JP5093087B2 - Method for enhancing immune response with polyethylene glycol and urea - Google Patents

Description

  The present invention relates to a method for enhancing the reaction between an antigen and an antibody. According to the present invention, in an immunoassay that utilizes a reaction between an antibody and an antigen (substance recognized by the antibody) that is commonly used in the field of clinical diagnosis, by enhancing the immune reaction between the antibody and the antigen, Provided is a method for detecting an antigen in a sample (a blood-derived component such as serum or plasma derived from a patient to be subjected to immunoassay or a biological sample such as urine) with sensitivity.

  A technique for promoting an immune reaction by coexisting a low concentration of polyethylene glycol is known (for example, Patent Document 1). On the other hand, with regard to urea, examples of use as a treatment agent for poorly water-soluble proteins and examples of use as a nonspecific antibody binding reducing agent in immune reactions for detecting autoantibodies related to infectious diseases have been reported (for example, Patent Documents 1 to 3).

Japanese Patent Laid-Open No. 7-181182 J. et al. Virol. Methods 52, 95-104, 1995 J. et al. Clin. Lab. Anal. 8, 16-21, 1994 J. et al. Med. Virol. 33, 100-105, 1991

  As described above, Patent Document 1 discloses that an immune reaction is promoted by coexisting polyethylene glycol. However, Patent Document 1 discloses a technique in which an extremely low concentration of polyethylene glycol, which is less than 1% (weight / weight), coexists. This is because coexistence of high-density polyethylene glycol causes protein precipitation, resulting in difficulty in generating an immune response, and the reproducibility of measurement results may be lost. Coexistence of glycol increases the reactivity of non-specific reactions as well as antigen-specific reactions, so when considering the S / N ratio (signal / non-specific signal ratio due to antigen-specific reactions), large performance (measurement) Accuracy) cannot be expected.

  On the other hand, with respect to urea, all non-patent literatures disclose only using urea as a non-specific antibody binding reducing agent.

  In an immunoassay using an immune reaction between an antigen and an antibody, measurement sensitivity is a very important factor. Accordingly, an object of the present invention is to provide a method for enhancing an antigen-antibody reaction that enhances the reaction between an antigen and an antibody and can improve the measurement sensitivity in immunoassay or the like to which the reaction is applied.

  Diseases are diagnosed by quantifying trace components contained in a sample by immunoassay or the like, but various techniques are used to detect trace components in a sample with higher sensitivity. As a result of intensive research on sensitivity enhancement in such immunoassay, the present inventor enhanced the immune reaction between an antigen and an antibody by using polyethylene glycol, which is an immune reaction promoter, together with urea, not alone. The inventors have found that measurement sensitivity in immunoassay and the like can be improved, and have completed the present invention. That is, the present invention is a method for enhancing an antigen-antibody reaction, in which an antigen and an antibody are reacted in the presence of polyethylene glycol and urea. In addition, the present invention is a reagent that enhances an antigen-antibody reaction, and includes either an antigen or an antibody, polyethylene glycol, and urea. Hereinafter, the method and reagent of the present invention will be described in detail by taking as an example the case of application to immunoassay.

  In the present invention, in an immunoassay using a reaction between an antigen and an antibody, the antigen and the antibody are reacted in the presence of polyethylene glycol and urea. In the present invention, there is no particular limitation on the order in which the antigen, antibody, polyethylene glycol and urea are mixed and coexist. For example, an antigen and an antibody may be mixed to start an immune reaction, and polyethylene glycol and urea may be rapidly added sequentially or simultaneously by mixing them in advance. Since the reaction between the antigen and the antibody occurs rapidly, in order to maximize the effect of enhancing the immune response according to the present invention (improving the measurement sensitivity when applied to immunoassay), the immunization between the antigen and the antibody It is preferable that both polyethylene glycol and urea coexist from the moment when the reaction starts. For this purpose, for example, it is possible to exemplify mixing both polyethylene glycol and urea in advance with either the antigen or the antibody and mixing it with the other of the antigen or the antibody. In this preferred example, the coexistence of polyethylene glycol and urea is particularly preferred in the sense that it can be carried out by a single operation of adding a certain amount of the mixed solution.

