JPS59131165A - Detecting reagent for body being analyzed - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to analytical systems using reagents molecularly labeled with radiant energy emitters, and more particularly to reagents useful for the detection of antigenic type reactants and their use for that purpose. It is about the method.

For the detection and classification of reactants in highly specific reactions, such as antigens in antigen-antibody reactions, techniques are known for labeling one of the reactants (eg, an antibody) with a radiant energy emitter.

This method is based on the fact that the product or complex of labeled antibody and antigen is distinguishable from unbound labeled antibody.

Current methods for detecting antigens with labeled antibodies have a number of significant drawbacks. In particular, the amount of antigen may be so small that it may not reach the detectable concentration of the label using current methods. Therefore, in many cases of serious diseases with an antigenic etiology, detection and treatment may be delayed and sometimes even fatal.

When the radiant energy emitter is a radioactive substance, the reagent containing the labeled antibody may pose a risk to the manufacturer and to clinical laboratory technicians who perform analyzes using the reagent. Moreover, the delivery of radioactive reagents of this type is increasingly subject to onerous limitations. The light emission from each radioactive atom is, of course, extremely weak.

Finally, when the half-life of radioactivity is short, as is often the case, radioactivity declines rapidly and shelf life is severely limited.

Labeling of antibodies is often carried out either as bound fluorescent dye molecules, e.g., dyes that, when directly excited by irradiation, exhibit fluorescence emission with a fairly high quantum efficiency in their absorption band, or as free dye molecules. This is accomplished by a fluorochrome that emits fluorescence with a substantially higher quantum efficiency when bound to the antibody than when present. Typical examples include: - Conventional dyes of this type include fluorescein, rhodamine, vironin, eosin, acridine, acriflavir,
Safranin, methylene blue, and a host of other dyes have been used with appropriate optical sensing equipment. The detection levels of fluorescent dyes attached to antibody-antigen complexes in the prior art are generally too low to detect individual particles. Traditionally, efforts have been made to improve this sensitivity by increasing dye loading (the number of bound dye molecules coupled to each antibody), resulting in a decrease in the specificity and sensitivity of the antibody-antigen reaction. . There are many possible reasons for this decline.

(1) If enough dye molecules are coupled to the antibody,
(2) the overall hydrophilicity and net charge of the dye-antibody molecule produced; changes alter its reactivity and solubility; (3) steric and hydrophilic stresses in the dye-antibody molecule, as well as possible changes in its vibrational behavior, cause distortions in the three-dimensional structure of the protein, resulting in (4) The chemical interaction at the dye-antibody binding site changes its specificity.
For example, it is possible to change the atomic bond even at a specific binding site that is a certain distance from the binding site.

The present invention overcomes these problems in the prior art with a novel and useful system that increases the number of fluorescent dye molecules bound to antibody molecules while not affecting antibody-antigen specificity. be. To this end, the present invention provides reagents in which an antibody molecule and a number of fluorescent dye molecules are covalently linked via a polymer backbone having reactive functional groups along the chain direction of the polymer backbone. be. That is, by attaching a polymer chain having a large number of fluorescent residues to a single site on an antibody, the degree of fluorescence emission from the bound molecule (under appropriate excitation) is significantly increased;
On the other hand, the influence on the specificity of the reactivity between the antibody and antigen is kept to a minimum.

Thus, a primary objective of the present invention is to increase dye loading in certain antibody immunofluorescence methods without compromising its specificity.

Another object of the invention is to improve the sensitivity of antibody immunofluorescence by increasing it by at least two orders of magnitude over the prior art.

The terms "first reactant" and "second reactant" (hereinafter referred to as "analyte") are used in a broad physical sense. The first reactant is an antibody or two or more reactive sites (or functional groups) that are sterically separated from each other, one site capable of binding to a carrier polymer, and this site capable of binding to a carrier polymer. It can be defined as any chain molecule located so as not to impede the specific reactivity with the light isomer. Typical examples of first reactants are biological ones, such as serum proteins;
those whose production is mediated biologically in response to the presence of the analyte in the form of an antigen;
Examples include those containing organic hiding agents or chelating agents. The analyte is a substance that reacts with a specific first reactant, preferably with high specificity;
Each analyte particle or analyte body has a plurality of reaction sites and can therefore be coupled to a plurality of first reactant molecules. That is, analytes are usually complex organic molecules of high molecular weight (e.g., 10,000 or more) such as enzymes, toxins, proteins, or polysaccharides and lipoproteins, and microorganisms such as bacteria, males, protozoa, etc. It can be thought of as a living or dead body, a substance that corresponds to a habten or an antibody but does not itself produce the biological production of antibodies. Of course, biological analytes or antigens of this type have specific reactivity with the corresponding biological antibodies.

