JPH10160735A - Sandwich measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sandwich measuring method in which a high-sensitivity measuring system can be constructed by a method wherein two kinds of polychlonal antibodies are used as a first antibody and a second antibody for a sandwich method. SOLUTION: A sandwich method is constructed in such a way that human MK is used as an antigen and that a rabbit and a chicken are used an immune animals. When epitopes on MK molecules recognized by two kinds of polyclonal antibodies derived from the rabbit and the chicken are compared, the two kinds of polyclonal antibodies recognize respectively different epitope groups on identical antigen molecules. When the polyclonal antibodies are used as a first antibody and a second antibody for the sandwich method, it is possible to establish the sandwich method similar to that of monoclonal antibodies. In addition, an antigen is a protein which is originally owened by an animal species, different animal species are immunized, and polyclonal antibodies are obtained from them. In addition, two kinds of animal species which are immunized are not limited to the rabbit and the chicken, and a combination of animal species in which epitopes are not overlapped can be used arbitrarily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a so-called sandwich assay in an immunoassay.

[0002]

2. Description of the Related Art An immunoassay is a method for quantitatively measuring an antigen or antibody using an antigen-antibody reaction. Among them, the immunoassay using the enzyme as a tracer is called "enzyme immunoassay" or "enzyme immunoassay". It is generally called enzyme immunoassay, and EIA has been established as an abbreviation.

[0003] For EIA, an unbound free fraction F (free form) of an enzyme-labeled antibody (or antigen) is combined with an antigen-antibody conjugate B (bound
form) which requires a BF separation operation, and those which do not. The former is heterogenous E
IA, the latter is called homogeneous EIA. The homogeneous method has the advantage that the operation is simple and results can be obtained in a short time, but has the disadvantage that the measurement sensitivity is low and the measurement range is narrow. There are two types of heterogeneous methods, a competitive method and a non-competitive method. The former is a method in which a fixed amount of a labeled antigen and a labeled antigen are competitively bound to a fixed amount of an antibody, and the enzyme activity contained in the bound (or free) form is measured. This method is not widely used due to its narrow measurable concentration range, poor sensitivity, and susceptibility to blood interfering substances. Therefore, the latter method is generally used for the so-called sandwich method or two-point measurement method. It is a target. The principle is that an immune complex consisting of an antibody insolubilized on a solid phase, an antigen to be measured in a liquid phase, and an enzyme-labeled antibody is formed on the solid phase, and the enzyme activity on the solid phase is measured and measured. This is a method to know the amount of antigen to be used.

[0004] In the sandwich method, when a polyclonal antibody is used as an immobilized antibody (also referred to as a first antibody) and an antibody in a liquid phase (also referred to as a second antibody), generally, the polyclonal antibody is composed of an antigen molecule. Since the antigen solution is a population of several monoclonal antibodies that react with the various different epitopes above, most of the epitopes on the antigen molecules will be in the liquid phase if the antigen solution and polyclonal antibodies in the liquid phase are reacted in advance. And the number of epitopes that can be reacted is extremely reduced. As a result, even if this complex is reacted with an immobilized antibody, an accurate measurement of the amount of antigen cannot be performed. That is, in general, when the antibody in the liquid phase is not a monoclonal antibody, the antigen to be measured and the antibody in the liquid phase cannot be reacted in advance without any operational restrictions.

[0005] Therefore, first, an antigen is bound to an immobilized antibody as a primary reaction, and a liquid phase antibody labeled with an enzyme or the like is added to a measurement system as a secondary reaction, and the antigen is trapped on the antigen molecule trapped by the immobilized antibody. A sandwich method in which a liquid phase antibody is bound to an epitope to which no immobilized antibody is bound has been used. Alternatively, there is also a method in which an antigen, a liquid-phase antibody, and a solid-phase antibody are almost simultaneously mixed to initiate a reaction, and a sandwich method is established by competing for epitopes. In this case, the operability is poor and the scramble for epitopes is still occurring.
The reaction efficiency is lower than when the following monoclonal antibodies are combined.

