JPS63313067A - Reagent for measuring antigen - Google Patents

Abstract

PURPOSE:To decrease a non-specific adsorption rate by conjugating glucose oxidase GOD which is a labeling body with the remaining antigen conjugated part Fab' of immunoglobulin (IgG) from which a crystallized section Fc is disconnected and using the same as a labeled antibody. CONSTITUTION:The IgG obtd. from the serum of an animal dosed with the antigen is adsorbed to a solid phase to prepare a solid phase antibody and the antigen is specifically adsorbed to this solid phase antibody; further the labeling body is conjugated to the IgG to prepare the labeled antibody. The adsorption quantity of the antigen is indirectly determined by adsorbing the labeled antibody prepd. in such a manner specifically to the antigen adsorbed to the solid phase antibody and investigating the quantity of the labeling body. Namely, the IgG conjugated with the two Fab' to the Fc is first obtd. from the serum of a rabbit. The IgG is cut as shown by an alternate long and short dash line to remove the FC and to obtain the Fab'. The Fab' conjugated with, for example, GOD is used as the labeled antibody. The IgG which is obtd. from the serum of a goat dosed with the antigen and is adsorbed to the solid phase is used as the solid phase antibody. The non-specific adsorption rate of the labeled antibody to the solid phase antibody is thereby lowered.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to an antigen measurement reagent used in an immunoassay method using an antigen-antibody reaction.

B1 Summary of the Invention The present invention provides a measurement reagent for use in the Sand-Deutsche method, which is an immunoassay method, in which the remaining antigen-binding site of immunoglobulin obtained from rabbit serum is removed from the crystallized site and used as a labeled antibody. By using immunoglobulin obtained from goat serum as the solid-phase antibody, the amount of nonspecific adsorption of the labeled antibody to the solid-phase antibody is suppressed, thereby improving measurement sensitivity and achieving good reproducibility. It was designed so that

C1 Conventional technology Antigen-antibody reactions have the characteristics that antigens and antibodies specifically bind and that the reaction occurs even at fairly low concentrations. It is possible to measure small quantities of antibodies. Such assay methods are called immunoassay methods and are broadly divided into competitive methods and non-competitive methods. A representative example of a competitive method is the first antibody solid phase method, and a representative example of a non-competitive method is the Sand-Deutsch method. The present invention is directed to the Sanderutsch method, and the specific contents of the Sanderutsch method and the shortcomings of the prior art will be described below.

First, an antigen, such as carcinoembryonic antigen (CEA), is periodically injected subcutaneously into a rabbit, and the immunoglobulin generated based on the antigen is obtained from the rabbit's serum, which is then transferred to a solid phase made of, for example, a polystyrene ball. Prepare solid-phase antibodies by adsorption.

On the other hand, a labeled antibody, such as glucose oxidase (hereinafter referred to as rGODJ), is bound to the immunoglobulin to prepare a labeled antibody. In order to investigate the amount of the antigen in the human body, first, the antigen to be measured is brought into contact with a solid-phase antibody, and the antigen is specifically adsorbed to the solid-phase antibody. Next, when a labeled antibody is applied to this, the labeled antibody is specifically adsorbed to the antigen. That is, the antigen is sandwiched between the in-phase antibody and the labeled antibody. Then, the amount of COD of the labeled antibody is measured. This measurement is performed by the so-called chemiluminescence method, in which hydrogen peroxide is generated by the action of glucose on the COD, and the amount of luminescence is measured by the action of luminol on the hydrogen peroxide. It can be determined indirectly from the measured amount of COD.

Reagents used in these immunoassays include rabbit-labeled antibody-rabbit homologous antibody, both labeled antibodies and solid-phase antibodies, prepared based on rabbit serum, or goat antibodies, both prepared based on goat serum. Labeled antibodies--goat solid-phase antibodies, etc. have been conventionally used.

D1 Problems to be Solved by the Invention By the way, considering the measurement principle of the sandwich method, it is ideal that the amount of labeled antibody directly adsorbed to the solid-phase antibody should be zero, but in reality, some of the labeled antibody will be non-specifically adsorbed to the same phase antibody. In order to improve measurement sensitivity and reproducibility, it is important to keep this non-specific absorption as low as possible.