  For example, immunoassay is performed in the order of (1) reaction between the target sample and the antibody immobilized on the carrier, (2) so-called B / F separation, and (3) reaction between the separated carrier and the antibody bound to the label. In the case of the so-called two-step sandwich method, a certain effect can be obtained if polyethylene glycol and urea coexist in the reaction of either (1) or (3). In order to maximize the effect of the present invention, it is preferable to coexist both reactions (1) and (3). In that sense, if the target sample, the antibody immobilized on the carrier and the antibody bound to the label are reacted in one step, a one-step sandwich method or a similar measurement method, polyethylene glycol and urea are used in the reaction. It is particularly preferable because the effect of the invention can be maximized by coexisting with each other, and the number of steps required for the operation can be reduced.

  The present invention can be applied to conventional immunoassays without any particular limitation. As an immunoassay to which the present invention can be applied, for example, measurement by a competitive method, a sandwich method, a fluorescence polarization method or the like using an enzyme label, an isotope label, a fluorescent label or a luminescent label can be exemplified. It can also be applied to binding measurement using a homogeneous measurement method or a surface plasmon resonance analysis method.

  The antibody in the present invention may be any polyclonal antibody or monoclonal antibody produced by humans, mice, rabbits, goats, etc. against an antigenic substance. The antigen is a partner to which the antibody specifically recognizes and binds, and may be an environmental hormone or the like in addition to a biological protein or hormone related to a specific disease. In some cases, the antibody itself can be the subject of the present invention as an “antigen”.

  Polyethylene glycol having various average molecular weights is commercially available, but there is no particular limitation on the molecular weight in the present invention. There is no particular limitation on the amount of polyethylene glycol to be used (the concentration of polyethylene glycol to coexist), and the concentration (final concentration) in the solution causing the reaction between the antigen and the antibody is 1 to 10% (weight / weight). It ’s fine. Since the final concentration of polyethylene glycol has an optimum range depending on its molecular weight, it is particularly preferable to determine it by conducting preliminary experiments under specific conditions prior to carrying out the present invention. For example, according to the knowledge of the present inventor, when polyethylene glycol having an average molecular weight of 6000 is used, it is possible to maximize the effects of the present invention by setting the final concentration to 3 to 7%. is there.

  As for urea, the concentration (final concentration) in a solution that causes a reaction between an antigen and an antibody may be 1 to 4 mol / liter, but it may be determined in consideration of the concentration of polyethylene glycol used. preferable. For example, according to the knowledge of the present inventor, when polyethylene glycol having a molecular weight of 6000 is used at a final concentration of 5%, the final concentration is 2 to 4 mol / liter, particularly preferably 3 mol / liter. It is possible to maximize the effects of.

  As described above, when polyethylene glycol and urea are mixed in advance and mixed with a sample or the like, the mixture is prepared at a concentration twice the final concentration described above, and 1: 1 with the sample. And so on. The mixed solution of polyethylene glycol and urea is added with about 10 mM Tris-HCl or the like so as to have a pH range of about pH 8 that does not hinder the reaction between the antigen and the antibody. It is preferable to add a surfactant (for example, Tween 20 or the like) of about 1% (weight / weight).

  When the method of the present invention is applied to, for example, immunoassay, a reagent set containing polyethylene glycol and urea is used in addition to various reagents for performing normal immunoassay. Polyethylene glycol and urea may be held in separate containers and used as the composition of the reagent set. However, since they do not cause alteration or the like when they coexist, it is preferable that both are mixed. . Specifically, a reagent containing polyethylene glycol and urea on the antigen or antibody involved in the antigen-antibody reaction can be exemplified. In addition, for example, together with components other than the antigen to be measured (for example, antibody bound to carrier and enzyme-labeled antibody), polyethylene glycol and urea are lyophilized in a single container, and the target sample is added. It can also be exemplified that the present invention can be carried out only by the above. With such a container, the present invention can be implemented by adding the target sample to the container, so that the ease and simplicity of operation can be improved and the reproducibility of the operation can also be improved.

  By allowing both polyethylene glycol and urea to coexist, it is possible to enhance the immune reaction between the antigen and the antibody. And by enhancing the immune reaction, it is possible to dramatically improve the measurement sensitivity in the immunoassay.

EXAMPLES The present invention will be described in more detail below with reference to examples, but these examples do not limit the present invention.
Example 1
As a target antigen in the immunoassay, human staphylococcal nuclease domain containing 1 (hereinafter referred to as “SND1”) was selected, and a reagent for the measurement was prepared.