On the other hand, two kinds of monoclonal antibodies recognizing two different epitopes on an antigen molecule are combined as an antibody, or either an immobilized antibody or an antibody in a liquid phase (labeled antibody). A method of using a crab monoclonal antibody, mixing an antigen and a labeled antibody, and reacting the mixed antibody with an immobilized antibody is disclosed in Japanese Patent Publication No. 2-39747. In the method shown here, after the antigen and the labeled antibody are mixed, the immobilized antibody is reacted without any particular time limitation, so that not only the efficiency of the immune reaction is high but also the operability is good. But it can be said that it is excellent.

However, when the antigen to be measured is an antigen originally possessed by humans or other animals (for example, albumin), these antigens have very high homology in the primary structure of amino acids among animal species. For example, it is known that it may be difficult to produce a high-titer monoclonal antibody against an antigen highly homologous to a mouse antigen, for example. On the other hand, in the case of polyclonal antibodies, immunized animals with low antigen homology can be selected in consideration of such antigen homology, so that polyclonal antibodies have higher titers than monoclonal antibodies. High antibodies are often obtained. If it is found that two types of polyclonal antibodies prepared based on such a concept recognize different epitopes on the same antigen molecule, these polyclonal antibodies are used as an immobilized antibody and a liquid phase antibody. Compared to the case where a monoclonal antibody is combined, stronger binding to an antigen can be expected, and it is considered that a sandwich method excellent in operability can be assembled. Therefore, when the antigen to be measured is an antigen originally possessed by the animal, the antigen and the liquid-phase antibody are mixed in advance in the same manner as the monoclonal antibody even in the case of the combination of the polyclonal antibodies, and then reacted with the solid-phase antibody. As a result of studying whether the method is feasible, the present invention has been reached.

[0008]

SUMMARY OF THE INVENTION The present invention is directed to a method for measuring an antigen to be measured using a polyclonal antibody as the first antibody and the second antibody when the antigen to be measured is a protein originally possessed by a living body. It is an object of the present invention to provide a sandwich method by which a complex obtained by reacting with a (labeled antibody) in advance can be reacted with a first antibody.

[0009]

Means for Solving the Problems The present inventors have found that most of the protein antigens that animals originally have in common have a high homology in the primary structure of amino acids among animal species. When focusing on the non-existent parts, there is often no agreement between animal species, so two types of polyclonal antibodies obtained by immunizing two different animals with such a protein antigen recognize the respective antibodies. When the epitopes of the respective antibodies were compared based on the prediction that the epitopes would not completely match, it was confirmed that they recognized different epitopes on the protein antigen molecule as considered by the present inventors. By using such two kinds of polyclonal antibodies for the first antibody and the second antibody of the sandwich method, it is possible to assemble an excellent sandwich system for the protein antigen. It found that there was the completion of the present invention.

That is, the present invention comprises the invention described in each claim.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Antigens applicable to the present invention include proteins originally possessed by animals and do not include foreign antigens such as viruses. Specifically, serum proteins such as albumin and globulin, cytokines of interleukins 1 and 2, G-CSF and midkine described in detail below.
Growth factors such as (MK). Viruses, bacteria, protozoa, etc. are completely foreign to animal species that do not have these,
Since the resulting antibodies will be heterogeneous polyclonal antibodies containing different antibodies having various specificities and affinities, even if two types of immunized animals are used, the antigenic molecules recognized by the polyclonal antibodies obtained from each will be Epitopes overlap and a similar assay cannot be assembled with a monoclonal antibody. In addition, the present invention cannot be applied to a protein produced in a living body if its epitope is in the sugar chain portion. Specifically, CA19
The present invention cannot be applied to so-called sugar chain antigens such as -9.