Non-biological analytes can be as simple as metal ions, complex molecules, etc. that are reactive with the corresponding ligand.

The carrier or backbone polymer selected has reactive sites distributed along the direction of the polymer, with additional chemically different reactive sites at the end of the chain. This carrier polymer molecule may be rigid or easily bent into a spherical configuration in water, as long as steric hindrance occurring around its expansion or vibration can be eliminated. Its net ionic charge (
(when combined with the dye molecules attached to its side chains) should be substantially identical in action, per unit volume, to that of the first reactant, especially if it is an antibody. The reactive sites dispersed within the chains of the carrier polymer molecules must have a low affinity for the first reactant and must not act as fluorescent occluding agents. Carrier? Preferably, the remer molecule has the same hydrophilicity as the first reactant. The backbone of the polymer molecule must have covalent bonding sites spaced far enough apart to avoid loss of luminescence of dye residues caused by perturbation effects of one dye molecule interfering with other dye molecules through space. Must be.

As used herein, the term "specificity" refers to a reaction in which the reactants react with the parenchyma alone and only to a very small or negligible extent with other reactants, such as antigens. - intended to describe antibody responses.

Polymeric backbone molecules suitable for the practice of this invention include polyethyleneimine (in which case a suitable molecular weight range is 1.200 to 60.000'), polypebuty-P such as polylysine, polyamides such as nylon-6, low molecular weight ( 1
00 to 1,0.000) Polymerized carboxylic acids and other polymeric substances in which reactive functional groups are continuous along the chain direction.

The polymeric framework material is dye labeled by reacting with fluorescent dye molecules. Each fluorescent dye molecule has a reactive group and thus covalently reacts with successive functional groups of the polymeric material. Prior to reaction with fluorescent dye molecules, the reactive end groups of the polymer molecules are temporarily occluded or chemically protected. Examples of carffnyl compounds suitable for the purpose of reaction with carbonyl compounds include, for example, benzaldehyde, glutaraldehyde, and the like.

Fluorescent dyes suitable for use in the present invention, including those in which they are functionalized, include, but are not limited to, the following examples:

Ac1d Violet 4 BL C
,1,/lfx 42575Acridine Br1
lliant Orange C, I, A 4
6005Acridine Orange
C61,1646005Acridine Ye
low C,1,A 56025A
criflavine C,I,
446000Auramine OC,1,4410
00Aurophosphine G
C,1,/1646035Benzo Flavin
e C,1,A 46035Ber
berine 5ulfate C,1
,475160Brilliant Phosphin
e C,1,A 46035Brilli
ant 5ulfo Flavine C,!
, 456205Chrysoidine
C,1,A 11270Coerulein
S C,1,A 45510C
oriphosphine OC,! , /% 460
20Coriphosphine Fuchsin
C,1,A 42755Euchrysine
2G O,I, /16.46
040Euchrysine 3RX
C,1,A46005Flavo Pho
sphine RC,1,A 46035Fluore
scein C, 1
, & 45350Geranine G
C,1,A 14930Met
hylene Blue
C,1,A 52015Morin
C,1,/l67566
ONNeutral Red
C, L /I6500400range e
C, 1, A 1
6230Phosphine 3RC,1,/164
6045Primuline
D.1. A 49000Pyronin
GS (Pyronin extra) C,
1, A645005Rhoduline Orange
e C, 1, flat 46005Rh
oduline violet
C,1,/1629100Rosole Red
B C, 1, A
438008afranin
C,1,/16502108calet
RC,I, 426105Sulpho Rhoda
mine B C, 1, A 4
5100Tertrazine OCJ, /l61
9140Thiazine Red RC,I,A
14780 Thiazol Yellow
C,1,A 19540Thiofla
vine 8 C,I
,*49010Thionin
After attaching the dye molecules through the reactive site groups of the C,1,A 52000 polymer, depending on whether the reactive end groups of the polymer were protected with monoaldehyde or polyaldehyde,
The operation will be slightly different. When a monoaldehyde such as benzaldehyde is used as a preservative, the dye is bound to the polymer, and then the benzaldehyde residue is removed by hydrolysis to make the terminal and end sites reactive again.

