JP4830114B2 - General-purpose high-sensitivity ELISA method and its reagent kit - Google Patents

Description

  The present invention relates to an ELISA method that can be used for general purposes and enables highly sensitive detection, and a reagent kit used in the method. In particular, the present invention is a highly sensitive and versatile ELISA method for the amino plate ELISA method, which eliminates the problems of conventional noise and overblocking phenomenon, and further increases the sensitivity to detect small samples. And a reagent kit used for the method.

  Enzyme-linked immunosorbent assay (ELISA) is a method for measuring the amount of an antigen using an antigen-antibody reaction, and has been widely used in biochemical tests and the like (see, for example, Non-patent Document 1). .

  In the ELISA kit currently on the market, the competition method or the sandwich method is mainly used because of its high sensitivity. Moreover, there are a direct method and an indirect method as general-purpose ELISA methods constructed by the measurer himself. The amino plate method belongs to the direct method.

  The competition method requires a large amount of purified antigen and is expensive. The measurement principle of the competition method is a competitive reaction, which leads to instability of the assay system. In particular, when a measurer constructs a competitive ELISA, it is difficult to set an optimum condition that can obtain highly reproducible results. Therefore, the competition method is not suitable for the purpose of constructing an assay system that can provide a stable result with high sensitivity while being low-cost, simple and versatile.

  In the direct method, an antigen (target object) that is a measurement object such as protein is directly immobilized on the surface of a plate, and then reacted with a labeled antibody to measure the amount of the antigen (target object) bound to the labeled antibody. Is the method. Although this method has advantages such as simple, versatile and low cost, it has a disadvantage that it is not suitable for measurement of a sample with a small amount of target because of its low sensitivity. One reason for this is that, since the target is directly attached to the surface of the plate, the epitope is surrounded by plate irregularities and other contaminants and cannot react sufficiently with the antibody. . In the direct method, since the target is only physically attached to the plate surface, it is difficult to concentrate, that is, increase the sensitivity by repeatedly adding the target to the plate and washing.

  In the sandwich method, an antibody that specifically reacts with a target is immobilized on a plate in advance, and the immobilized antibody captures an antigen (target), which is then labeled with a labeled antibody (secondary antibody). In this method, the amount of the antigen (target substance) bound to the labeled antibody is measured. This method is generally highly sensitive because it uses the reaction specificity for an antigen (target). However, this advantage can be fully enjoyed when there is a sufficient amount of sample containing the antigen (target). That is, it is necessary that there is a sufficient amount of the sample containing the target so that the target can be concentrated on the plate by repeatedly adding and washing the sample to the plate. In addition, the sandwich method uses a specific antigen-antibody reaction and thus lacks versatility and has a disadvantage of high cost (for example, the sandwich ELISA kit is 30 to 100,000 in plate units (96 wells per plate). Yen and expensive).

  On the other hand, in the amino plate method, by using a plate (amino plate) that has been surface-treated so as to be covalently bonded to an amino group, an antigen having an amino group (target) is specifically captured by the plate, Then, this is reacted with a labeled antibody, and the amount of antigen (target substance) bound to the labeled antibody is measured. For this reason, it has an advantage that a target having an amino group such as a peptide or protein (including an enzyme and an antibody) can be detected with high sensitivity. In addition, there is an advantage that it can be used universally for a target having an amino group and is low in cost. That is, the amino plate method is a method that combines the versatility and low cost that are the advantages of the direct method described above and the high sensitivity that is the advantage of the sandwich method.

  However, since the labeled antibody used to label the antigen (target) is also a protein, it binds not only to the target but also to the plate surface, and the resulting signal (non-specific reaction signal) is: There is a problem that the detection sensitivity of a target signal (specific reaction signal) is reduced due to noise.