Looking at the non-specific adsorption rate of conventional measurement reagents, in the case of rabbits it is approximately 4.8xlO-' to 36.8x10
−”%, approximately 7.1×lO−” for goats ~1
The value is quite high at 6.0xlO-'%. Note that the nonspecific adsorption rate is the ratio of the amount of nonspecific adsorption to the total amount of labeled antibody. Considering these circumstances, we investigated the combination of animal species and found that if we use a reagent consisting of a rabbit-labeled antibody and a goat homologous antibody, which were prepared based on the intuition of rabbits and goats, respectively, the non-specific adsorption rate could be reduced. It was found that it could be suppressed to about 1.5X10-2 to 1.7X10-2%.

However, even in this case, sufficient measurement sensitivity cannot be obtained, and in order to perform highly reliable measurements, it is required to further suppress the nonspecific adsorption rate. The present invention was made in response to these demands, and its purpose is to provide a measurement reagent with a low nonspecific adsorption rate.

Means for Solving E6 Problems In the present invention, first, immunoglobulin (hereinafter referred to as rIgGj) is obtained from the serum of a rabbit to which an antigen has been input. This Ig
As shown in FIG. 1, G is formed by binding two antigen-binding sites (hereinafter referred to as rFab'J) to a crystallization site (hereinafter referred to as "FC"). Here, Fc is a region that has no binding strength with antigens and has the property of crystallizing at low temperatures.
ab' is a site with a portion at its tip to which an antigen can be specifically adsorbed. Then, IgG is cleaved as shown by the dashed line to remove Fc to obtain Fab', and this Fab' has, for example, COD (glucose oxidase).
used as a labeled antibody. In addition, solid-phase antibodies are obtained from goat serum injected with antigens.
gG adsorbed onto a solid phase is used.

F. Example (1) Example 1 The antigen α-fetoprotein (hereinafter referred to as ``FPj'') was regularly subcutaneously injected into rabbits, and COD was bound to FPLb' obtained based on the serum of the rabbits. AFP was used as a labeled antibody, and AFP was regularly injected subcutaneously into a goat, and IgG obtained based on the goat serum was used.
is adsorbed onto a polystyrene ball and used as a solid phase antibody. Note that AFP is used for diagnosis of liver cancer.

The preparation method of Example 1 will be explained below.

(a) Preparation of IgG Rabbit serum 2!1i was collected and saturated with ammonium sulfate 2!1i
2 was added drop by drop, and the mixture was slowly stirred at a temperature of 4°C for 2 to 3 hours. The resulting cloudy reaction solution was put into a centrifuge tube and centrifuged at 3000 rpm for 15 minutes, then the supernatant was discarded and the precipitate was mixed with 0.005 oQ/
Phosphate buffer (pH
6,0) Add and dissolve 2al. Thereafter, the dissolved solution was added to a column (ix9C) cm (trade name: Sephacryl 300, manufactured by Pharmacia), which had been equilibrated with the above mixed solution, to 6 MO
,/hour to elute and place 11 in the test tube.
Aliquot 1Q each. IgG in the liquid that was subsequently collected
Collect the portion with an absorbance of 280 nm that corresponds to the absorbance of
Obtain TgG.

(b) Preparation of Fab' The (gG prepared in (a) above) was
(0.07 moQ/Q acetate buffer (p II 4 , 0) containing 1/Q sodium chloride at a temperature of 4°C.
Dialysis was carried out for 18 hours, and a pevenone solution was added to the obtained IgG so that the weight ratio of IgG pepsin was 00.3.
, 1 m (!!) 11. Shake for 18 hours in a constant temperature bath at a temperature of 37°C. Then, add this solution to O1 but oQ.
A column (IX90crx, trade name: Sephacryl 200,
(manufactured by Pharmacia) at a rate of 6 m12/hour, and the eluate is fractionated into test tubes. Next, a portion of the fractionated liquid with an absorbance of 280nx corresponding to the absorbance of P(ah')* is collected to obtain F(2Lb'),
get. This F(ab')y is a conjugate of two Fab's. Furthermore, the obtained F(ab')t is 0, I
MoQ/Q mercaptoethylamine is added at 0.05xC and reacted at a temperature of 37°C for 90 minutes to cleave the bonds between Fab'. Thereafter, this reaction solution was desalted by passing it through a column of trade name Sephadex G25 (IX30c shoulder, manufactured by Pharmacia) at a rate of 6xQ/hour, and the eluate was fractionated into test tubes in 13112 portions. A concentrated solution of Fab' is obtained.