The terminal portion is thereby functionalized for covalent bonding with the antibody molecule.

If a polyaldehyde, such as glutaraldehyde (propanedial), is used (preferably in large excess) to protect the terminal reactive sites of the polymer, there is no need to remove the protected glutaraldehyde residues; can be used as a binding agent for covalent bonding of a polymer and an antibody or other first reactant.

The dye/polymer conjugate is then combined with a first reactant, such as an antibody, to produce a dye/polymer/antibody conjugate. Since the dye/holimer/antibody molecules are preferably electrically neutral, this reagent is neutralized if necessary. Such neutrality minimizes inhibition of reaction specificity between the first reactant and the analyte, particularly in antibody-antigen reactions. The W of the reagent can be varied in a range such that protein denaturation occurs, ie, between about 4 and 10, for antibody-antigen type reactions, using known buffers. The excess charge on the reagent molecule is in weakly ionizable groups, such as carboxy or amino groups; the pK of such groups is about 4 to 10.

When the dye/polymer/antibody complex, or reagent, is mixed with a solution containing the antigen specific for the antibody in the complex, the antigen binds. Analytes reactive with the reagents of the present invention have several binding sites, so each analyte has two binding sites.
can bind more than one reagent molecule. dividing the liquid stream (or using other known techniques to separate molecules from each other)
When viewed and observed by irradiation with light in the absorption band of the dye in the complex, fluorescence from each reagent molecule in the liquid stream successively passing through the irradiated region is detected. By placing a threshold on the measurement of the amplitude of each detected fluorescence pulse, the low level signal from a single unbound reagent molecule can be reduced from the amplitude obtained from multiple reagent molecules bound to a single analyte. can be distinguished between each point source that produced a large signal,
The presence of analytes can be identified. Depending on this method,
Naturally, it is impossible to distinguish between a reagent molecule group bound to an analyte and a crosslinked reagent molecule group. Therefore, when producing the reagent of the present invention, it is important to protect against crosslinking by using an appropriate agent that temporarily protects the reactive group. Prior to use, the reagents are preferably subjected to a separation procedure such as silica gel chromatography to completely separate crosslinked reagent molecules. Following reaction of reagent and analyte, physical removal of unreacted reagent facilitates detection of analyte bound to reagent molecules.

Many functionalized dyes are commercially available, such as fluorescein isothiocyanate.

For example, by nitration of fluorescein with f10r3 and reduction of the nitrate with generator hydrogen derived from zinc and hydrochloric acid, a non-chromogenic extra amine group is added to the fluorescein molecule using known techniques. Fluorescein can be functionalized.

When thiophosdene is then added to this, isothiocyanate is produced. Of course, other techniques are also known for producing functionalized dyes. For example, converting the dye to the isothiocyanate type or combining it with other groups
', 2--F Lomonityl side button, etc. can also be introduced.

Commercially available antibodies are preferably fractionated and purified, for example, by using 5epharose to separate immunoglobulin-m from r-globulin. The latter 7-lactone is mixed with the dye-labeled polymer and the reaction is stopped with appropriate buffering, for example with ethanolamine or trimethylaminomethane hydrochloride. This latter reaction is an amino-aldehyde reaction that stops further binding of the dye-labeled polymer to other antibodies. The mixture must then be fractionated, for example using a 5 ephadex column, to separate the antibody/polymer/dye molecules according to the number of polymer molecules bound to each antibody molecule. Discard the antibody-only fraction or the antibody and two or more polymer fractions. The former is useless because it is unlabeled, and the latter is less specific and sensitive than the selected fraction.