As a method of solving this problem, after immobilizing the antigen (target) on the amino plate and before reacting with the labeled antibody, the surface of the plate is treated with a blocking agent such as casein or BSA, and the labeled antibody and A method of suppressing (blocking) non-specific binding of the phospholipid has been used. However, although this blocking treatment can suppress nonspecific binding between the amino plate and the labeled antibody (reduction in noise), it also suppresses the specific binding between the antigen (target) and the labeled antibody. It is also known that the detection signal (specific reaction signal) decreases (this is referred to as “overblocking phenomenon”) (see Non-Patent Document 1).
Tijssen P, Eiji Ishikawa Translation: “Enzyme immunoassay”, Tokyo Chemical Doujin, 8-345 (1992)

An object of the present invention is to provide an ELISA method capable of highly sensitive detection with a small amount of sample while being versatile and low-cost. More specifically, in the aforementioned amino plate ELISA method, the solidification rate of the antigen (target) on the amino plate surface is improved and / or due to non-specific reaction between the amino plate and the labeled antibody. It is an object of the present invention to provide an ELISA method having a higher sensitivity with a small amount of sample by significantly suppressing noise generated by the above. Furthermore, the present invention provides a reagent kit used in such a high-sensitivity ELISA method, particularly as an antigen-immobilized buffer and / or blocking agent used when an antigen (target) is immobilized on a plate. An object of the present invention is to provide a reagent kit containing a specific reagent.

  The inventors of the present invention have been diligently studying day and night in order to achieve the above object. By using TBS (Tris-buffered saline) as an antigen-immobilizing buffer in the aminoplate ELISA method, an aminoplate can be obtained. The solid phase of the antigen (target) on the surface is remarkably increased, and as a result, a high detection signal (specific reaction signal) can be obtained, and by using polyethyleneimine as a blocking agent, amino It was found that non-specific binding between the plate and the labeled antibody was suppressed and noise was reduced, and as a result, a high detection signal (specific reaction signal) was obtained. It was confirmed that an amino plate ELISA method capable of measuring a small amount of sample could be realized.

  The present invention has been completed based on such findings and includes the following aspects.

(I) ELISA reagent kit (I-1) An ELISA reagent kit comprising at least one of the following reagents:
(a) Tris-buffered saline (TBS) as a buffer for immobilizing an antigen,
(b) Polyethyleneimine as a blocking agent.
(I-2) The ELISA reagent kit according to (I-1), which is an ELISA reagent kit for the aminoplate ELISA method.
(I-3) The ELISA reagent kit according to (I-1) or (I-2), further comprising an amino plate.

(II) Aminoplate ELISA method (II-1) Aminoplate ELISA method comprising the following steps (1) to (4):
(1) (a) a step of immobilizing an antigen on an amino plate in the presence of an antigen-immobilizing buffer;
(2) (b) a step of treating the antigen-immobilized plate with a blocking agent,
(3) reacting an antigen immobilized on the plate with a labeled antibody; and
(4) identifying the formed antigen-labeled antibody and measuring the antigen-labeled antibody,
-Using TBS as a buffer for immobilizing antigen (a)
-(B) Polyethyleneimine is used as the blocking agent, or
-Aminoplate ELISA method, characterized in that (a) TBS is used as the antigen-immobilizing buffer and (b) polyethyleneimine is used as the blocking agent.
(II-2) (a) The aminoplate ELISA method according to (II-1), wherein 0.005 to 0.5 M TBS is used as a buffer for immobilizing an antigen.
(II-3) (b) The aminoplate ELISA method according to (II-1) or (II-2), wherein 0.1 to 3% by weight of polyethyleneimine is used as a blocking agent.

  According to the aminoplate ELISA method using the antigen immobilization buffer (TBS) provided by the present invention, the solidification rate of the antigen on the aminoplate is improved, so that the detection signal (antigen and labeled antibody Signal generated by a specific reaction) can be increased (high sensitivity) (see Experimental Example 1). Further, according to the aminoplate ELISA method using the blocking agent (polyethyleneimine) provided by the present invention, it is possible to suppress nonspecific binding between the aminoplate and the labeled antibody, thereby reducing noise. In particular, the measurable range in the low concentration region can be expanded and the detection limit can be lowered (higher sensitivity) (see Experimental Example 2). That is, according to the aminoplate ELISA method of the present invention using TBS as an antigen-immobilizing buffer and / or polyethyleneimine as a blocking agent, the amount of target substance (antigen amount) to be measured is determined. Highly sensitive detection is possible using a small amount of sample as a specimen.

  Sandwich ELISA is usually sold as a kit in plate units (1 plate 96 well), and the price is high (30,000 yen to 100,000 yen) (high cost). In addition, since the antibody is preliminarily immobilized on the plate, it cannot be used for detection other than a specific antigen that reacts with the antibody (non-generic). For example, in the case of a small amount of antigen sample such as collected from a mouse fetus, one plate having a cost of several tens of thousands of yen must be purchased to measure only a few wells.