There have been reported several prior examples in which one kind of antigen is immunized to two kinds of animal species as in the method of the present invention, and measurement is performed using the obtained antibodies. For example, Tokuhei 6-92
972 discloses an example in which an antibody obtained by immunizing a different animal species with an antibody on a solid phase and a labeled antibody is used in an immunoassay for measuring HTLV-III virus. In this case, first, since the antigen to be measured is a foreign antigen, an antibody that can be used in the same manner as the monoclonal antibody as in the method of the present invention cannot be obtained. In addition, a so-called two-step sandwich method in which washing is performed after reacting an antigen with an immobilized antibody is performed in an actual usage form. Further, in the present invention, the antibody obtained by immunizing the antibody on the solid phase side and the labeled antibody with a different animal species is used, when using only an antibody obtained from one kind of animal species, Since a non-specific reaction to the antibody of the animal species may be a problem, it is performed as a method for avoiding the non-specific reaction. In other words, the subject of the invention of the preceding example is a method for removing a non-specific reaction and is not aimed at reducing the reaction operation or the like, so that the two kinds of antibodies obtained as described above are ultimately used. Not used in coexistence.

Further, Japanese Patent Application Laid-Open Nos. 63-118656 and 5-203647
Shows an example in which antibodies derived from different animal species are combined and used in the same state as in the present invention. In the case of these prior examples, foreign antigens such as viruses are also mentioned as antigens to be measured, and two kinds of animal species were immunized with the protein antigen originally contained in the living body described in the present invention. Obviously, in this case, it is not assumed that a polyclonal antibody which can be used in the same manner as a monoclonal antibody can be obtained. Furthermore, there is no example of reacting an antigen with a liquid phase antibody and then reacting with a solid phase antibody as in the method of the present invention, and all three antigens, liquid phase antibodies and solid phase antibodies are reacted simultaneously. Examples are only shown. Here, the above three cases are allowed to react at the same time to coexist, and after the antigen and the liquid phase antibody are reacted in advance, they are reacted with the solid phase antibody at an arbitrary time, and finally,
When people coexist, there is a big gap in the content. The method of simultaneously reacting the above three can be carried out with most antibodies. However, even if the later antigen is reacted with the liquid phase antibody and then reacted with the solid phase antibody at any time, there is no problem at all. This is because the antibody for which no measurement is performed is limited to a combination of antibodies having substantially no epitope overlap.

[0014] Therefore, it is not claimed that a combination of a polyclonal antibody alone and a measurement system similar to that in the case of a combination of a monoclonal antibody can be constructed. Only those using mouse monoclonal antibodies in either the solid or liquid phase have been described. This is thought to be due to the fact that the efficiency of the reaction is improved by utilizing the non-overlapping property of the mouse monoclonal antibody, and there is a combination without overlapping epitopes when using an animal antibody other than the mouse monoclonal antibody. It is clear that we have not confirmed that.

That is, when the same antigen derived from a living body is immunized to different animal species, the epitopes may not overlap as in the case of a monoclonal antibody. These inventions do not mention that the same measurement system as in the case of using a monoclonal antibody can be assembled between antibodies.

As an object of antigen measurement, midkine (mid
kine: MK) and pleiotrophin: pleiotrophin: PT
The present invention will be described in more detail by taking N) as an example. MK was discovered as an expression product of a gene whose expression is induced early during the differentiation process of embryonic tumor cells by retinoic acid (Kadom
atsu, K. et al .: Biochem. Biophys. Res. Commun., 15
1: 1312-1318, 1988). PTN found in neonatal rat brain as a heparin-binding protein capable of neurite outgrowth
(Rauvala, H .: EMBO J., 8: 2933-2941, 1989). MK and PT
N showed 45% homology in primary structure. MK and PTN form independent families as heparin-binding growth factors (Muramatsu, T. et al .: Dev. Growth Differ., 3
6: 1-8, 1994). Human MK and mouse MK have an amino acid primary structure homology of 87%, and human MK and chicken MK have a homology of 65%, indicating a difference in amino acid primary structure between species. When human MKs are immunized to mice or chickens, antibodies against the same amino acid primary structure as the MK of each animal species are not produced, and antibodies against different primary structure parts are produced. It is thought that. As a result, polyclonal antibodies obtained from each animal species recognize different epitopes on the antigen molecule, even if a method that does not differ from a normal polyclonal antibody production method is used as a method for producing antibodies. It is thought that.

