JP2000266750A - Immunoassay of midkine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To greatly improve immunoreaction efficiency in comparison with that under a physiological condition by setting ionic strength of an immunoreactive buffer solution used for a reaction between Midkine(MK) and an antibody within a predetermined value range higher than a usual one. SOLUTION: A preferable measurement system can be constructed because a polyclonal antibody and a monoclonal antibody are used alike. As an immunoreaction buffer solution, a phosphoric acid buffer solution, tris- hydrochloric acid buffer solution, and the like can be used. For regulating ionic strength, a tris-hydrochloric acid buffer solution with ignorable strength can be used conveniently. Although a salt such as NaCl and KCl may be used for regulating ionic strength, KCl is desirable. As to a salt concentration, an ionic strength ranging 0.3-1.5 is used, however, an ionic strength ranging from 0.4 to 0.6 provides the most stable result. An MK measuring immunoreaction buffer solution improving ionic strength accomplishes improvement important for high sensitive immunomeasurement when it is used in either of two reactions between MK and an antibody carried out in a two-step sandwich method or used in a one-step method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for improving the sensitivity and stabilization of MK immunoassay used for quantitatively measuring midkine (hereinafter referred to as MK) in a clinical sample. The present invention relates to a method for significantly improving the efficiency of an immune reaction with an antibody, and at the same time, reducing nonspecific reactions to achieve both high sensitivity of the reaction and improvement in measurement reproducibility.

[0002]

2. Description of the Related Art MK is a heparin-binding growth factor rich in basic amino acids having a molecular weight of 13,000 and found as a product of a gene whose expression is induced by retinoic acid.
(Kadomatu, K. et al.,: Biophys. Res. Commu., 151: 1312-1.
318) It has been suggested that MK is involved in maintaining survival of nerve cells and elongating neurites, and is also deeply involved in processes such as ontogenesis, tissue repair, and progression of cancer. In addition, the expression of MK has been examined in many human cancers, and the expression of MK at a higher frequency is recognized in cancerous parts more frequently than in non-cancerous parts. From such a background, measuring MK in human body fluid may provide an effective clue for diagnosis and treatment of various diseases including cancer. However, the concentration of MK in human body fluid is as low as several tens to several hundreds pg / ml (healthy persons),
In order to assemble a practical measurement system, it is important to assemble a measurement system that provides as high a sensitivity as possible and a result with good reproducibility. Therefore, the present inventors have studied various conditions for measuring MK with higher sensitivity and reproducibility in an immunological assay using a sandwich assay,
It has been found that the reactivity between MK and the antibody is significantly improved when the ionic strength of the buffer for the immune reaction is higher than the strength normally used. Moreover, the ionic strength showing the highest reactivity is around 0.5, which is much higher than the usual physiological condition of 0.15, and the improvement of the response at that time is under physiological conditions
When the ionic strength is more than 4 times, and when the ionic strength is 0.3 or more, the fluctuation range of the response due to the fluctuation of the ionic strength becomes small, and further, the reaction to the non-specific solid phase is reduced, and the blank level is reduced. We also found that the reproducibility of the results was improved, and completed the immunological assay for MK of the present invention, which provides highly sensitive and reproducible measurement results.

[0003]

