the table below. And stored refrigerated until use.

5) Preparation of hydrogen peroxide solution 200 μl of 35 wt% hydrogen peroxide solution was dissolved in 1 liter of deionized water to obtain hydrogen peroxide solution. Stored refrigerated until use.

6) Preparation of substrate solution 0.18 g of luminol (manufactured by Tokyo Chemical Industry) and 0.1 g of 4- (cyanomethylthio) phenol were dissolved in 1 liter of Tris / HCl buffer solution of 0.1 M (mol / L), pH 8.5. . Stored in the dark and refrigerated until use.

3. Immune reaction operation In a 12 × 75 mm test tube, 300 μL of immunoassay buffer and 10 μL of standard β2-microglobulin solution (concentration 0, 15, 200 ng / mL) {clinical test drug “Gurazyme β2-produced by Sanyo Chemical Industries Ltd.” The one in “Microglobulin-EIA TEST” was used. } And one anti-β2-microglobulin polyclonal antibody-bound bead, and allowed to react at 37 ° C. for 10 minutes. After removing the reaction solution with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added and washed in the same manner. Next, 300 μL of enzyme-labeled antibody solution was added to the washed beads and reacted at 37 ° C. for 10 minutes. The reaction solution was removed with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added and washed twice in the same manner. The enzyme activity was measured for the washed beads.

4). Enzyme activity measurement procedure Set the test tube (12 x 75 mm) containing the washed beads in a sample holder of the luminescence reader BLR-201 manufactured by Aloka, and add chemiluminescence reaction by adding 200 µL of the substrate solution and 200 µL of hydrogen peroxide. Started. The amount of luminescence for 10 seconds was measured 40 seconds after the start of the luminescence reaction, and the amount of luminescence showing enzyme activity was determined. One β2-microglobulin polyclonal antibody-bound bead was added and allowed to react at 37 ° C. for 10 minutes. After washing the beads three times with 500 microliters of physiological saline, 150 microliters of peroxidase-labeled anti-β2-microglobulin polyclonal antibody solution was dispensed into a test tube and reacted at 37 ° C. for 10 minutes. After washing the beads three times with 500 microliters of physiological saline, 100 microliters of each of the substrate solution and hydrogen peroxide solution is added to the test tube, and the photomultiplier tube is used to emit light for 40 to 45 seconds after the addition. It measured with the used luminometer (Aroca company make, BLR-201).

5. Measurement Results Table 2 shows the results of measuring standard β2-microglobulin solutions (concentrations of 0, 15, 200 ng / L) using the enzyme-labeled antibody solutions 1 to 9 according to the above measurement procedure. The numerical values in parentheses in Table 2 are values obtained by dividing the S / N ratio, that is, the light emission amount by the light emission amount of 0 g / mL as standard.

  It can be seen that the enzyme-labeled antibody solutions 1 to 5 clearly have a lower light emission amount of 0 ng / mL than the enzyme-labeled antibody solutions 6 to 9, and the nonspecific reaction is suppressed. In particular, when compared with the S / N ratio at a standard of 15 ng / mL, the labeled antibody solutions 1 to 5 achieve an S / N ratio of 50 or more necessary for highly sensitive measurement. Moreover, the light emission amount differences of 15 ng / mL and 200 ng / mL and 0 ng / mL are almost equal, and the specific reaction is at the same level. It can be said that it is a simple measuring reagent.

<Example 2>
In this example, when the casein (A) for immunoassay reagent of the present invention is applied to an enzyme-labeled antibody-containing immunoassay reagent, the nonspecific reaction is low even when it deteriorates over time. is there.

1. Degradation treatment of enzyme-labeled antibody solution The enzyme-labeled antibody solutions 1 to 9 prepared in Example 1 were allowed to stand at 25 ° C for 10 days and subjected to degradation treatment by heating. These enzyme-labeled antibody solutions were designated 1 ′ to 9 ′, respectively.