It is particularly advantageous to use dialdehydes when coupling primary amine-containing multifunctional polymeric backbones to antibodies.

Because the reaction of the aldehyde imparts heterogeneous functionality to the polymer, the fluorescent dye reacts selectively to other functional sites on the polymer chain, and the unbound aldehyde residues are later conjugated to the antibody. That is, the dialdehyde acts as a protecting group and as a functional group in subsequent reactions.

In one embodiment of the invention, in the case of a -class amine-containing polymeric backbone, the functional groups continuously present in the polymeric material are reacted with a covalently reactably functionalized fluorescent dye. . Prior to reaction with the functionalized fluorescent dye molecules, the primary amine of the polymeric material is reacted with a dialdehyde to protect the reactive amine end groups of the polymer while providing functionality for subsequent covalent attachment to the antibody. .

Glutaraldehye P is preferred as the dialdehyde.

The following formula illustrates a method for preparing a suitable antigen detection reagent having a polyethyleneamine skeleton.

R4(NCH2CH2”1nNHCH2CH2NH2+
0HC(CH2)3CHO→OHC(CH2) 3H
C-NCH2CH2(NC)! 2 CH2”]nNH
CH2”.

NC'S F F- NHNH C=S RC-8 →OHC(CH2) 3HC=NCH2CH2NCH2
CH2[NCH2CH2)nNCH2・”10antibodyNH C=8 → (antibody)-N=CH(CH2)3HC=NC'H2C
H2N -...In the formula, η is H or [CH2NH2NH
)nCH2CH2NH2, R, is H2NCH2CH2-
, F is fluorescein.

The reagent of the present invention can be produced by, for example, polyethyleneimine 2
00 (molecular weight 20,000) with glutaraldehyde in the presence of sodium cacodylate, reacting the resulting imine with fluorescein isothiocyanate, and reacting this dye-labeled polymer with anti-Echo 12 virus antiserum. This is done by

Typical antigens detected by the method of the present invention include hepatitis B antigen, Echo 12 to 1rus antigen, Hoof-M
Mention may be made of the disease virus antigen, and the swine vesicle virus antigen. However, the method of the invention is not limited to the detection of the antigens named above, but is generally applicable to all antigens, as long as suitable antibodies are available.

The present invention involves reacting a -class amine-containing multifunctional polymer backbone with a dialdehyde, reacting this balmy multifunctional polymer backbone with a functional fluorescent dye molecule, and dye labeling and labeling.
The present invention includes a method for producing an antigen detection diagnostic agent, which is characterized by producing an IJ polymer and then reacting this dye-labeled polymer with an appropriate antibody.

As an example of this method, the functional fluorescent dye molecule is fluorescein isothiocyanate, the dialdehyde P is glutaraldehyde, the -class amine-containing polyfunctional polymer backbone is polyethyleneimine with a molecular weight of 2000, A method may be mentioned in which the number of fluorescent dye molecules is about 120.

The invention also encompasses a method for detecting an antigen in a biological fluid or tissue, characterized in that the sample of the biological fluid or tissue is mixed with the aforementioned diagnostic reagents. This method of the invention is particularly applicable to the detection of hepatitis B antigen in blood samples.

Next, the present invention will be explained in more detail with reference to the following examples. This example is merely illustrative of the invention;
It is not intended to limit the spirit or scope of the invention. It goes without saying that many modifications of the present invention, both in terms of materials and methods, are possible and are encompassed by the present invention, as will be obvious to those skilled in the art.

Example 1 The preparation of a polymer/dye complex is carried out as follows.

Polyethyleneimine 200 (molecular weight 20,000) 21
n9 in 0.1M sodium cacodylate solution 1 m/1c
Add 25% glutaraldehyde aqueous solution Q, 1 mA' to the solution dissolved in p147.0 while stirring vigorously. The resulting reaction mixture was stirred for approximately 5 minutes, then 8 epha
Excess glutaraldehye P is removed by passing through a column of dex G-25 (0,9×15 cm ) (silica gel). The column was eluted with O-I M % PH7-2 sodium cardylate aqueous buffer, and the eluate was
Fluorescein isothiocyanate 50■0.5
M, pH 9,5 sodium cacodynate aqueous buffer 1.5rIL7! Dissolve and add. Stir the mixture constantly during the addition and continue stirring for about an additional 16 hours. During this time, protect the mixture from light. 5ephadex()
-25 (silica gel) column (0.9X 30.Oa
m) to remove excess dye and column to 0.1M.