Based on the above concept, human MK obtained by the method described in Example 1 of Japanese Patent Application No. 7-255354 was used as an antigen.
The rabbits and chickens were used to assemble the sandwich method for immunized animals. Comparison of the epitopes on the MK molecules recognized by the obtained rabbit and chicken polyclonal antibodies revealed that the two types of polyclonal antibodies recognized different epitope groups on the same antigen molecule as thought by the present inventors. Was found. Using such a polyclonal antibody as the first and second antibodies in the sandwich method, a sandwich method similar to the monoclonal antibody was established. Even when rabbits and chickens were similarly immunized with PTN, the epitopes on the PTN molecules recognized by the polyclonal antibodies obtained from each were different from each other.

The expression "recognizing different epitopes on the same antigen molecule or not substantially overlapping epitopes" as used herein means that the concentration of polyclonal antibody from each animal species coexisting in the reaction solution is: Normal,
In the concentration range used when measuring the amount of antigen,
This means that each polyclonal antibody can perform the measurement without any inhibition of the reaction of the other antibody with the antigen without any problem, and does not mean that there is no overlapping portion at all.

The term "antigen" as used herein refers to a protein originally possessed by an animal species, and can be used to immunize different animal species to obtain polyclonal antibodies from each species, and to determine the presence or absence of overlap in epitope measurement. It is sufficient that the sandwich method of the present invention can be established by confirming the above, and is not limited by the presence or absence of information on the primary structure of the amino acid.

Furthermore, two kinds of animal species used for immunization are also
The combination is not limited to rabbits and chickens, and any combination of animal species having substantially no overlapping epitopes can be used.

Among such combinations of two animal species, it is particularly preferable to use one animal species as a chicken. In practicing the present invention, it is an animal species to be considered for use first. This is because chicken antigens are generally considered to have a large difference in primary structure from mammalian animal antigens. This is because an IgG antibody (IgY) can be obtained, and further, the IgY antibody has no merits for rheumatoid factor, and thus has a great merit as a material for a clinical diagnostic agent.

On the other hand, it should be noted that conventionally, no immunized animal has been selected for the purpose of eliminating the overlapping of epitopes because of the lack of such recognition.

Furthermore, for example, even when a human antigen is to be measured, if a human antigen can be measured by a combination of polyclonal antibodies obtained by immunization with an antigen derived from another animal species, the human antigen derived from another animal species can be measured. It is also possible to select an antigen as the immunogen. For example, mouse antigens can be selected as immunogens to measure human antigens.

At this time, at least the polyclonal antibody used for the solid phase is preferably used after affinity purification using the antigen used for immunization in order to improve the specificity and reaction efficiency of the reaction. The polyclonal antibody to be used does not necessarily need to be subjected to affinity purification. For example, in a sandwich method using a third antibody, the antiserum can be used as it is. However, when a liquid-phase polyclonal antibody is labeled and used, it goes without saying that it is better to use this polyclonal antibody after affinity purification.

When the liquid-phase polyclonal antibody is directly labeled with a radioisotope, an enzyme, or the like, the immune reaction is reduced to 1%.
A so-called one-step sandwich method, which is performed in stages, is possible with the antibody combination of the present invention.

Further, when the liquid phase antibody is not labeled,
The reaction is carried out in the coexistence of the solid-phased antibody, the antigen, and the antibody in the liquid phase.
A method for further reacting the antibody can be used.

When the polyclonal antibody of the present invention is used, regardless of whether the one-step sandwich method is used or the third antibody (labeled antibody) is used,
The antigen and the liquid phase antibody can be mixed with each other and then reacted with the immobilized antibody immediately, or can be incubated for a certain period of time and then reacted with the immobilized antibody to give substantially the same results.

Examples of the insoluble solid used in the solid phase include inorganic carriers such as glass, silica gel, silicate carriers such as bentonite, magnetic substances, and organic carriers such as
Examples include plastics, polymer gels (polysaccharides such as dextran and agarose or polyacrylamide gels), filter paper, and the like. Among these, preferred are glass (glass beads and glass tubes) to which antibodies can be easily and stably bound, and easy to handle, and plastic (plastic beads, plastic tubes, plastic trays).