2. Confirmation of performance of enzyme-labeled antibody solutions 1 ′ to 9 ′ For enzyme-labeled antibody solutions 1 ′ to 9 ′, standard β2-microglobulin solutions (concentrations 0 ng / mL, 15 ng / mL, 200 ng / mL) were used in the same manner as in Example 1. The measurement results are shown in Table 3. The measurement results before the deterioration treatment and the difference before and after the deterioration treatment are shown together in Table 3.

  The difference in the amount of luminescence of 0 ng / mL before and after the treatment is hardly observed in the enzyme-labeled antibody solutions 1 ′ to 5 ′, whereas the enzyme-labeled antibody solutions 6 ′ to 9 ′ show an increase in the amount of luminescence and deteriorated. It can be seen that non-specific reaction increased. That is, it can be said that the enzyme-labeled antibody solutions 1 'to 3' are less likely to reduce the effect of preventing non-specific reactions over time.

<Example 3>
This example shows that the nonspecific reaction is suppressed by removing high-molecular casein from the immunoassay (reaction) reagent with an increased nonspecific reaction.
1. Removal of polymer casein in enzyme-labeled antibody solution The enzyme-labeled antibody solution 9 ′ prepared in Example 2 was concentrated to a volume of 1/50 using an ultrafiltration membrane (manufactured by Toyo Roshi Kaisha, Ultrafilter Q0500). In the same manner as the casein fraction of Example 1, this solution was subjected to gel filtration column method to remove a molecular weight portion larger than 900,000 daltons. The collected fraction was concentrated with an ultrafiltration membrane to a casein concentration of 0.1% by weight to obtain enzyme-labeled antibody solution 10.

2. Performance confirmation of enzyme-labeled antibody solution 10 The results of measuring standard β2-microglobulin solution (concentrations 0 ng / mL, 15 ng / mL, 200 ng / mL) in the same manner as in Example 1 for enzyme-labeled antibody solution 10 are shown in Table 4. . Moreover, the measured value about enzyme-labeled antibody solution 9 ′ was transcribed.

  It can be seen that the light emission amount of 0 ng / mL, which was high in the enzyme-labeled antibody solution 9 ', is decreased in the enzyme-labeled antibody solution 10 and that nonspecific reaction can be reduced by removing the polymer casein.

<Example 4>
This example is an example showing that the casein (A) for immunoassay reagent of the present invention does not have a high molecular weight even after long-term storage.

1. Storage of enzyme-labeled antibody solution The enzyme-labeled antibody solutions 1 to 9 and 10 prepared in Example 1 and Example 3 were stored in a refrigerator (2 to 10 ° C) for 6 months.

2. Analysis of high molecular weight casein After storage, the enzyme-labeled antibody solution was concentrated to 1/10 volume using an ultrafiltration membrane (Toyo Filter Paper, Ultrafilter Q0500), and then gel filtration HPLC (column: Shodex GS manufactured by Showa Denko) -620HQ) to determine the ratio (% by weight) of casein (a) greater than 900,000 daltons (Da) to total casein in the casein solution. The results are shown in Table 5.

  In the enzyme-labeled antibody solutions 1 to 5 and 10 according to the present invention, the ratio of casein having a high molecular weight is 1% by weight or less based on the total casein, and the ratio at the time of preparation is substantially maintained. It was found that the ratio was increased at ˜9.

<Example 5>
This example is an example of an immunoassay reagent for insulin measurement prepared by a method of removing high molecular weight casein with diatomaceous earth.

1. Preparation of Reagent 1) Preparation of Anti-Insulin Polyclonal Antibody-Binding Beads Anti-insulin polyclonal antibody-binding beads were prepared and stored in the same manner as described in Example 1 using an anti-insulin polyclonal antibody (manufactured by Dako Japan).

2) Preparation of peroxidase-labeled anti-insulin polyclonal antibody A peroxidase-labeled anti-insulin polyclonal antibody was prepared in the same manner as described in Example 1 using an anti-insulin polyclonal antibody (manufactured by Dako Japan) and stored frozen (−30 ° C.). .

3) Preparation of buffer for immune reaction 1 g of bovine serum albumin (manufactured by Miles), 0.85 g of sodium chloride, and 0.05 g of sodium azide were dissolved in 100 mL of phosphate buffer (0.02M, pH 7.2). A buffer for immunoassay was used. Stored refrigerated until use.