In the sandwich assay method, when measuring a substance to be measured having at least two epitopes (hereinafter referred to as an antigen), an antibody immobilized and an antibody in a liquid phase are used. This method measures the amount of antigen by sandwiching (= sandwiching) the antigen.
At this time, actually, the antibody in the liquid phase is labeled (labeled) with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like, and the amount of the antigen to be measured is evaluated from the amount of the label. In addition, the liquid-phase antibody itself is not labeled, and the liquid-phase antibody is indirectly labeled by further reacting with a tertiary antibody such as a labeled anti-liquid-phase antibody that specifically reacts with the liquid-phase antibody. There is also a method for evaluating the amount of antigen to be measured from the amount of antibody. These measurement systems perform an immune reaction twice, that is, a so-called two-step measurement system and perform only once.
It can be divided into two types of step measurement systems. In the two-step measurement system, first, an antigen is bound to a solid-phase antibody as a primary reaction, and then unreacted antigen is removed by washing or the like. Then, a liquid-phase antibody labeled as a secondary reaction is added to the measurement system, This is a method in which a liquid phase antibody is bound to an antigen molecule trapped by a phase antibody. In a one-step measurement system, antibodies that recognize different epitopes are combined as antibodies, and the binding reaction between the antigen and these antibodies is fixed. This method is performed simultaneously in the presence of a phase antibody and a liquid phase antibody. Since the one-step measurement system has a small number of measurement operations and high reaction efficiency, almost one-step measurement systems are used when monoclonal antibodies capable of selecting antibodies having different epitopes are used. In the case of MK, it is possible to set up a one-step measurement system even with a combination of polyclonal antibodies by the method described in JP-A-10-160735. At this time, the buffer for the immunoreaction is two types for the primary reaction and the secondary reaction in the case of the two-step measurement system, and is one type in the case of the one-step measurement system. When an unlabeled antibody is used as a liquid phase antibody in the case of a one-step measurement system, a step of further reacting a labeled anti-liquid phase antibody against the liquid phase antibody is required. The buffer for use does not include the buffer used in such a process, but refers to the buffer for immunoreaction used for the reaction between the MK to be measured and its antibody. Further, for example, there is a method in which an antibody to be solid-phased is labeled with biotin or the like and trapped with a streptavidin solid phase after an antigen-antibody reaction.In this case, too, the binding process between the biotinylated antibody and the streptavidin solid phase is also used. The reaction buffer used in the above is not included in the present invention. The present invention relates to a buffer for an immunoreaction for MK measurement, which significantly improves the efficiency of an immunoreaction found as a result of examining a buffer for an immunoreaction for MK measurement and improves the reproducibility of the reaction itself. .

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the method of the present invention will be described in more detail. Usually, the ionic strength of the buffer solution for immunoreaction used in a measurement system assembled using a polyclonal antibody is generally set to around 0.15 of physiological conditions. (P.Tijssen, translated by Eiji Ishikawa, Enzyme Immunoassay, Biochemical Experimental Method 11, Tokyo Kagaku Doujin, p114) On the other hand, non-specific It is added for the purpose of suppressing the binding reaction, and there is no known example in which the reaction efficiency of the target antigen-antibody reaction itself is improved as the ionic strength is increased as in the present invention. For example,
In the measurement of human growth hormone (hGF), (Hashida,
S.et al., Use of inorganic salts tominimize serum i
nterference in a sandwich enzyme immumoassay for h
umangrowth hormone using Fab'-horseradish peroxid
Clin. Clim. Acta, 135 : 263-273, 1983) When measuring a biological sample such as serum as a sample as described in There is described an example in which 0.4 M NaCl is added to an immunoreaction buffer for the purpose of suppressing the original antigen-antibody reaction from being inhibited by adsorption to a solid phase. in this case,
The purpose is to restore the response reduced by inhibition to the state before inhibition by adding more salt under physiological conditions, and the antigen-antibody reaction itself is amplified by the addition of salt. Absent. Therefore,
The response when adding a high concentration of salt does not exceed the response when measuring hGF in a buffer under physiological conditions without serum proteins. The method of reducing non-specific reactions by adding such a salt is that as the ionic strength increases, the charge of a protein that is easily non-specifically adsorbed to the solid phase is shielded by the presence of ions, and the adsorption power decreases. It is based on However, a minority, such as hGF, is extremely sensitive to serum components, and it is known that increasing the ionic strength of the reaction system usually reduces the binding strength of the antibody to the antigen to be measured. (Eisen, HN 1980 Immunology: An Introductio
n to Molecular and Cellular Principles of the Immu
ne Responses. 2nd edition.Harper and Row Publ.,)
At present, the ionic strength of the commonly used immunoassay system is considered to be a physiological condition as a result of considering both the effect of reducing non-specific reactions and the condition that does not reduce the reactivity of the original antigen-antibody reaction as much as possible. Is often set near 0.15.