As a method of binding the first antibody to the insoluble solid, a method of chemically binding glass to the antibody,
For example, a method using a silane coupling agent and, if necessary, a crosslinking agent (JP-A-54-108696), or a method of physical adsorption (US Pat. Nos. 4,289,092 and 3,682,617),
A method of physically adsorbing antibodies to plastic ("Enzyme Immunoassay" by P. Tidsen) or a method of efficiently immobilizing antibodies by reacting avidin immobilized on a solid phase with biotin chemically bound to antibodies. Etc.

The enzymes used for labeling in the present invention include, for example, β-D-galactosidase, peroxidase, glucose oxidase, alkaline phosphatase, catalase, acetylcholinesterase, glucoacylase, malate dehydrogenase, glucose-6-
Phosphate dehydrogenase, urease and the like.

Many enzyme labeling methods such as glutaraldehyde method, periodic oxidation method, maleimide method, pyridyl disulfide method, carbodiimide method, mixed anhydride method and the like have been developed. It is preferable because the maleimide group and the thiol group respectively introduced efficiently react under mild conditions to form a stable crosslink.

[0032]

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

Example 1 Preparation of rabbit and chicken anti-human MK antibodies and preparation of chicken anti-human MK antiserum Recombinant human MK (hereinafter referred to as human MK) obtained according to the method described in Example of Japanese Patent Application No. 7-255354. Rabbit anti-human MK antiserum and chicken anti-human MK antiserum were obtained by immunizing rabbits 6 times in total every 2 weeks and immunizing chickens in total 8 times every 2 weeks.

For immunization with human MK, in the case of rabbits, 400 μg of human MK is mixed with an equal amount of Freund's complete adjuvant for the first immunization, and the second and subsequent times are used in an amount of 40 μg / hour.
A mixture of 0 μg of human MK and Freund's incomplete adjuvant was used. 100 μg per chicken
, Except that human MK was used.

Antisera from rabbits and chickens were salted out with ammonium sulfate, and then an IgG fraction was obtained with a protein A column. Further, a human MK affinity column in which human MK was immobilized on Affigel 10 (Bio Rad) was used. Affinity purified.

Example 2 Preparation of rabbit anti-human MK antibody plate The rabbit anti-human MK antibody prepared in Example 1 was dissolved in PBS 0.1% sodium azide (pH 7.2) to a concentration of 10 μg / ml, and
The solution was dispensed at 50 μl / well into a ysorp plate (NUNC) and adsorbed at room temperature for 16 hours. After washing with PBS-Tween 20 washing solution, a buffer solution containing 0.5% BSA added to the antibody diluent was added at 150 μl /
After performing a blocking operation at 37 ° C. for 2 hours by dispensing each well, the solution was stored at 4 ° C. until measurement.

Example 3 Peroxidase (hereinafter referred to as POD) labeling of rabbit anti-human MK antibody and chicken anti-human MK antibody The rabbit anti-human MK antibody prepared in Example 1 was conjugated with 0.1 M phosphate buffer, 5 mM EDTA (pH 6.0). After dialysis against 200 mg / ml of antibody, 200 μl was aliquoted. 2-mercaptoethylamine (hereinafter referred to as 2MEA) was added to the above antibody solution to a final concentration of 10 mM, reacted at 37 ° C. for 2 hours to generate a thiol group, and then desalted using a G25 column.

On the other hand, peroxidase (POD) (Boehrin
Ger Mannheim (EIA Grade) 1 mg was dissolved in 200 μl of 0.1 M phosphate buffer (pH 7.5), and then Sulfo-SMCC (PIERCE
Was added and gently mixed at room temperature for 90 minutes for reaction to introduce a maleimide group into the POD, followed by desalting with a G25 column.

A coupling reaction between a rabbit anti-human MK antibody having a thiol group and a POD having a maleimide group introduced was performed at room temperature for 2 hours. Then Centriplus50 (AMICON
After unconcentrated POD was removed with an AcA34 column, the mixture was concentrated to 1 ml with Centriplus50 to obtain a rabbit anti-human MK antibody-POD labeled antibody.