  In contrast, in the aminoplate ELISA method of the present invention, a reagent used as an antigen-immobilizing buffer and a blocking agent can be obtained at low cost, and the measurement target (target) has an amino group. Since it can be immobilized on a plate as long as it has one, a single plate (for example, one plate 96 well) can be used for detection of many types of targets (general versatility). One plate can be used as much as possible (inexpensive). In addition, the aminoplate ELISA method of the present invention can obtain a sensitivity as high as a sandwich ELISA by a single reaction with a labeled antibody (high sensitivity).

  That is, according to the present invention, an inexpensive, versatile and highly sensitive ELISA method and an ELISA reagent kit used therefor can be provided.

I. ELISA reagent kit The ELISA reagent kit of the present invention is a reagent suitably used in the aminoplate ELISA method, and the content component thereof is either one of the following (a) or (b), or It is characterized by including both:
(a) Tris-buffered saline (TBS) as a buffer for antigen immobilization
(b) Polyethyleneimine as a blocking agent.

  In order to achieve higher sensitivity, it is preferable to include both (a) and (b).

(a) Buffer for antigen immobilization In the present invention, Tris-buffered saline (TBS) used as a buffer for antigen immobilization dissolves Trizma (registered trademark) base (SIGMA) and sodium chloride in water. For example, 10 × TBS (0.5 M Tris Base, 9% NaCl, pH 7.6) can be prepared as follows.

<10 × TBS (0.5M Tris Base, 9% NaCl, pH7.6)>
Trizma (registered trademark) base (SIGMA) 61 g
NaCl 90 g
Distilled water 1000 ml
Trizma (registered trademark) base and sodium chloride are dissolved in distilled water at the above ratio, and adjusted to pH 7.6 using hydrochloric acid.

  The concentration of TBS used as a buffer for immobilizing an antigen in the present invention is usually a 1/100 to 1-fold dilution (based on the above 10 × TBS (0.5M Tris Base, 9% NaCl, pH 7.6)). 0.1 to 10 × TBS), preferably 1/50 to 1 / 10-fold diluted concentration (0.2 to 1 × TBS). In the present invention, the concentration is usually 0.005 to 0.5M, preferably 0.01 to 0.05M, corresponding to the concentration (M) of Tris Base.

  The TBS used as the antigen-immobilizing buffer in the present invention is not limited to the above pH 7.6, and for example, one in the range of pH 5 to 11, preferably pH 6 to 9.5 can be used. Such pH can be adjusted using hydrochloric acid as described above.

  The antigen immobilization buffer is a buffer used when immobilizing the target (antigen) to be measured on the amino plate, and the above TBS is used as the antigen immobilization buffer. By this, the immobilization rate (solid phase immobilization rate) of the antigen to the amino plate can be increased. The TBS only needs to coexist in the system when the target (antigen) is immobilized. Usually, the above-mentioned coexistence state can be realized by preparing a sample containing a target (antigen) to be injected into a well of an amino plate using the TBS. However, the present invention is not limited to this.

  The ELISA reagent kit of the present invention, particularly the reagent kit for aminoplate ELISA, can contain the TBS as an antigen-immobilized buffer in an individually packaged form. In this case, TBS may be in a concentrated state in consideration of storage stability, regardless of the concentration used. In this case, it is possible to dilute and use distilled water, ion-exchanged water, sterilized water, or the like so that the above concentration is obtained during use.

(b) Blocking agent
The blocking agent is prepared by treating the amino plate in advance before adding the labeled antibody to the well of the amino plate on which the target (antigen) is immobilized and reacting with the target (antigen). It is a reagent used in order to suppress non-specific binding.

The polyethyleneimine used as a blocking agent in the present invention is usually used in the form of an aqueous solution. The concentration is not particularly limited, but can be usually 0.1 to 3% by weight, preferably 0.5 to 2% by weight, more preferably 0.7 to 1.5% by weight.

  The ELISA reagent kit of the present invention, particularly the reagent kit for aminoplate ELISA, can contain the polyethyleneimine as a blocking agent in the form of individually packaged, as in the above TBS. In this case, the polyethyleneimine may be in a concentrated state in consideration of storage stability regardless of the concentration used. In this case, it is possible to dilute and use distilled water, ion-exchanged water, sterilized water, or the like so that the above concentration is obtained during use.