  First, cDNA of SND1 (Genbank accession number NM — 04390.2) was cloned from human prostate cancer cell LnCap using RT-PCR according to a conventional method. After removing the stop codon from the cloned cDNA, polyhistidine-tag SND1 prepared by adding a codon encoding histidine 6 residue was introduced into baculovirus transfer vector pFASTBac-1 (manufactured by Invitrogen), and Bac- Baculovirus was prepared according to the protocol using the to-Bac system (Invitrogen).

  The baculovirus prepared as described above was used to infect Sf21 according to a conventional method, followed by culture to prepare an expression culture supernatant containing polyhistidine-tag SND1. The culture supernatant was dialyzed with TBS (Tris buffer saline; 10 mM Tris-HCl, 150 mM NaCl, pH 7.4), and then HisTrap HP metal chelate column (manufactured by GE Healthcare Biosciences, Cat. NO. 17-5248). -01) was used for purification according to the protocol.

  FIG. 1 shows an SDS-PAGE image of a purified product of polyhistidine-tag SND1 prepared by a purification operation. The concentration of the purified SND1 was measured with a BCA protein quantification kit (Pierce Bioctechnology, Cat. No. 23225) to obtain the SND1 concentration.

The monoclonal antibody was prepared according to a conventional method. First, Balb / C mice (7-week-old female) were immunized intraperitoneally with SND1 (50 μg) together with Freund's complete adjuvant. Next, the B cell obtained from the mouse and the mouse myeloma cell line PAI were fused according to a conventional method to obtain a monoclonal antibody-producing strain. Two monoclonal antibodies capable of measuring SND1 by sandwich ELISA (Enzyme-Linked Immunosorbent Assay) were selected from the monoclonal antibodies produced by the obtained cells. The reactivity of the selected monoclonal antibody with purified SND1 or human prostate cancer cell LnCap is shown in FIG.
Example 2
After physically adsorbing one of the monoclonal antibodies selected in Example 1 to 100 ng / carrier on a water-insoluble spherical carrier (manufactured by EVA having a particle diameter of about 1.5 mm with ferrite kneaded inside), Blocking treatment was performed with BSA.

A buffer (3) containing the other of the monoclonal antibodies selected in Example 1, in which 12 carriers were placed in a magnetically permeable container (capacity 1.2 ml) and then labeled with 0.2 μg / mL alkaline phosphatase. % BSA, 5% water-soluble gelatin, 10 mM Tris buffer, pH 8.0) and lyophilized.
Example 3
Using the reagent prepared in Example 2 and using purified SND1 added to calf serum (FBS) as an object sample, the effect on urea measurement was verified. 10 mM Tris-HCl containing urea and 0.1% Tween 20 (pH 8) were mixed with the target sample at a ratio of 1: 1, and 150 μL of the mixed solution was added to the container. After causing an immune reaction for 10 minutes at 37 ° C., B / F separation is performed to separate and remove the free labeled antibody from the carrier, and 4 methylumbelliferyl phosphate, which is a substrate for alkaline phosphatase, is removed. In addition, the production rate Rate (nM / sec) of 4-methylumbelliferone was measured. The measurement was automatically carried out using a commercially available immunoassay device (trade name AIA-600II, manufactured by Tosoh Corporation). In this apparatus, 150 μL of a target sample is added to a magnetically permeable container, the carrier is stirred with a magnet at 37 ° C. for 10 minutes or 40 minutes, and an immune reaction is caused in that state. In addition, this apparatus has a production rate per unit time (nM / second) of an enzymatic reaction degradation product (4-methylumbelliferone) from 20 to 295 seconds after adding 4-methylumbelliferyl phosphate which is a substrate of alkaline phosphatase. ).
The results are shown in FIG.

As shown in FIG. 3, in the presence of 1 mol / liter (M) of urea, a slight increase in reactivity was observed in the immune reaction for 40 minutes. Any of the SND1-specific binding was reduced. The increase in reactivity at a coexisting urea concentration of 1 M in an immune reaction of 40 minutes is also a slight increase of 33% compared to the case of non-coexistence, and it can be seen that high sensitivity by improving the reactivity cannot be achieved by urea alone.
Example 4
In the same manner as in Example 3, the influence of polyethylene glycol on the measured value was verified. As the polyethylene glycol, PEG 6000 (manufactured by Naiarai Tesque, average molecular weight: 7400 to 12000) was used. The results are shown in FIG.