Preparation of a chicken anti-human MK antibody-POD labeled antibody was carried out in the same manner as described above.

Example 4 Confirmation that the epitopes recognized by the rabbit anti-human MK antibody and the chicken anti-human MK antibody are substantially different from each other (1) Using a rabbit anti-human MK antibody plate as a solid phase,
Chicken anti-human MK antiserum is 1,000 to 10,000 in advance
500 pg / m of human MK with antigen dilution diluted 0 times
and used for measurement. This antigen solution was dispensed at 50 μl / well into a rabbit anti-human MK antibody plate and reacted at room temperature for 60 minutes. After washing with PBS-Tween 20, a 5,000-fold dilution of rabbit anti-chicken IgG-POD (PIERCE) was dispensed at 50 μl / well and reacted at room temperature for 30 minutes. After washing, 100 µl / w of TMB chromogenic substrate (DAKO S1600)
After the reaction was performed at room temperature for 30 minutes, the reaction was stopped with 100 μl of 2N sulfuric acid, and the absorbance at a wavelength of 450 nm was measured. The result is shown in FIG.

If the epitope site in the human MK antigen molecule is saturated with the antibody in the chicken anti-human MK antiserum, no reaction occurs with the solid phase rabbit anti-human MK antibody, and the color development level decreases. However, as shown in FIG.
Despite the relatively low antigen concentration of 500 pg / ml, the concentration of chicken anti-human MK antiserum was higher than the actual concentration used 1,
It is evident that the color development level hardly decreases even when added at a 000-fold dilution.

(Part 2) Using a rabbit anti-human MK antibody plate as a solid phase, dispensing 50 μl / well of an antigen diluent containing 50 ng / ml of human MK as an antigen for measurement, and reacting at room temperature for 15 minutes. went. After washing, a rabbit anti-human MKPOD-labeled antibody was
Anti-human in the diluted antibody solution or the labeled antibody solution
An antibody solution containing MK chicken antiserum at 50-fold dilution and 100-fold dilution was dispensed into a rabbit anti-human MK antibody plate at 50 μl / well, and each was reacted at room temperature for 15 minutes. After washing, 100 μl / well of TMB chromogenic substrate (DAKO S1600) was dispensed, and the reaction was carried out at room temperature for 15 minutes. The reaction was stopped with 100 μl of 2N sulfuric acid, and the absorbance at a wavelength of 450 nm was measured. As a result, as shown in FIG. 2, the chicken anti-human MK antiserum had no effect on the reaction of the rabbit anti-human MKPOD-labeled antibody with the antigen.

(Part 3) Chicken anti-human MK antiserum was added to 2,000
Human MK was diluted to 0.25, 0.
5, prepared to be 1.0 ng / ml. After dispensing 50 μl / well of these solutions to a rabbit anti-human MK antibody plate,
The reaction was performed for minutes. At that time, a comparison was made between the case where the reaction was started immediately after the preparation and the case where the reaction was started after incubation at room temperature for 90 minutes after the preparation. After washing, rabbit anti-chicken IgG-POD (PIERCE) was diluted 5,000-fold,
μl / well was dispensed and reacted at room temperature for 15 minutes. After washing
Dispense 100 μl / well of TMB solution (DAKO S-1600) at room temperature
The reaction was performed for minutes. Stop the reaction with 100 μl of 2N sulfuric acid,
The absorbance at 0 nm was measured. Table 1 shows the results.

[0045]

[Table 1]

From Table 1, it was confirmed that the same result was obtained for the absorbance regardless of the presence or absence of the incubation time between the antigen and the liquid phase antibody. This result indicates that the antigenic determinant recognized by the anti-human MK chicken antiserum does not substantially overlap the antigenic determinant recognized by the anti-human MK rabbit antibody.

As described above, according to the present invention, a one-step sandwich method can be assembled in the same manner as a monoclonal antibody using polyclonal antibodies derived from two animals that mutually recognize different epitopes on the same antigen molecule. is there.