(C) Other components The ELISA reagent kit of the present invention can contain an amino plate as another component in addition to the above-mentioned antigen-immobilizing buffer and / or blocking agent.

  Here, the amino plate is an ELISA plate in which an electrophilic functional group capable of forming a covalent bond with an amino group of a peptide or protein is bound to the surface directly or via a spacer. As (amino) or immobilizer plate (amino), commercially available and widely used ones can be widely used. Usually, these plates have a plurality of wells corresponding to reaction vessels. The amino plate used in the present invention is not particularly limited in the number of wells, but can usually have 8 to 96, preferably 96 wells per plate.

  Commercially available amino plates include Immobilizer Amino Plate Clear Strip and Immobilizer Amino Plate White (Nunc) used in the experimental examples described below; Immobilizer Amino 96-well plate, Immobilizer Amino 96-well plate, Immobilizer Amino Rockwell Module plate (above, Nalge Nunc International KK); Immobilizer Amino F96 Clear / Plate, Immobilizer Amino F96 Shiro / Plate, Immobilizer Amino F96 Black / Plate, Immobilizer Amino F8 Module Clear / Plate, Immobilizer Amino C8 Rockwell Clear / Plate (above And Cosmo Bio). Here, the transparent plate (clear plate) is a plate suitable for an absorbance measurement system, while the white and black opaque plates are plates suitable for a luminescence measurement system and a fluorescence measurement system, and are used for measurement. It can be appropriately selected depending on the detection system.

  In addition, the ELISA reagent kit of the present invention includes a plate cover glass, a labeled antibody, a detection reagent (for example, a reagent that develops color, emits light, or fluorescence by reacting with the labeled antibody), a reaction terminator (for example, the labeled antibody and the detection reagent). 1), a buffer solution, and a specification describing the aminoplate ELISA operation using the ELISA reagent of the present invention.

II. Aminoplate ELISA method The aminoplate ELISA method provided by the present invention is a method having the following steps (1) to (4), comprising the following (a) an antigen-immobilizing buffer and / or (b ) As a blocking agent, the above-mentioned TBS and / or polyethyleneimine is used:
(1) (a) a step of immobilizing an antigen on an amino plate in the presence of an antigen immobilization buffer (antigen immobilization step),
(2) (b) a step of treating the antigen-immobilized plate with a blocking agent (blocking treatment step),
(3) a step of reacting an antigen immobilized on the plate with a labeled antibody (labeling step), and
(4) A step of identifying the formed antigen-labeled antibody and measuring the antigen-labeled antibody (detection step).

  The ELISA method of the present invention comprises the above (1) antigen immobilization step only (a) a method using TBS as an antigen immobilization buffer, or (2) only a blocking treatment step (b) as a blocking agent. Although it may be a method using polyethyleneimine, preferably (1) antigen immobilization step (a) using TBS as an antigen immobilization buffer, and (2) blocking treatment step (b) In this method, polyethyleneimine is used as a blocking agent.

  In addition, only when (1) antigen immobilization step is used (a) when TBS is used as an antigen immobilization buffer, (2) blocking treatment is performed using bovine serum albumin (for example, 0.5% BSA) which is a conventionally known blocking agent. ) Or casein (for example, 0.5%), or a commercially available blocking reagent (for example, “EZ-Block” (manufactured by Atto)). In addition, when (b) polyethyleneimine is used as the blocking agent only for (2) the blocking treatment step, (1) the antigen immobilization step is performed by using PBS (phosphate buffered saline) or SBS (sodium), which is a conventionally known buffer solution. bicarbonate buffered saline).

  (1) When the antigen immobilization step is performed in the presence of TBS as a buffer for antigen immobilization (a), the concentration is 10 × TBS (0.5M Tris Base, 9% NaCl as described above). , pH 7.6), usually 1/100 to 1-fold diluted concentration (0.005 to 0.5M TBS), preferably 1/50 to 1 / 10-fold diluted concentration (0.01 to 0.05M TBS). it can. Moreover, as the pH, pH 5-11, Preferably pH 6-9.5 can be mentioned. The TBS only needs to be present in the system when the target (antigen) is immobilized on the surface of the amino plate. Usually, the above-mentioned coexistence state can be realized by preparing a sample containing a target (antigen) to be injected into a well of an amino plate using the TBS. However, the present invention is not limited to this.