(c) Preparation of maleimide COD 0, I MoQ/(l of phosphate buffer with 0.3t
Prepare four test tubes containing nQ each, add approximately 3 mg of COD to each, and add succinimidyl-4-maleimidobutyrate (GMBS) so that the molar ratio of COD and GMBS is 1:50. . The solution obtained in this way was applied to a column (IX30C
I, manufactured by Pharmacia) at a rate of 12 MQ/hour for desalting treatment, and the eluate was fractionated into test tubes in 1 iQ portions to obtain maleimide COD.

(d) Preparation of labeled antibody The maleimide COD obtained in (c) above and the Fab' obtained in (b) above were mixed in equal moles, and at a temperature of 30°C, 1
After standing for a period of time, it is left to stand overnight at a temperature of 4°C. Then, this solution was mixed with 0,1
Sephacryl 300 column (I
0 am, Pharmacia) to 6xQ/h o u
The eluate was poured into a test tube at a rate of 1M.
A labeled antibody in which COD is bound to Fab' is obtained by fractionating each Q amount. Then, this labeled antibody was added with 0.1% NaN5.0.
Add 1% bovine serum albumin (BSA) and store at 4°C.

(e) Preparation of solid-phase antibody Based on goat serum, IgG was obtained in the same manner as in (a) above, and 0, l JI9/Shoulder Q I
0,111012/Q phosphate buffer containing gG (
A polystyrene ball (hereinafter referred to as rPBJ) with a diameter of 6.5*x, which becomes a solid phase, is immersed in a solution (pH 7.5) at a temperature of 4.
Incubate overnight at ℃. Then add 0 polystyrene balls,
Washed three times with phosphate buffer (pH 7,5) at 1 txoQ/σ, followed by 0, 13!012/Q NaCl2
．． 0 containing 0.1% BSA and 0.1% NaNs
After washing three times with OlmoQ/Q phosphate buffer (pH 7,0), PB was immersed in the mixture at a temperature of 4°C.
Save with .

PB thus coated with IgG.

That is, a homologous antibody is obtained.

(2) Example 2 A labeled antibody-solid phase antibody combination was obtained in exactly the same manner as in Example 1, except that carcinoembryonic antigen (hereinafter referred to as rcEAJ) was used as the antigen. Note that CEA is mainly used for diagnosis of colorectal cancer.

G. Comparative Example (1) Comparative Example 1 AFP as an antigen was periodically injected subcutaneously into a rabbit, and IgG obtained based on the rabbit serum and conjugated with COD was used as a labeled antibody, and a solid phase antibody The same material as in Example 1 was used.

Here, to explain the preparation of labeled antibodies, the above (a)
) The IgG obtained in section 0. O05mo (xQ's ED
Add 0,1 txo (1/Q phosphate buffer (pH 6,0) 2xf2 containing TA, and add this solution and S-acetylmercaptosuccinic acid at a molar ratio of S-acetylmercaptosuccinic acid to IgG. D M F (N-dixetylforxa buttocks 1d
Add to e), dissolve and stir at room temperature for 30 minutes. This solution was then diluted with 0,1 xoQ/Q Tris-HCl (p
H7, 0) 0, 1 xQ, 0, 1 Shoulder oQ/ (!
EDTA (pH 7,0) 0, 02II112 and 1 mob, /ρ hydroquinamine aqueous solution (pt(
7,0) Add to the mixture of O, I mQ and keep at a temperature of 30°.
C for 4 minutes, and then the reaction solution was diluted with 0.005
io(!/12 including EDTA 0,1 moQ/1
The sample was desalted by passing it through a Sephadex 025 column (1x30cx, manufactured by Pharmacia) equilibrated with phosphate buffer (pH 6, 0) at a rate of 12xQ/hour, and eluted. SII group-bound [gG is obtained by dividing the solution into test tubes in portions of lz&, which is collected and concentrated in a water-cooled collodion bag. The SH-IgG thus obtained and the maleimide COD obtained in section (c) above were mixed in equimolar amounts, and the same molar method as described in section (d) above was used to prepare SI (-IgG with maleimide COD). Obtain bound labeled antibody.