Example 5 Comparison with Conventional Method for Measurement of Human MK A measurement system assembled using only rabbit anti-human MK antibody (= comparative example), rabbit anti-human MK antibody and chicken anti-human MK antibody The colorimetric sensitivity in human MK measurement was compared and examined using the two types of measurement systems (the method of the present invention).

In Comparative Examples and the present invention, MK was diluted using the following antigen diluting solutions to obtain 10.0, 2.0, 0.2 and
Antigen dilutions at each stage of 0 ng / ml were prepared.

Comparative Example: 50 mM Tris-HCl, 0.3 M NaCl, 0.
A solution consisting of 5% BSA and 0.1% sodium azide (pH 8.0). -The present invention: a solution containing chicken anti-human MK antiserum at a 5,000-fold dilution ratio in the above solution.

The antigen diluent in each of the above steps was used as a rabbit anti-human
The reaction was started by dispensing 50 μl / well into the MK antibody plate. The reaction was performed at room temperature for 30 minutes, and after washing four times with PBS-Tween 20, the following labeled antibody solutions were added to the present invention and the comparative example, respectively, and the reaction was further performed at room temperature for 15 minutes.

The present invention: a rabbit anti-chicken IgG-POD labeled antibody (PIERCE catalog No. 31401) was added to PBS 0.01% Micr
ocide I A 4,000-fold dilution in a 0.5% BSA solution.・ Comparative Example: Rabbit anti-human MK antibody-POD labeled antibody was added to PBS 0.
01% Microcide I 400 times diluted in 0.5% BSA solution.

After the completion of the reaction, the plate was washed four times with PBS-Tween20.
100µl / wel of TMB solution (DAKO S1600) as enzyme color solution
After the reaction at room temperature for 30 minutes, 2N sulfuric acid 100μl / we
The reaction was stopped with II, and the absorbance at a wavelength of 450 nm was compared.

Table 2 shows the results.

[0055]

[Table 2]

Example 6 Assembly of One-Step Sandwich Measurement System Using Chicken Anti-Human MK Antibody-POD Labeled Antibody The concentration of human MK was calculated from the absorbance of OD280, and based on this, the antigen (human MK) concentration was determined. 10, 4, 2, 1, and 0.5 ng / ml of the standard solution were added to 50 mM Tris-HCl 0.3 M NaCl 0.5% BSA 0.01% Mic
It was prepared by dilution with an antigen dilution solution of rocide I pH 8.0. To each of the standard solutions (20 μl), 100 μl of the antigen dilution solution containing the chicken anti-human MK antibody-POD labeled antibody prepared in Example 3 was added and mixed well. Rabbit anti-human MK antibody plate, 50 μl of the mixture of the above standard solution and POD-labeled antibody
The reaction was carried out at room temperature for 90 minutes. PBS-Twe
After washing four times with en20, a TMB solution (DAKO
S1600) was dispensed in 100 μl / well portions, and allowed to react at room temperature for 30 minutes. Finally, the reaction was stopped with 100 μl / well of 2N sulfuric acid, and the absorbance at a wavelength of 450 nm was measured. Standard solution is 1/6 at the time of reaction
The antigen concentration in the reaction solution is the highest concentration
Although the concentration was as low as 1.67 ng / ml, a good calibration curve as shown in FIG. 3 was obtained.

Example 7 Assembly of a measurement system for human PTN by the sandwich method According to the method described in Example 1 of Japanese Patent Application No. 7-255354, human PTN was used.
N cDNA (Yue-Sheng, L., et al. (1990) Science 250, 1
690-1694) to obtain recombinant human PTN (human PTN). Immunization of rabbits and chickens with PTN was performed in the same manner as in Example 1. The antiserum from chicken was used after affinity precipitation with a human PTN affinity column prepared by immobilizing human PTN on Affigel 10 (BioRad) after salting out with ammonium sulfate. On the other hand, the antiserum from the rabbit was used as it was.