  The preparation of the sample containing the target (antigen) to be injected into the well of the amino plate is not limited, but the protein concentration is usually about 3 pg / ml to 50 ng / ml, preferably about 50 pg / ml to 10 ng / ml. It can be prepared appropriately.

  As described above, the amino plate used here is an ELISA plate in which an electrophilic functional group capable of forming a covalent bond with an amino group is bound to the surface directly or via a spacer. .

  The antigen immobilization step can be performed by injecting a sample containing the target (antigen) into the well of the amino plate and reacting in the presence of TBS (the electrophilic functional group of the amino plate and the target). A covalent bond with the amino group of the product (antigen). The reaction conditions are not particularly limited, and include normal conditions of standing or shaking at room temperature for 10 minutes to several hours, preferably about 30 minutes to 2 hours.

  (2) When the blocking treatment step is performed using (b) polyethyleneimine, the concentration of the aqueous solution is usually 0.1 to 3% by weight, preferably 0.5 to 2% by weight, more preferably 0.7 to A range of 1.5% by weight can be mentioned.

  The blocking treatment is usually carried out after washing the obtained amino plate (antigen-immobilized plate) with water or the like after the above-described (1) antigen-immobilized step. Here, as the washing solution, a buffer solution containing a surfactant such as Tween 20 in addition to water such as distilled water, ion exchange water, and sterilized water can be used.

  The blocking treatment conditions are not particularly limited, but the antigen-immobilized plate is left in an aqueous solution containing polyethyleneimine, usually at room temperature for 10 minutes to several hours, preferably about 30 minutes to 2 hours, or shaken. The method of processing can be mentioned.

  After the (2) blocking treatment step, the treated amino plate (blocking treatment plate) is usually washed with water, and then (3) a labeling step in which the antigen on the amino plate reacts with the labeled antibody, and (4 ) A detection step of identifying the formed antigen-labeled antibody and measuring the antigen-labeled antibody can be performed. In addition, as above-mentioned, the said washing | cleaning can be performed using the buffer solution containing water and surfactant, such as distilled water, ion-exchange water, and sterilized water.

  The (3) labeling step and the (4) detection step after the (2) blocking treatment step can be carried out according to a conventional ELISA method (see, for example, the aforementioned Non-Patent Document 1), Depending on the type of target to be measured and the purpose of measurement, an optimum method can be adopted based on the common general knowledge of those skilled in the art.

  (3) Any known enzyme can be used as the labeling agent in the labeling step without discrimination. For example, representative examples include alkaline phosphatase and peroxidase (for example, horseradish peroxidase). Examples of labeled antibodies bound to these labeling agents include antibodies that bind to the antigen (protein) to be measured. The labeled antibody may be either a polyclonal antibody or a monoclonal antibody, and commercially available antibodies can also be used.

  (4) The detection step is a step of detecting the antigen-labeled antibody formed in the above (3) labeling step. According to a conventional method, a detection system (absorbance detection) according to the type of substrate of the labeling agent used for the labeled antibody. System, fluorescence detection system, chemiluminescence detection system). Thus, the presence or absence of the antigen to be measured (target substance) in the test sample or its content can be measured from the presence or absence of the absorbance, fluorescence or chemiluminescence and its intensity.

  EXAMPLES Hereinafter, although an experiment example and an Example are shown and this invention is demonstrated, this invention is not restrict | limited by this Example etc. In the following experimental examples and the like, “%” means “% by weight” unless otherwise specified.

Experimental Example 1 Influence of Buffer on Immobilization of Antigen on Plate In amino plate ELISA, the following three kinds of buffers were used, and the influence of each buffer on the immobilization of antigen (target) on amino plate. I investigated.
(I) TBS: Tris-buffered saline (SIGMA) (pH 7.6)
2-amino-2- (hydroxymethyl) -1,3-propanediol (1.39 g / l), tris (hydroxymethyl) aminomethane hydrochloride (6.06 g / l), and sodium chloride (9 g / l) Contained aqueous solution (ii) PBS: phosphate buffered saline (Wako Pure Chemical Industries, Ltd.) (pH 6.5)
An aqueous solution containing 8.1 mmol / l Na2HPO4, 1.47 mmol / l KH2PO4, 137 mmol / l NaCl and 2.68 mmol / l KCl (iii) SBS: sodium bicarbonate buffered saline (pH 9.8)
An aqueous solution containing 10.6 g / l sodium bicarbonate.