[0005] The present inventors have also set up an immunoassay for MK in which how the ionic strength of the buffer used for the immune reaction between MK and an antibody affects the efficiency and specificity of the reaction. After examining whether it is
As the ionic strength is increased as described above, the response increases as well, and it is found that the blank level, which is an index of a nonspecific reaction, decreases with an increase in the ionic strength. MK with good measurement reproducibility
Has been completed. The effect is
Even when the measurement system was a one-step measurement system, it was observed at any stage of the two immunoreactions in the two-step measurement system. It was also confirmed that the response increased in response to the fluctuation. Specifically, when the salt was NaCl, the response increased 3.2 times when the ionic strength varied from 0.15 to 0.3, and when the salt was 0.4, the response was 4.1 times 0.15. Although the response gradually decreased after 0.4, the decrease was gradual, and 0.15
The response was lower than in the case of 1.
It was after three or more. However, even if the response is below physiological conditions, the signal / noise ratio, which is related to the sensitivity of the immune response, exceeds the physiological condition of 0.15 until the ionic strength reaches 1.5 because the blank is greatly reduced. In that sense, 1.5 was within the range of effective ionic strength. However, practically, in the case of NaCl, the range of 0.4 or more and 0.6 or less has a small fluctuation range of the response due to the fluctuation of the ionic strength and there are few secondary problems such as salt precipitation. Is determined to be a range of ionic strength suitably used in the MK measurement. Also
Similar effects are observed with salts other than NaCl. For salts that generate monovalent ions, KCl and KI, for salts that generate divalent ions, ammonium sulfate (NH ₄ ) ₂ SO ₄ and potassium sulfate K ₂ SO ₄ The effect was confirmed. This indicates that the magnitude of the ionic strength itself is important, not the phenomenon that this phenomenon appears only in specific ions such as sodium or potassium ions or chloride ions.
On the other hand, a large variation in response due to ionic strength poses a problem in terms of measurement reproducibility. In other words, from the viewpoint of practicality, it can be said that the reproducibility of the measurement is easier to ensure if the response does not fluctuate even if the ion intensity of the measurement system fluctuates somewhat. In this regard, KCl is the most suitable salt for the MK measurement system studied in this study because KCl has a wide range of ionic strength showing the same response and has a lower blank level than other salts. When KCl is used, the range of ionic strength that gives a highly reactive and stable response is based on Tris-HCl buffer.
It is 0.3 to 0.6. However, the actual use concentration of KCl differs depending on how many times the analyte to be measured is diluted. This is because, in practical use, the ionic strength of the specimen itself also becomes a problem, and the concentration not affected by the ionic strength is the concentration actually used. For example, serum, plasma, saliva, cerebrospinal fluid,
When various body fluids such as urine are used for the specimen, KCl may be added to the immune reaction buffer in the range of 0.4 to 0.5. Because the ionic strength of the body fluid is considered to be in the range of 0 to 0.15, if the dilution ratio of the sample with the buffer for immunoreaction is about 10 times, the use of This is because the change in the ionic strength of the buffer during the immune reaction due to the change in the ionic strength can be almost ignored. However, when the dilution ratio of the sample can be made extremely high, it is not necessary to stick to the above range of ionic strength.
However, since the range of fluctuation of the response is large between 0.15 and 0.3, it is important to arbitrarily select an ion intensity in a range higher than 0.3 where the range of fluctuation is small in order to stabilize the measurement result. In addition, the effect of the reaction temperature and the pH of the reaction solution on the range of ionic strength was examined.The range of ionic strength showing a good response was the fluctuation of the reaction temperature between 20 ° C and 37 ° C and the range of pH between 7 and 9.2. Reaction liquid
The previous range can be judged to be almost universal because it was not affected by the fluctuation of pH.