As shown in FIG. 4, both non-specific binding and SND1-specific binding increased due to the increase in the coexisting polyethylene glycol concentration in any of the immune reaction times of 10 minutes or 40 minutes. The rate of increase was 11.1 when the coexisting polyethylene glycol concentration was 5% in the 10-minute reaction showing the maximum Rate value in SND1-specific binding, and 4% in the 40-minute reaction, compared with the absence of polyethylene glycol, respectively. It was 0 times and 4.1 times. However, the zero samples not containing SND1 also increase to 19.9 times and 33.2 times, respectively, so the antigen-specific signal / non-specific noise ratio (S / N ratio) does not improve, and polyethylene It can be seen that, with only glycol, high sensitivity cannot be achieved even though the reactivity can be improved.
Example 5
Polyethylene glycols having different average molecular weights, PEG 4000 (manufactured by Naiarai Tesque, average molecular weight 3000 to 3700), PEG 6000 (manufactured by Naiarai Tesque, average molecular weight 7400 to 12000), PEG 20000 (manufactured by Naiarai Tesque, average molecular weight 15000 to 25000) ) Was used to measure a zero sample not containing SND1 and a target sample containing 1 μg / mL in the same manner as in Example 3. The results are shown in FIG.

As shown in FIGS. 5A and 5B, the improvement in reactivity was observed in any polyethylene glycol, but it was revealed that the effect differs depending on the molecular weight of polyethylene glycol. Further, as shown in FIG. C, it can be seen that the addition of polyethylene glycol alone does not improve the S / N ratio.
Example 6
In Example 3, the reactivity when polyethylene glycol (PEG 6000) was further coexisted at a coexisting urea concentration of 2 M, which showed the maximum S / N ratio in an immune reaction for 10 minutes, was verified. The measurement method is the same as in Example 3. The results are shown in FIGS.

As shown in FIGS. 6A and B, it can be seen that both SND1-specific reactivity and non-specific reactivity are increased by the coexistence of polyethylene glycol. However, as shown in FIG. 6C, the improvement of the S / N ratio becomes dramatic due to the coexistence of 2.5% of polyethylene glycol. Further, as shown in FIG. 7, compared with the reactivity when urea and polyethylene glycol are not coexisting, the zero sample measurement value not containing SND1 is slightly reduced (0.6 times) in the presence of urea 2M alone, The measured value of the SND1-containing sample does not vary (0.9 times). As a result, only a slight increase in S / N ratio (1.6 times) is recognized, and high sensitivity cannot be achieved only by coexistence of urea. On the other hand, when polyethylene glycol coexists with urea, a decrease in the measured value of the zero sample not containing SND1 is not observed (0.9 times), but the increase in the measured value of the sample containing SND1 is large (4.0). As a result, an increase in the S / N ratio of 4.2 times is recognized, indicating that high sensitivity has been achieved.
Example 7
In the coexisting polyethylene glycol (PEG 6000) concentration of 5% in which the antigen-specific reactivity showed the maximum value in the immune reaction for 10 minutes in Example 4, the reactivity when urea was further coexisted was verified. The results are shown in FIGS.

As shown in FIGS. 8A and 8B, non-specific reactivity decreases due to the coexistence of urea, and SND1-specific reactivity increases until the coexisting urea concentration reaches 2M. As is clear from FIG. 8C, the S / N ratio is dramatically improved when the coexisting urea concentration is 3M. Moreover, as shown in FIG. 9, compared with the reactivity when urea and polyethylene glycol do not coexist, the measurement value of the SND1-containing sample is dramatically improved (10.7 times) in the coexistence of polyethylene glycol alone, but SND1 is increased. The zero sample measurement value which is not included also increases (20.9 times), and as a result, the improvement of the S / N ratio (0.5 times) is not recognized. On the other hand, when 3M urea coexists, the measured value of the SND1-containing sample is dramatically improved (8.4 times), and the increase of the measured value of the zero sample can be suppressed (1.6 times). . By coexistence of polyethylene glycol and urea, a 5.2-fold increase in S / N ratio is recognized as a result, and it can be seen that high sensitivity is achieved.
Example 8
The reactivity when polyethylene glycol (PEG 6000) was further allowed to coexist at the coexisting urea concentration of 2 M in which the S / N ratio showed the maximum value in the immune reaction for 40 minutes in Example 3 was verified. The results are shown in FIGS.