(1) Experimental method
(1) First, each of the above buffer solutions is poured at a rate of 100 μl / well into wells of an amino plate (Immobilizer Amino Plate Clear Strip: Nunc) (injected into 3 wells for each buffer solution) at 26 ° C. And left for 60 minutes while shaking at 100 rpm (pretreatment with buffer).
(2) Next, the wells were washed with 0.05 M TBS containing 0.1% Tween 20, and then the antigen anti-GOAT-IgG (SIGMA) diluted to 1 μg / ml with each buffer was added at a rate of 100 μl / well. The mixture was poured again and left for 60 minutes while shaking at 100 rpm under the condition of 26 ° C. (antigen immobilization treatment). The anti-GOAT-IgG used here as an antigen is labeled with horseradish peroxidase (HRP).

(3) Next, each well was washed with 0.05 M TBS containing 0.1% Tween 20, and then 100 μl of TMB (3,3 ′, 5,5′tetramethylbenzidine: (SIGMA)) which reacts with HRP and shows blue color The solution was added at a ratio of / well and allowed to stand for 15 minutes.
(4) Pour reaction stop solution (ELISA Stop-solution: phosphoric acid aqueous solution 1mol / l) at a rate of 100μl / well to stop the above TMB reaction, and measure the absorbance (450nm) of the reaction solution in each well. , Measured with MICROPLATE READER (BIO RAD).

  In the above, (2) Aminoplate was pretreated with each buffer in advance before antigen-immobilization treatment (with buffer pretreatment), but the system without such pretreatment (without buffer pretreatment) The same experiment was conducted.

(2) Results of the experiment are shown in FIG. The horizontal axis indicates the type of buffer used, and the vertical axis indicates the absorbance (450 nm) obtained by the ELISA method. Note that (+) and (−) after the buffer name on the horizontal axis mean “with buffer pretreatment” and “without buffer pretreatment”, respectively.

  As can be seen from this result, a remarkably high signal was detected when TBS was used as a buffer solution, and it was confirmed that the presence of TBS improved the solidification rate of the antigen to the amino plate. On the other hand, for all the buffer solutions, there was no effect due to the presence or absence of the buffer solution treatment prior to antigen immobilization. Therefore, when immobilizing an antigen (target) on an amino plate, the use of TBS as a buffer can increase the solid phase of the antigen on the plate, resulting in higher sensitivity. You can see that is possible.

  According to this method, the detection signal (the signal based on the specific reaction between the antigen and the labeled antibody) can be increased by improving the solid phase rate of the antigen. In particular, the measurable range in the low concentration region where the influence of noise (background) is large can be expanded, and the detection limit can be lowered (see FIG. 2). That is, according to the ELISA method of the present invention using TBS as a buffer solution, high-sensitivity detection is possible using a small amount of sample with a small amount of target antigen as a specimen.

Experimental Example 2 Evaluation of blocking performance In the amino plate ELISA method, 10 types of reagents [glycine, nicotinamide, monoethanolamine, sodium glutamate, gelatin, casein, polyvinyl alcohol (above, Wako Pure Chemical Industries), bovine serum albumin (BSA) Polyethyleneimine (SIGMA)] was used to examine the blocking performance of each reagent. Of these reagents, casein and BSA are known reagents conventionally used as blocking agents in the aminoplate ELISA method.

  Each reagent was prepared and used at a concentration of 0.1 to 1% by weight, and casein and BSA were used at a commonly used concentration (0.5%).

(1) Experimental method
(1) Diluted to each concentration (3.12 ng / ml, 1.56 ng / ml, 781 pg / ml, 390 pg / ml, 195 pg / ml, 97 pg / ml, 48 pg / ml) using TBS Mouse-IgG (DAKO) was poured into each well of an amino plate (Immobilizer Amino Plate White: manufactured by Nunc) at a rate of 100 μl / well, and TBS was injected at a rate of 100 μl / well at 26 ° C. Under the condition, the sample was left for 60 minutes while shaking at 100 rpm (antigen immobilization treatment). As TBS, 2-amino-2- (hydroxymethyl) -1,3-propanediol 1.39 g / l and tris (hydroxymethyl) aminomethane hydrochloride 6.06 g / l were used as TBS. An aqueous solution containing was used.