Elute with pH 7.0 sodium cacodylate aqueous solution, 2-
Collect the Fraxidan.

The resulting “polymer/dye complex” was treated with Folin-C1o
Analyze using the caulteau protein analysis method.

In this analytical method, polyethyleneimine gives a straight line and is suitable for quantifying the amount of polymer present.

The extinction coefficient of fluorescein inthiocyanate at 495 nm is 73 x 10r3, and this value is reduced to 75% upon conjugation. By quantifying both the polymer and dye in a given complex sample, the degree of dye binding can be determined.

The degree of binding is determined by the dye concentration in the initial reaction mixture. Limited fractionation is performed by filtration. The composite prepared according to this example contains approximately 70 moles of dye molecules per mole of polyethyleneimine molecules.

Example n Proceed as in Example 1 except that the molecular ratio of dye and polyethyleneimine is continuously varied to obtain a polymer/dye composite containing 65 and 80 molecules of dye, respectively, per molecule of polyethyleneimine 200. .

Example ■1 The procedure is as in Example 1, except that 0.1 ml of a 25% benzaldehyde solution in dioxane is used instead of glutaraldehyde, and the reactive terminal site of each polyethyleneimine molecule is coupled to a benzaldehyde residue. A polymer/dye complex similar to that in Example 1 is obtained.

Example 2 A polymer/dye composite containing about 130 dye molecules per polyethyleneimine molecule is obtained by the same procedure as in Example 1, except that polyethyleneimine 600 (molecular weight 60.0[110]) is used instead of polyethyleneimine 2000.

Example V Polylysine (molecular weight 8.0
00 to 20. OD O, polylysine/rhodamine with multiple dye molecules attached to the backbone molecule of the complex, operated under the same conditions as in Example 1 except that lissamine rhodamine-B isothiocyanate was used instead of fluorescein isothiocyanate. Obtain the complex. The structure of lissamine rhodamine B is given by Trotman: Dyeing a
nc5. Chemical Technology
of Textile Fibers', 45th edition (C
, Griffin & Co.

(Published in London), page 679.

EXAMPLE ■ A similar procedure in Example V using the sulfonyl chloride of lissamine rhodamine B gives the corresponding rhodamine/polymer complex.

Example ■ To form a reagent of the invention (e.g. dye/7'?Lima1/antibody complex), anti-Echo virus antireagent (25m9) was mixed with 0.1 M, pl (7,0 cacodylate). Dissolve in aqueous sodium solution and add, with stirring, 1.1 μ of the polymer/dye complex from Example 1. Stir the resulting mixture for 10 minutes and add Tris/chloride 11n9 to inhibit cross-linking between antibody molecules. .Stir 3 more times
for 5 minutes, then the mixture was adjusted to 0.1 M, pH
8,5) 5eph equilibrated with lis/chloride buffer
adex G-200 column (0.9X 60cnt
) (Silica sol). Elute the column with the same buffer and collect 2 ml fractions.

Measure the optical density of reagents and fractions at 280 nm and 495 nm. For spectral difference analysis,
The amount of antibody in each fraction is measured to determine the amount of dye bound to one molecule of antibody. Since the number of dye molecules per polymer molecule is known, the average number of polymer molecules per antibody molecule can be calculated. By this quantitative method, antibody 1
It can be seen that 1.2 to 1.6 polymer molecules are bound per molecule. - The immunoreactivity of the dye/polymer/pile body complex is measured by red blood cell collection. By this method, the dye/polymer/antibody conjugate was shown to retain 70% of the activity of the unconjugated antibody.

dye/? The fluorescence of the remer/antibody complex is A
.

Obtained using a Bowan fluorometer. Fluorescence has a concentration of 0.001
or 1. It is measured by the ratio to 3 ml of fluorescein isothiocyanate standard solution. Excitation is measured at 495 nm and emission at 526 nm. The complex is diluted to give the same optical density at 495 nm as a known dilution of fluorescein isothiocyanate.