The concentration of human PTN was calculated from the absorbance of OD280, and based on the concentration, an antigen dilution solution (50 mM Tris-HCl 0.15
Rabbit anti-human P to M NaCl 0.5% BSA 0.1% sodium acid chloride pH 8.0
Solution with TN serum diluted 200 times) 100, 50, 25,
Each human PTN solution at 0 ng / ml at each stage was prepared and left for 30 minutes. Standard antigen solutions at each of the above concentrations and anti-human PTN
After mixing rabbit antiserum, human PTN solutions of each concentration were dispensed at 50 μl / well into chicken anti-human PTN antibody plates prepared in the same manner as in Example 2 and reacted at 37 ° C. for 60 minutes. Was. After washing 4 times with PBS-Tween20, goat anti-rabbit
IgG-POD labeled antibody (DAKO No.448) was added to PBS 0.01% Microcide I
What was diluted 3000 times in 0.5% BSA solution was added at 50μl / wel
The reaction was further performed at room temperature for 20 minutes. After the reaction, the plate was washed four times with PBS-Tween 20, and a TMB color developing solution (S1600, DAKO) was dispensed in 100 μl / well portions and reacted at room temperature for 30 minutes. The reaction was stopped with 100 μl / well of 2 N sulfuric acid, and the absorbance at a wavelength of 450 nm was measured. Table 3 shows the obtained results. As in the case of Example 6, in the case of PTN, a standard curve was obtained without any problem even if the antigen and the antiserum were mixed in advance (FIG. 4).

From the above results, according to the method of the present invention, even in the case of a combination of polyclonal antibodies, the first antibody and the second antibody can coexist without any restrictions as in the case of the monoclonal antibody. It is possible to construct a more sensitive measurement system by combining the following antibodies.

In this example, a rabbit antibody was used as the first antibody.
Although the MK antibody is used and the chicken anti-human MK antibody is used as the second antibody, the combination may be reversed. In this case, it is needless to say that an anti-rabbit IgG antibody is used as the third antibody.

[0061]

[Table 3]

[0062]

According to the present invention, it is possible to carry out a reaction by the sandwich method similar to the case of combining a monoclonal antibody while being a combination of polyclonal antibodies,
In addition, a highly sensitive measurement system can be assembled.

[Brief description of the drawings]

FIG. 1 is a diagram showing that epitopes on MK molecules recognized by rabbit anti-human MK antibody and chicken anti-human MK antibody do not substantially overlap.

[Figure 2] Chicken anti-human MK antibody and rabbit anti-human MK antibody
It is a figure which shows whether the reaction with MK is inhibited.

FIG. 3. Immobilized rabbit anti-human MK antibody and chicken anti-human MK
It is a figure which shows the calibration curve at the time of performing an immunoreaction with a POD label antibody in one step.

[FIG. 4] A PTN antigen and a rabbit anti-PTN antiserum are mixed and reacted with a chicken anti-PTN immobilized antibody.
FIG. 3 is a diagram showing a calibration curve obtained by reacting a POD-labeled antibody.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Munehiro Oda 540, Narita, Odawara-shi, Kanagawa Meiji Dairies Co., Ltd.Cell Engineering Center Co., Ltd. Block 6, Block 35 (72) Inventor, Hisako Muramatsu, Block 2845, Kuroishi, Heijiro, Tenpaku-ku, Nagoya-shi, Aichi Pref.

Claims (5)

[Claims] The present invention relates to two different animal species which naturally have, as a biological component, the same species of protein having a portion having no primary structural homology with a protein antigen having at least two immunologically distinct epitopes. A polyclonal antibody obtained by immunization with each of the proteins, wherein the polyclonal antibodies which are substantially different from each other in the epitope on the protein antigen recognized by the polyclonal antibody are used as the first antibody and the second antibody. Sandwich measurement method. 2. The sandwich assay method according to claim 1, wherein a third antibody against the second antibody is used. 3. The protein antigen is midkine.
The sandwich measurement method according to claim 1 or 2, wherein 4. The method according to claim 1, wherein the protein antigen is pleiotrophin (pleitrophin).
otrophin). 5. The method according to claim 1, wherein the two different animal species are a combination of chicken and rabbit.