  (2) Next, after washing each well with 0.05M TBS containing 0.1% Tween20, various reagents (blocking agent candidates) were added at a rate of 200μl / well and shaken at 26 ° C and 100 rpm. And left for 60 minutes.

  (3) After washing with 0.05 M TBS containing 0.1% Tween20, 100 μl / well of labeled antibody anti-mouse-IgG-HRP (VECTOR) diluted to 30 ng / ml with TBS is added, and at 100 ° C. under 26 ° C. conditions. Left for 60 minutes with shaking.

  (4) Next, after washing, ECL Western blotting solution (Amersham) was added at a rate of 100 μl / well and allowed to stand for 15 minutes.

  (5) After the reaction, the luminescence intensity (428 nm) of the reaction solution in each well was measured with a fluoroscan (Dainippon Pharmaceutical).

(2) Experimental results From the measured luminescence intensity (signal intensity), the optimum concentration of each reagent (blocking agent candidate) was determined. As a result, the optimum concentration of glycine is around 0.5%, nicotinamide is around 0.8%, monoethanolamine is around 4 mM / L, sodium glutamate is around 0.5%, gelatin is around 0.5%, polyethyleneimine is around 1%, polyvinyl alcohol The optimum concentration of was found to be around 0.7%.

Therefore, the luminescence intensity obtained by using each of these reagents as a blocking agent at this optimum concentration was compared with the case where a conventionally known blocking agent (BSA, casein, used at 0.5% each) was used. As a result, only when polyethyleneimine ( PEI ) was used, it was found that stronger emission intensity was obtained than when the above-mentioned conventionally known blocking agent was used, and the blocking performance was excellent. FIG. 3 shows the result of comparing the blocking performance of polyethyleneimine ( PEI ) and BSA (average value of n = 3). When each group ( PEI use group, BSA use group) was discriminated by ANOVA, P was 0.01 or less, and two groups could be discriminated significantly. In response, a factor analysis was performed using a post-hoc test. Two asterisks are attached to P values of 0.05 or less, and one asterisk is attached to values of 0.1 or less. FIG. 4 shows the result of comparing the blocking performance of PEI and casein. 3 shows the measured average value of n = 3, and FIG. 4 shows each value of n = 3. In each figure, the horizontal axis represents the antigen concentration used (log (ng / ml)), and the vertical axis represents the emission intensity (428 nm) obtained by the ELISA method used.

In addition, in FIGS. 5 to 9, the results of comparing the blocking performance (luminescence intensity) of polyethyleneimine ( PEI ) and other reagents (glycine, nicotinamide, monoethanolamine, sodium glutamate and polyvinyl alcohol) (n = 3) Value). As can be seen, all other reagents were inferior to PEI .

  As can be seen from the above results, polyethyleneimine has better blocking performance than known blocking agents and attenuates the specific reaction (specific reaction signal) between the antigen (target) and the labeled antibody. Therefore, it was found that the non-specific binding of the labeled antibody to the plate was suppressed and it was effective in reducing noise (noise removal).

According to this method, as shown in FIG. 10, by reducing the noise, the measurable range of the low density region can be expanded and the detection limit can be lowered (higher sensitivity). That is, according to the ELISA method of the present invention using polyethyleneimine as a blocking agent, high-sensitivity detection is possible using a small amount of sample with a small amount of target antigen as a specimen.
Example Detection of leukemia inhibitory factor (LIF) Cerebrospinal fluid (CSF), amniotic fluid (AF), serum (SE and SD) collected from a mouse fetus mixed with an equal amount of 50% ethylene glycol The aminoplate ELISA method of the present invention was performed using Specifically, Nunc Immobilizer Amino plate (Nalge Nunc International) was used as an amino plate, 0.05 M Tris-buffered saline (TBS) was used as an antigen immobilization buffer, and 1 wt% as a blocking agent. The conventional direct ELISA method (Harlow E, Lane DP. Antibodies .: A laboratory manual. New York, Cold Spring Harbor laboratory; 1988) was used except that polyethyleneimine was used. Specifically, the specimen was prepared with the above TBS so as to have a predetermined concentration, and this was injected into each well of the amino plate and treated at room temperature for 1 hour (immobilization). Next, each well of the obtained plate was washed with distilled water, and then a 1% by weight polyethyleneimine aqueous solution was injected thereto, followed by treatment at room temperature for 30 minutes for blocking. Next, each well of the obtained plate was washed with distilled water, and then a 1% by weight polyethyleneimine aqueous solution was injected thereto, followed by treatment at room temperature for 30 minutes for blocking.