The quantum efficiency of the polyethyleneimine-containing complex with 80 dye molecules was determined to be 4% compared to 100% for free fluorescein isothiocyanate.

The reagent thus obtained is passed through a 5 ephadex column, leaving behind the fraction containing the reagent whose polymer molecules are bound to one antibody, and discarding the others. When this fraction is mixed with a solution containing anti-Echo virus as the analyte, an antibody-antigen reaction occurs and the virus particles bind to two or more antibody complexes.

Example ■1 To prepare other reagents of the present invention, the benzaldehyde residue of the complex in Example ■ was dissolved in 1% hydrochloric acid bath solution.
- removal by gentle hydrolysis for about 3 hours and then dialysis of the solution to remove excess acid. The solution is then evaporated under reduced pressure to remove water and the resulting polymer/dye complex is dissolved in hot acetone. To this acetone solution, 10 equivalents of thiophosgene are further added along with acetone and the mixture is refluxed for 4 hours. The acetone is then evaporated to yield the functionalized polymer/dye complex.

The functionalized polymer/dye complex was dissolved in water and buffered with 0.5 M Na2CO3 to pH 9.5, which added approximately 2.5
Add ml of anti-Echo virus antiserum. The mixture is stirred constantly for about 16 hours, during which time the mixture is protected from light.

It is then passed through an 8 ephadex column and the column is then eluted with O-IM + aqueous sodium cacodylate solution pH 7-0 to remove excess dye.

A 2 ml fraction containing the polymer/dye/antibody complex is collected. The ratio of polymer to antibody in this complex is 1:1
It is.

Example ■ A reagent of the invention that can be used to detect the presence of a polyvalent metal (in this case nickel is identified) is produced from the metal glue gun P as follows.

Polymer/dye composite 1Q my prepared in Example 1
benzyl P-aminobenzyl diisonitrosoethane (i.e., benzyl P-aminobenzyl diisonitrosoethane) as a ligand.
Add 0.1■ of aminoglyoxime), and add 0.1■ of this mixture to 1
Stir for a minute. The reaction is terminated by adding 100 ml of ethanolamine buffered to pH 9 as a carbonate.

The mixture is dialyzed to remove excess ethanolamine and benzylglyoxime. When this reagent is applied to the dried residue of a nickel-containing solution on a glass slide, about four molecules of polymer/dye/sorgane P are bound to one nickel atom. This slide P is lightly washed with water to remove excess reagent. Microscopic examination of this slide under illumination with the absorption band of fluorescein reveals the presence of nickel, with fluorescence amplitudes from each point source indicating the presence of bound complexes.

Example
A solution of 0-8 mol dissolved in 0.1 M) lisamine hydrochloride is prepared. A dye/polymer/antibody conjugate is prepared according to Example 2 using polyethyleneimine 200 as the backbone, fluorescein isothiocyanate as the dye and a commercially available antibody to the Australian antigen.

The ratio is adjusted so that there are approximately 70 molecules of dye per molecule of polyethyleneimine in the complex. Fractions containing polyethyleneimine molecules in a 1:1 ratio of antibody to antibody are separated using an 8 ephadex column.

The concentration of the resulting 7-lactone is 34 o mq of protein.
/ ml % i.e. labeled antibody 2.2 X i O−
It is 9 moles. This fraction was used at an antibody:antigen ratio of 100.
:i, 10:1 and 1:1, and each mixture is irradiated to bring its fluorescence into an excited state.

The 100:1 mixture exhibited approximately the same fluorescence as the labeled antibody itself, with only very low intensity emission observed. The 10:1 mixture has a slightly higher radiation source, and the 1;1 mixture
The mixture showed more bright sources. Generally, as the proportion of antigen increases, the number of sources exhibiting high brightness increases, and in the latter case the sources are believed to indicate that the antigen is bound to multiple antibodies.

Although certain modifications may be made to the methods and products described above without departing from the scope of the invention, the foregoing description is intended to be illustrative only and not limiting. There's no size.