As shown in FIGS. 10A and 10B, the non-specific reactivity increases more rapidly with the coexistence of polyethylene glycol than the coexistence of 4% polyethylene glycol, and the SND1-specific reactivity increases with a peak at the coexistence of 5%. . However, as is clear from FIG. 10C, although the S / N ratio is maximized by coexistence with 1% polyethylene glycol, the rate of increase is lower than that during the 10-minute immune reaction. Further, as shown in FIG. 11, compared with the reactivity in the absence of urea and polyethylene glycol, when the urea 2M and polyethylene glycol 1% coexist, the reduction of the zero sample measurement value is large (0.13 times), and SND1 Combined with the increase in the measured value of the contained sample (1.57 times), as a result, an increase of 11.4 times in the S / N ratio is recognized, indicating that high sensitivity has been achieved.
Example 9
In the coexisting polyethylene glycol (PEG 6000) concentration of 4% in which the antigen-specific reactivity showed the maximum value in the immune reaction for 40 minutes in Example 3, the reactivity when urea was further coexisted was verified. The results are shown in FIGS.

As shown in FIGS. 12A and 12B, non-specific reactivity is drastically decreased due to the coexistence of urea, and SND1-specific reactivity is observed to increase in reactivity at the peak of 1.5% coexistence. As is apparent from FIG. 12C, the S / N ratio was dramatically increased by the coexistence of 3M urea. As shown in FIG. 13, when 3M urea and 4% polyethylene glycol coexist in comparison with the reactivity in the absence of urea and polyethylene glycol, the measured value of zero sample is reduced (0.34 times). Together with the increase in the measured value of the SND1-containing sample (2.27 times), as a result, an increase in the S / N ratio of 6.6 times is recognized, indicating that high sensitivity has been achieved.
Example 10
A 1 / 2-fold dilution series sample was prepared based on a 1000 ng / mL solution of SND1, and an immune reaction was performed for 10 minutes and 40 minutes according to the method of Example 3. In order to enhance the immune response, immunoassay is performed under the conditions of (1) coexistence of polyethylene glycol and urea, (2) coexistence of 2M urea only, or (3) coexistence of 4% polyethylene glycol (PEG6000) and 3M urea. did. The results are shown in FIG.

As shown in FIGS. 14A and 14B, it can be seen that the slope of the graph increases (reactivity increases) when polyethylene glycol 4% and urea 3M coexist in any of the immune reaction times of 10 minutes and 40 minutes. . When only urea 2M coexists, the slope of the graph does not increase compared to the case of non-coexistence, but rather the graph shifts to the lower value side, and the reactivity decreases overall. I understand.
Example 11
In the same manner as in Example 10, a 1 / 2-fold dilution series sample was prepared based on a 50 ng / mL solution of SND1, and an immune reaction was performed for 40 minutes. In order to enhance the immune response, immunoassay is performed under the conditions of (1) coexistence of polyethylene glycol and urea, (2) coexistence of 2M urea only, or (3) coexistence of 4% polyethylene glycol (PEG6000) and 3M urea. did. The results are shown in FIGS.

  As shown in FIG. 15, when 4% polyethylene glycol and 3M urea coexist, the slope of the graph increases (reactivity increases), and non-specific reactions are reduced compared to the case of non-coexistence. I understand. When only urea 2M coexists, a decrease in non-specific reaction is observed, but the slope of the graph does not increase compared to the case of non-coexistence, rather the graph shifts to the lower value side and the reaction It can be seen that the sex is generally decreased.

  FIGS. 16A, 16B, and 16C show (measurement value average + 2 × standard deviation) for detection sensitivity (measurement value average of minimum measurement points that do not overlap with (measurement value average−2 × standard deviation) of adjacent measurement values). + 2 × standard deviation) is indicated by a broken line. Table 1 shows the detection sensitivity calculated from the measured value, the execution sensitivity calculated from the graph created by the power approximation of the measured value, and the concentration corresponding to 20% CV (coefficient variation) of the measured value, zero sample measured value average + 2 X Summarizes three types of sensitivity evaluations, ie, detection limits calculated from a correlation equation when a measured value having a concentration corresponding to a standard deviation value of 10 ng / mL or less is linearly regressed. In any of the sensitivity evaluation methods of FIG. 16 or Table 1, it can be seen that a sensitivity improvement of 10 times or more is recognized by the immune reaction enhancement method in which polyethylene glycol and urea coexist.