4) Preparation of enzyme-labeled antibody solution After adding 0.2 g of caseinate sodium salt (manufactured by Sigma-Aldrich) to 100 mL of phosphate buffer (0.1 M, pH 6.0), heating and dissolving at 95 ° C. with stirring, the pore size was 0. Filtration through a 45 μm membrane filter. Further, 500 μg of peroxidase-labeled anti-insulin polyclonal antibody was added in the amount of protein, 0.05 g of sodium azide and 0.05 g of 4- (methylthio) phenol were added and dissolved by stirring to prepare an enzyme-labeled antibody solution.

5) Warming treatment of enzyme-labeled antibody solution and diatomaceous earth treatment The prepared enzyme-labeled antibody solution was stored at 40 ° C. for 5 days and heat-treated (labeled antibody solution 11). 20 g of diatomaceous earth (trade name: Celite No. 545, purchased from Wako Pure Chemical Industries, Ltd.) per 100 mL of labeled antibody solution 11 was added and stirred at room temperature (about 25 ° C.) for 10 minutes. After stirring, the mixture was filtered with a filter paper (Toyo Filter Paper: 5A) using a Buchner funnel to obtain an enzyme-labeled antibody solution 12.

2. Confirmation of high molecular weight casein in enzyme-labeled antibody solution In the same manner as in Example 1, enzyme-labeled antibody solution 11 and enzyme-labeled antibody solution 12 were subjected to gel filtration HPLC (column: Shodex GS-620HQ manufactured by Showa Denko). As a result of analysis, the ratio (% by weight) of casein (a) larger than 900,000 daltons in the solution to the total casein was as follows.
Enzyme labeled antibody solution 11: 8.4%
Enzyme-labeled antibody solution 12: 0.6%

3. Test of measurement performance According to the immunoreaction operation and the enzyme activity operation method described in Example 1, a standard insulin solution (concentration 0, 25, 250 μIU / mL) was measured using the reagent prepared above. The standard insulin solution is 0,250 μIU / mL and 25 μIU / mL diluted from them in the clinical test drug “Sphereite Insulin Calibrator Set” manufactured by Sanyo Chemical Industries.

4). Measurement Results Table 6 shows the results of measuring standard insulin solutions (concentrations of 0, 25, 250 μIU / mL) using enzyme-labeled antibody solutions 11 and 12. The numerical values in parentheses in Table 6 are values obtained by dividing the S / N ratio, that is, the light emission amount by the light emission amount of standard 0 μIU / mL.

  The enzyme-labeled antibody solution 12 clearly has a lower light emission amount of 0 μIU / mL than the enzyme-labeled antibody solution 11, indicating that non-specific reaction is suppressed. In comparison with the S / N ratio, it is clear that the enzyme-labeled antibody solution 12 can be measured with higher sensitivity than the enzyme-labeled antibody solution 11.

  The casein (A) of the present invention can be applied to immunoassay reagents for various uses. For example, reaction reagents such as immune reaction buffers, storage reagents containing antigens or antibodies, carrier blocking reagents, specimen dilutions It can be applied to a reagent for cleaning and a cleaning reagent used for B / F separation.

Claims (5)

The content (% by weight) of casein (a), which is a fraction having a molecular weight greater than 900,000 daltons, is 1 or less based on the weight of casein (A) having a molecular weight of 75,000 to 375,000 daltons. Casein (A) for immunoassay reagent characterized by being. An immunoassay reagent comprising the casein (A) according to claim 1. The immunoassay reagent according to claim 2, wherein the content (g / liter) of casein (A) is 0.01 to 30 based on the volume of the immunoassay reagent at 25 ° C. The immunoassay reagent according to claim 2 or 3, further comprising a labeled ligand. The content (% by weight) of casein (a), which is a fraction with a molecular weight greater than 900,000 daltons, is 1 or less based on the weight of casein (A) with a molecular weight of 75,000-375,000 daltons. A method for producing an immunoassay reagent containing (A), comprising the step of adsorbing and removing (a) after heating the solution containing (A) at 20 to 60 ° C. A method for producing an immunoassay reagent.