Attorney Asamura Hiroshi Procedural Amendment 1122/16/1980 Mr. Commissioner of the Patent Office 1, Detection Reagent for Displayed Analyte in the Case 3, Person Making the Amendment Relationship with the Case Patent Applicant 4, Agent 5, Amendment Order Date: Showa, Month, Day 6, Number of inventions increased by amendment 1 (1) The scope of claims is amended as shown in the attached sheet.

(2) In the 1IJI specifications, page 3, lines 8-9, ``Regarding reagents and their purposes.'' is corrected to ``Relating to reagents.''

(3) In the same book, page 7, line 13, “stereoscopic” is “configural”
Correct.

(4) "By the method of the present invention" in the second line from the bottom of page 19 of the same book is corrected to "by using the reagent of the present invention."

(5) In the same book, page 20, lines 2-3, "method of the present invention" is corrected to "method in which the reagent of the present invention can be used."

(6) In the same book, delete ``The present invention'' from the bottom two lines on page 20, and ``The present invention is characterized by...'' from the last line to the second line from page 21.
...This method of the invention is capable of detecting antigens in biological fluids or tissues.

This method is corrected to ``.

9. List of attached documents I have also submitted a request for application examination at the same time.

(Delete claim 3) "2. Claim (1) A chain-like structure comprising a plurality of individual molecules of the first reactant, each of which has a plurality of reaction sites. is a molecule,
a solution of a first reactant, wherein substantially only one of the reaction sites is capable of specifically reacting with the analyte, the reaction allowing some of the molecules to couple to the analyte; A plurality of multifunctional polymeric backbone molecules, substantially each of which is covalently bonded at another site that does not interfere with the first reaction, and the backbone molecule is bonded thereto at a separate site that does not interfere with the first reaction. 1. A reagent reactive with an analyte, comprising: a polymer backbone molecule having a limited number of fluorescent dye molecules coupled thereto so as not to substantially interfere with the specificity of the analyte.

(2) Produce a protected polymer by reacting a polyfunctional primer molecule with a Cal-Nyl compound to block one or more reactive sites at the end of the polymer backbone; The formula of the molecule is reacted with a side reactive site in the remer backbone to produce a dye-labeled polymer molecule, and then one molecule of the dye-labeled/9-mer can be specifically reacted with the analyte by one of the terminal reactive sites. A method for producing a reagent reactive with an analyte comprising steps of covalently coupling with a reactant molecule. ”

Claims (3)

[Claims] (1) a chain molecule comprising a plurality of individual molecules of the first reactant, each of which has a plurality of reaction sites;
a solution of a first reactant, wherein substantially only one of the reaction sites is capable of specifically reacting with the analyte, the reaction allowing some of the molecules to couple to the analyte; A plurality of multifunctional polymeric backbone molecules, substantially each of which backbone molecules are sterically separated from only a corresponding one of said first reactant molecules and said reaction site, and are sterically separated from said reaction site. covalently attached at discrete sites that do not substantially interfere with the specificity of the reaction, and the scaffold has a limited number of fluorescent dye molecules thereon that does not substantially interfere with the specificity of the reaction. A reagent reactive with an analyte, characterized by comprising a polymer skeleton molecule coupled thereto; (2) A protected polymer is produced by reacting a polyfunctional polymer molecule with a cardanyl compound to block one or more reactive sites at the end of the polymer backbone, and the dye functionalized molecule is added to the protected polymer molecule. reacting with a lateral reactive site in the polymer backbone of the dye to produce a dye-labeled polymer molecule, which is then shared by one of the terminal reactive sites with a reactant molecule capable of specifically reacting with the analyte. A method for producing a reagent that is reactive in an analyte body, including steps of coupling the reagent to the analyte. (3) each of the reactant molecules has a plurality of reactive sites, substantially one of which includes a reactant molecule capable of specifically reacting with a corresponding one of the analytes; Each also has one multifunctional polymeric backbone molecule covalently coupled thereto, which polymeric backbone molecule in turn is responsible for the specificity of the reaction between the analyte and reactant coupled thereto. An analyte in a solution comprising the step of mixing with the analyte a reagent having a limited number of fluorescent dye molecules that does not substantially interfere with the analyte. Detection method.