実施例１２
水不溶性の球状担体（内部にフェライトを練り込んだ粒子径約１．５ｍｍのＥＶＡ製）に、実施例１で選択したモノクローナル抗体の一方を１００ｎｇ／担体となるよう物理的に吸着させた後、ＢＳＡでブロッキング処理を行った。磁力透過性の容器（容量１．２ｍｌ）に１２個の担体を入れた後、緩衝液（３％ ＢＳＡ、５％水溶性ゼラチン、１０ｍＭトリス緩衝液、ｐＨ８．０）を加え、凍結乾燥した。

Example 12
After physically adsorbing one of the monoclonal antibodies selected in Example 1 to 100 ng / carrier on a water-insoluble spherical carrier (manufactured by EVA having a particle diameter of about 1.5 mm with ferrite kneaded inside), Blocking treatment was performed with BSA. After 12 carriers were placed in a magnetically permeable container (capacity 1.2 ml), a buffer solution (3% BSA, 5% water-soluble gelatin, 10 mM Tris buffer, pH 8.0) was added and freeze-dried.

  1% BSA containing 0.2 μg / mL alkaline phosphatase-labeled anti-SND1 antibody (one of the monoclonal antibodies selected in Example 1), 10 mM Tris buffer, 150 mM NaCl, 0.1% Tween-20, pH 8.0 The measurement was performed using the same immunoassay apparatus as in Example 3. First, 150 μL of the target sample was added to the container and reacted at 37 ° C. for 20 minutes. Then, the unreacted substance was B / F separated, and 100 μL of the labeled anti-SND1 antibody solution described above was added. After reacting at 37 ° C. for 20 minutes, B / F separation was performed to separate and remove the free labeled anti-SND1 antibody from the carrier, 4 methylumbelliferyl phosphate was added, and the substrate was added for 20 to 295 seconds. The production rate (nM / sec) per unit time of the enzymatic reaction degradation product (4-methylumbelliferone) was measured.

  The target sample is a 1 / 2-fold dilution series sample prepared based on a 1000 ng / mL solution of SND1, and (1) a solution containing polyethylene glycol and urea, (2) a solution containing only 2M urea, or (3) 4 One of the solution containing 1% polyethylene glycol (PEG6000) and 3M urea and the target sample were mixed at a ratio of 1: 1, and then 150 μL of the sample was added to a container to carry out the measurement. The results are shown in FIG.

  As shown in FIGS. 17A and 17B, when polyethylene glycol and urea do not coexist or when only urea coexists, the sensitivity is greatly inferior to the so-called “one-step measurement” described in the above-described embodiment. I understand. On the other hand, as shown in FIG. 17C, when polyethylene glycol and urea coexist, high sensitivity (detection sensitivity 15.6 ng / ml) is achieved, but detection sensitivity (1.56 ng) in so-called “one-step measurement” is achieved. / Ml), the value was about 10 times lower. As in Example 11, a list of sensitivities is shown in Table 2. However, the detection sensitivity could not be calculated because there was no concentration at which CV was 20%.