When performing an immunoassay for MK, 0.5
As a result, the antigen-antibody reaction was promoted at a very high ionic strength compared to 0.15 under physiological conditions, resulting in a response four times or more higher than that under normal conditions. Since the binding of the labeled antibody to the solid phase decreases and the blank level decreases, the finding that the double effect results in a highly sensitive and highly reproducible measurement system is newly found. is there. In addition, we measured human MK or mouse MK using another antibody, anti-mouse MK antibody, that this phenomenon was not a phenomenon that happened to be observed in the reaction between the currently used anti-human MK antibody and human MK. In this case, it was confirmed that the same phenomenon was observed. Furthermore, even if the antibody was derived from a different animal species from the rabbit antibody and the chicken antibody, no difference was observed between the immunized animals in the effect of ionic strength on the reaction between the obtained antibody and the MK antigen. Interestingly, in the two-step measurement system, the response increased with increasing ionic strength in both the first and second steps,
When the antibody reacts with the immobilized antigen prepared by directly immobilizing the MK antigen, the reactivity of the antibody to the solid phase antigen decreases as the ionic strength increases, as in the case of measuring the normal antigen. I was In other words, the phenomenon discovered this time is observed when the MK antigen on the liquid phase or on the antibody reacts with the antibody, in other words, when the MK antigen with a high degree of conformation reacts with the antibody. In the case of solid phase immobilization, it was not recognized. It is considered that these facts suggest that the conformation of MK changed from a normal state due to the increase in ionic strength, and as a result, the reactivity with the antibody was improved. The change in conformation is thought to occur due to the change in the charge on the MK surface, and it is speculated that the change in the charge occurs with an increase in ionic strength. That is, it is considered that what is important is a change in ionic strength and does not depend on the type of ion.

[0007] In the embodiment of the present invention, either polyclonal antibody or monoclonal antibody can be used as the antibody, but according to the method described in JP-A-10-160735,
Since a polyclonal antibody can be used in the same manner as a monoclonal antibody, a more desirable measurement system can be set up. When labeling a liquid phase antibody with a radioisotope, a method of binding radioactive iodine to the antibody using chloramine T is generally used. When labeling the enzyme, peroxidase (hereinafter POD), alkaline phosphatase, β-galactosidase and the like can be used as the labeling enzyme, but their selection is based on the final colorimetric
What should be done is what to select for the detection means such as light emission and fluorescence. For example, POD for commonly used colorimetry
Often select. Various methods are known for labeling an enzyme with an antibody.
kawa E et.al., Ann NY Acad Sci 420 74-89; 1983) can introduce enzymes into the hinge region of an antibody that is less likely to affect the antigen-antibody reaction under mild conditions. Cheap. As a method for immobilizing an antibody, a method for physically immobilizing an antibody regardless of the material of the solid phase is generally used. As the material of the solid phase, a known material that has been used for sandwich measurement, such as a magnetic material such as glass, plastic, or ferrite, can be used. Some of them have introduced a functional group such as an amino group, in which case the antibody can be immobilized by a chemical method. The buffer for the immune reaction used in the method of the present invention includes a phosphate buffer,
Buffers usually used for an immune reaction, such as a Tris-HCl buffer and a borate buffer, can be used. However, since the adjustment of the ionic strength changes depending on the type of the buffer, a Tris-HCl buffer, which can almost ignore the ionic strength of the buffer, is easily used. For example, when a condition for increasing the concentration of the buffer using a phosphate buffer is selected, it is necessary to set the concentration of KCl or the like again while paying attention to the ionic strength of the phosphate. To adjust the ionic strength, a salt such as NaCl or KCl may be used, but KCl is preferably used for the reasons described above. The concentration may be arbitrarily set between 0.3 and 1.5, but the range between 0.4 and 0.6 gives the most stable result. Since KCl and NaCl are monovalent strong electrolytes, it goes without saying that the ionic strength can be replaced by the molar concentration. The immunoreaction buffer for MK measurement with increased ionic strength of this method can provide a high response regardless of whether the two-step sandwich reaction between MK and the antibody is used or the one-step sandwich method. It is possible to realize an important performance improvement in performing a highly sensitive immunoassay such as an increase and a decrease in a blank level.