  Subsequently, each well of the obtained plate was washed with distilled water, and then goat anti-LIF antibody (SIGMA) labeled with N-hydroxysuccinimidyl 6- (biotinamido) hexaniate [biotine (long arm) -NHS] (Vector). ) (Biotin-labeled anti-LIF antibody) TBST solution was injected, reacted at room temperature for 90 minutes, and further reacted with extra-avidin-peroxidase (1: 3000) (SIGMA) for 15 minutes at room temperature. This was further reacted with Amersham ECL Western Blotting Detection Reagents (GE Healthcare), and the resulting chemiluminescence signal was measured with Fluoroskan Ascent FL (Thermo Electron Corporation).

  As a result, it was possible to grasp the fluctuation of leukemia inhibitory factor (LIF) concentration during embryonic period of mice from a small amount of specimen prepared from mouse fetus.

In Experimental example 1, the result of having investigated the influence (evaluation of immobilization efficiency) of the buffer solution in the antigen aminoplate immobilization is shown. Each graph represents an average of absorbance (450 nm) of each buffer, and error bars represent standard deviation. The test was a t-test. The schematic diagram which shows the relationship between the increase in the signal by the antigen-immobilization rate improvement and the expansion of the measurable range in the low concentration region (decrease in detection limit = higher sensitivity) is shown. In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine 1% and BSA 0.5% (average value of n = 3) are shown. The vertical axis represents the emission intensity (signal) (428 nm), and the horizontal axis represents the antigen concentration (log (ng / ml)) (the same applies to the following drawings). In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine ( PEI ) 1% and casein 0.5% (each value of n = 3) are shown. In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine ( PEI ) 1% and glycine 0.5% (each value of n = 3) are shown. In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine ( PEI ) 1% and nicotinamide 0.5% (each value of n = 3) are shown. In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine ( PEI ) 1% and monoethanolamine 4 mM / L (each value of n = 3) are shown. In Experimental Example 2, the results of comparing the blocking performance of 1% polyethyleneimine ( PEI ) and 0.5% sodium L-glutamate (each value of n = 3) are shown. In Experimental Example 2, the results of comparing the blocking performance of polyethyleneimine ( PEI ) 1% and polyvinyl alcohol 0.7% (each value of n = 3) are shown. The schematic diagram which shows the relationship between the reduction | decrease of the noise by blocking performance improvement, and the expansion of the measurable range of a low concentration area | region (decrease of a detection limit = high sensitivity) is shown.

Claims (5)

ELISA reagent kit for aminoplate ELISA method comprising the following reagents (a) and (b) in individually packaged form :
(a) Tris-buffered saline as a buffer for antigen immobilization,
(b) Polyethyleneimine as a blocking agent The ELISA reagent kit according to claim 1, further comprising an amino plate. An amino plate ELISA method comprising the following steps (1) to (4):
(1) (a) a step of immobilizing an antigen on an amino plate in the presence of an antigen-immobilizing buffer;
(2) (b) a step of treating the antigen-immobilized plate with a blocking agent,
(3) reacting an antigen immobilized on the plate with a labeled antibody; and
(4) identifying the formed antigen-labeled antibody and measuring the antigen-labeled antibody,
-Aminoplate ELISA method, characterized in that (a) Tris-buffered saline is used as a buffer for immobilizing an antigen, and (b) polyethyleneimine is used as a blocking agent. (a) The aminoplate ELISA method according to claim 3 , wherein 0.005 to 0.5M Tris-buffered saline is used as a buffer for immobilizing an antigen. (b) The aminoplate ELISA method according to claim 3 or 4 , wherein 0.1 to 3 wt% polyethyleneimine is used as a blocking agent.

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