FIG. 1 is a diagram showing the results of electrophoresis of SND1 prepared in Examples. The protein is stained with Coomassie Brilliant Blue. FIG. 2 shows the results of electrophoresis of SND1 (3 ng / lane) and LnCap cells (7000 cells / lane) prepared in the Examples, transfer to a polyvinylidene fluoride membrane, and alkaline phosphatase-labeled anti-SND1 antibody (selected in the Examples) It is a figure which shows the result at the time of performing Western blotting by 2 types). Detection was performed with CDP-STAR (manufactured by PerkinElmer). CBB shows a stained image obtained by staining all proteins electrophoresed with Coomassie brilliant blue. 3A and 3B show the results of measurement at various urea (Urea) concentrations for 10 minutes and C and D for 40 minutes, respectively. A and C are FBS (zero sample) not containing SND1, B is FBS containing 2 μg / mL of SND1, and D is FBS containing 1 μg / mL of SND1. Each point represents the measured value average ± 2 × standard deviation. 4A and 4B show the results of measurement at various polyethylene glycol concentrations for 10 minutes and C and D for 40 minutes, respectively. A and C are FBS (zero sample) not containing SND1, B is FBS containing 2 μg / mL of SND1, and D is FBS containing 1 μg / mL of SND1. Each point represents the measured value average ± 2 × standard deviation. FIG. 5 is a diagram showing the difference in reactivity between three polyethylene glycols having different average molecular weights. A is a zero sample, B is a sample containing 1 μg / mL SND1, and C is an S / N ratio obtained by dividing the measurement result of a sample containing 1 μg / mL SND1 by the zero sample measurement result. FIG. 6 is a diagram showing the results of verifying the reactivity in 10 minutes of immune reaction when 2M urea and polyethylene glycol coexist. A is the measurement result of zero sample, B is the measurement result of FBS containing 2 μg / mL of SND1, and C is the S / N ratio, and each point is the average of the measured values ± 2 × standard deviation. FIG. 7 shows measurement results of a zero sample, measurement results of a sample containing 2 μg / mL of SND1, and S in the case of coexistence of urea 2M, coexistence of urea 2M and polyethylene glycol 2.5%, and coexistence of urea and polyethylene glycol. / N ratio is shown. FIG. 8 is a diagram showing the results of verifying the reactivity in 10 minutes of immune reaction when 5% polyethylene glycol and urea coexist. A is the measurement result of zero sample, B is the measurement result of FBS containing 2 μg / mL of SND1, and C is the S / N ratio, and each point is the average of the measured values ± 2 × standard deviation. FIG. 9 shows measurement results of a zero sample, measurement results of a sample containing 2 μg / mL of SND1, and S in the case of coexistence of 5% polyethylene glycol, coexistence of 5% polyethylene glycol and 3M urea, and coexistence of urea and polyethylene glycol. / N ratio is shown. FIG. 10 is a diagram showing the results of verifying the reactivity in 40 minutes of immune reaction when 2M urea and polyethylene glycol coexist. A is the measurement result of zero sample, B is the measurement result of FBS containing 2 μg / mL of SND1, and C is the S / N ratio, and each point is the average of the measured values ± 2 × standard deviation. FIG. 11 shows a measurement result of a zero sample, a measurement result of a sample containing 2 μg / mL of SND1, and an S / N ratio in the case of coexistence of 2M urea and 1% polyethylene glycol, and in the absence of urea and polyethylene glycol. It is a figure which shows the result of having verified the reactivity in 40 minutes of immune reactions when 4% polyethyleneglycol and urea coexist. A is the measurement result of zero sample, is the measurement result of FBS containing 1 μg / mL of SND1, and C is the S / N ratio, and each point is the average of measured values ± 2 × standard deviation. FIG. 13 shows the measurement result of the zero sample, the measurement result of the sample containing 1 μg / mL of SND1, and the S / N ratio in the case of coexistence of 4% polyethylene glycol and 3M urea and in the absence of urea and polyethylene glycol. FIG. 14 is a diagram showing the reactivity when an SND1 sample prepared in a 1/2 dilution series based on 1000 ng / mL is measured with A: 10-minute immune reaction and B: 40-minute immune reaction. . The measurement was performed by triplicate measurement, and an average value ± 2 × standard deviation value was shown. FIG. 15 is a diagram showing the reactivity when an SND1 sample prepared in a 1/2 dilution series based on 50 ng / mL is measured by an immune reaction for 40 minutes. The measurement was performed in 5-fold measurement, and the average value ± 2 × standard deviation value was shown. FIG. 16 shows the measurement results in the case of A: non-existence of urea and polyethylene glycol, B: coexistence of 2M urea, C: coexistence of 4% polyethylene glycol and 3M urea. Value average -2 × Measured value average of points not exceeding standard deviation + 2 × standard deviation value is indicated by a broken line. FIG. 17 shows a result of so-called two-step measurement using A; non-existence of urea and polyethylene glycol, B: coexistence of 2M urea, C; coexistence of 4% polyethylene glycol and 3M urea. The measurement value average of the next sample−2 × the measurement value average of the points not exceeding the standard deviation + 2 × standard deviation value is indicated by a broken line.

Claims (5)

  A method for enhancing an antigen-antibody reaction, wherein an antigen and an antibody are reacted in the presence of polyethylene glycol and urea.   2. The method of claim 1, wherein the antigen or antibody is mixed with polyethylene glycol and urea and then reacted with the antibody or antigen.   The method according to claim 1 or 2, wherein the concentration of polyethylene glycol in the solution causing the reaction between the antigen and the antibody is 1 to 10% (weight / weight).   The method according to claim 1 or 2, wherein the concentration of urea in the solution causing the reaction between the antigen and the antibody is 1 to 4 mol / liter.   A reagent that enhances an antigen-antibody reaction, comprising either an antigen or an antibody, polyethylene glycol, and urea.

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