[0008]

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

Example 1 Effect of NaCl Concentration in Immune Reaction Buffer on One-Step ELISA Measurement of Human MK Human MK used in this study was a recombinant human MK obtained by the method described in JP-A-09-95454. Was. Rabbit anti-MK antibody, its peroxidase (hereinafter POD) -labeled antibody, and antibody-immobilized plate were prepared by the method described in JP-A-10-160735. The chicken anti-MK antibody was purified in the same manner as the rabbit antibody except that purification with Protein A was not performed, and the POD-labeled antibody and the antibody-immobilized plate were also prepared in the same manner as the rabbit antibody. 50 mM Tris-HCl 0.
^{5% BSA 0.01% Microcide TM I} (Amresco Inc. CODE E448) pH8.
2 was prepared, and NaCl was added therein to 0.8 M in 0.05 M steps, 0.8 M
It was added to 1.5M in 0.1M increments. Human MK O, 100ng /
ml (PBS 0.5% BSA 0.01% Microcide I) solution was diluted 10 times with Tris-HCl buffer adjusted to each salt concentration, and human MK was diluted.
After preparing a 0.10 ng / ml solution, it was used for the following measurements. The prepared human MK antigen standard solution was dispensed into test tubes in an amount of 20 μl. Then, POD-labeled chicken anti-human MK was added to the above Tris-HCl buffer.
The solution to which the antibody was added was dispensed in 200 μl portions. After mixing, 100 μl of each solution was dispensed into a rabbit anti-human MK antibody plate.
The reaction was performed at room temperature for 30 minutes. After washing 5 times with PBS-Tween20 washing solution, tetramethylbenzidine is used as an enzyme coloring solution.
A solution (hereinafter referred to as TMB) (100 μl, DAKO Co., S1600) was dispensed in 100 μl portions and reacted at room temperature for 30 minutes. Thereafter, the reaction was stopped with 100 μl of 2N sulfuric acid, and the absorbance at a wavelength of 450 nm was measured. Table 1 shows the obtained results. It can be confirmed that the response fluctuates greatly with the increase in the NaCl concentration, and that the response sharply increases particularly with an increase in the concentration from the physiological condition of 0.15M. In addition, the blank level decreases as the ionic strength increases.
Even at 5M, the S / N ratio shows a better value than at 0.15M.

[0010]

[Table 1]

Example 2 Comparison between NaCl and other salts The same measurement as in Example 1 was carried out for KCl, ammonium sulfate (NH ₄ ) SO ₄ , and potassium sulfate K ₂ SO ₄ in addition to NaCl. In order to compare the influence of each salt on the blank level, the measurement was performed in the same manner as in Example 1 except that the concentration of the labeled antibody was 2.5 times that in Example 1. The obtained results are shown in FIGS. 1A, 1B, 1C, and 1D. Although there was a difference in the pattern of response increase depending on the type of salt, all the salts examined showed a response higher than the physiological condition (NaCl 0.15) at an ionic strength higher than 0.15 (FIGS. 1A, B, C, D). In particular, KCl is a salt that is preferably used as a high ionic strength modifier of the present invention because it maintains a high response at almost the same level in the widest range of ionic strength and also shows the lowest level in the blank level. This was found (FIG. 1B).

Example 3 Influence of Ionic Strength of Immune Reaction Buffer during Two-Step Reaction The reaction performed in Examples 1 and 2 is a step in which MK antigen reacts with a solid phase antibody, and the solid phase antibody is reacted with MK antigen. The reaction was divided into two steps, in which the labeled antibody was reacted in this state, and the effect of the KCl concentration in the immune reaction buffer in each reaction step was examined. Influence of KCl concentration in the buffer for immunoreaction in the first step Example 1 was carried out in the same manner as in Example 1 except that KCl was added to the same Tris-HCl buffer in increasing concentrations and no labeled antibody was added.
After dilution of the MK antigen in the same manner as described above, the mixture was dispensed into a rabbit anti-human MK antibody plate and reacted at room temperature for 30 minutes. After washing, Tris-HCl Buffer containing 0.45 M of KCl was used as a buffer for the second reaction, and a chicken anti-human MK-POD-labeled antibody was added. After dispensing the plate, the plate was reacted at room temperature for 30 minutes. After washing, TMB
The enzyme coloring solution was dispensed, reacted at room temperature for 30 minutes, stopped with sulfuric acid, and the coloring levels were compared. Influence of KCl concentration in buffer for immunoreaction in the second step In the same reaction as in 1, the KCl concentration in the first buffer for reaction was fixed at 0.45 M and the amount of MK antigen was 10 times the amount of solid phase antibody. After a sufficient amount of the antigen was bound, the KCl concentration in the second reaction buffer was gradually increased, and the measurement was performed.

FIG. 2 shows the results. In both the first reaction and the second reaction, the response was improved as the KCl concentration was increased to 0.55M. That is, when the immobilized antibody reacts with the MK antigen in the liquid phase, or when the MK antigen immobilized on the immobilized antibody reacts with the labeled antibody in the liquid phase, the ion of the immune reaction buffer is It was confirmed that by setting the intensity higher than the physiological condition, the reaction efficiency between the MK antigen and the antibody was improved.

Example 4 Influence of Ionic Strength on the Reactivity of Physically Immobilized MK Antigen with Antibody MK antigen was diluted with PBS to a concentration of 10 μg / ml in Polysorp ^™ P
Late (NUNC) was dispensed in 50 μl aliquots, sealed, and incubated at room temperature for 16 hours to immobilize the MK antigen on the plate. KCl was gradually added to the base immunoreaction buffer used in Example 1 at a gradually increasing concentration, and a chicken anti-human MK-POD labeled antibody was added to each buffer by dilution, and then the MK-immobilized plate was added. And reacted at room temperature for 30 minutes. After washing 5 times with PBS-Tween20 buffer, the TMB solution (DAKO
S1600) was dispensed, reacted at room temperature for 30 minutes, stopped the reaction with sulfuric acid, and measured the absorbance at 450 nm. At this time, the same reaction was performed by dispensing the above reaction solution also on a plate on which bovine serum albumin (BSA) was immobilized as a blank. FIG. 3 shows the obtained results.

In this case, the reactivity of the chicken anti-human MK antibody to the immobilized MK antigen decreased as the KCl concentration increased. From the above, in the measurement of MK, the reactivity between the MK antigen and the antibody is improved with an increase in the ionic strength when MK is present in the liquid phase or when it is bound on the antibody, MK It was found that such an effect was not exerted when was directly immobilized on a solid phase, and that an immune reaction was suppressed with an increase in ionic strength, similarly to a normal antigen. The difference between the two states is considered that the former is a state in which the conformation of the MK molecule can be changed, and the latter is a state in which it is difficult to change. In other words, the effect of enhancing the immune response due to the high ionic strength in the present MK measurement was presumed to be that the conformation of MK changed with the increase in ionic strength, and as a result, the reactivity with the antibody was improved. It is probable that the effect did not appear when the formation was hard to change. From the above examination, it can be said that the effect of the method of the present invention is due to the fact that the MK antigen itself changes due to an increase in ionic strength, and the effect is not limited to the type of ion, even though the effect depends on the type of ion. .

[0016]

According to the present invention, when MK is measured by a measurement system using an immunoreaction, the ionic strength of an immunoreaction buffer used for the reaction between MK and an antibody is set to a higher than usual range of 0.3 to 1.5. , The efficiency of the immune response is significantly improved compared to physiological conditions, and non-specific responses are reduced,
Measurement with high sensitivity and good reproducibility is possible.

[Brief description of the drawings]

FIG. 1 shows the results of examining the response and blank level when the ionic strength of an immunoreaction buffer was varied using various salts when MK was measured by the one-step sandwich method.

FIG. 2 shows the results of examining the response and blank level when the ionic strength of the immune reaction buffer for the first reaction and the second reaction was varied using KCl when measuring MK by the two-step sandwich method. Yes (A: first reaction buffer, B:
Second reaction buffer).

Fig. 3 Response when the ionic strength of the immune reaction buffer was varied using KCl when reacting chicken anti-human MK-POD-labeled antibody with the MK antigen solid phase in which the MK antigen was directly immobilized on a plate. And the blank level. In this case, a BSA solid phase is used for the blank.

Claims (2)

[Claims] In an immunoassay for quantitatively measuring Midkine (MK), the ionic strength of an immunoreaction buffer for reacting an anti-MK antibody and MK is adjusted to 0.3 to 1.5.
MK immunological assay method characterized by setting 2. The method according to claim 1, wherein the ionic strength of the reaction system is adjusted with potassium chloride.