(2) Comparative Example 2 A labeled antibody-solid phase antibody combination was obtained in exactly the same manner as in Example 1, except that the animal species combination for the labeled anti-salt-solid phase antibody was rabbit-rabbit.

(3) Comparative Example 3 A combination of labeled antibody and solid phase antibody was obtained in exactly the same manner as in Example 1, except that the animal species combination of the labeled antibody and solid phase antibody was goat-goat.

(4) Comparative Examples 4 to 6 CE was used instead of AFP as the antigen for Comparative Examples 1 to 3.
The labeled antibody-solid phase antibody combinations using A are designated as Comparative Examples 4 to 6, respectively.

Table 1 shows the labeled antibody-solid phase antibody combinations on each side of the Examples and Comparative Examples obtained as described above.

Table 1H, Comparative Test (1) Non-Specific Adsorption Examples 1.2 and Comparative Examples 1-6 were compared for non-specific adsorption of labeled antibodies to in-phase antibodies.

The operating procedure is as follows.

First, the labeled antibody was dissolved in 0.1 ioc/(0.1 ioc) of Na Cff solution containing 0.1% BSA and 0.1% NaN3.
, 01 txo (1/Q phosphate buffer (pH 7,0) C
Hereinafter, this mixed solution is referred to as "Liquid A") 100 times diluted with 0.1 mQ of the solution, and 0.2 mQ of Solution A was added to this.
Immerse the solid-phase antibody in the solution containing ff(l) and let it stand overnight at room temperature.Then, take out the solid-phase antibody, wash it three times with Solution A, and transfer it to a new test tube. 0.01 containing glucose of 0,5 m'oQ/Q
MoQ/Q acetate buffer (pH 5.1) was added at a concentration of 0.31, and the mixture was allowed to stand at a temperature of 37° C. for 2 hours. Thereafter, 0.1xσ of this solution was sampled and 2X10-'moQ
Add 0.5 °C of carbonate buffer (pH 9,8) of 0.2 oQ/Q containing luminol of /Q, and further add 6X10-31
1oQ/(l of potassium ferricyanide aqueous solution 0,5 m
After adding Q and waiting 15 seconds, the amount of luminescence for 30 seconds is integrated. This amount of luminescence corresponds to the amount X1 of labeled antibody nonspecifically adsorbed to the solid phase antibody. On the other hand, since the total ff1x of labeled antibodies is known in advance, the nonspecific adsorption rate is (X +/X
2) Calculated as x 100. The experimental results thus obtained are shown in Table 2.

Table 2 As can be seen from Table 2, the combination of labeled antibody and solid-phase antibody was rabbit-derived Fab'-goat-derived IgG (Examples 1 and 2)
In the case of 1gG derived from rabbit - 1gG derived from goat (Comparative Examples 1 and 4), the nonspecific adsorption rate is significantly lower.

'Also, even if Fab' is used as the labeled antibody, if the animal species combination of the labeled antibody and in-phase antibody is rabbit-rabbit (Comparative Example 2.5) or goat-goat (Comparative Example 3.6), , the non-specific adsorption rate is 1 gG from rabbit - 1 gG from goat.
It is comparable to the gG combination. Therefore, it is thought that not only the use of Fab' as a labeled antibody but also the combination of animal species has a large effect on nonspecific adsorption.

(2) Antigen measurement range and detection limit Example 1.2 and Comparative Examples 1 to 6 were compared with respect to antigen measurement range and detection limit. The detection limit was determined using the F test (n=1
0). The operating procedure is as follows. The same applies when the antigen is CEA.

The solid-phase antibody obtained based on the antigen AFP was
AFP standard solution diluted with solution 0, A solution 0 for IRQ
．． '2rQ is added to the solution and left to stand at room temperature for 6 hours. After that, the solid-phase antibody was taken out and washed three times with solution A, and the labeled antibody obtained based on the antigen AFP was diluted with solution A to the optimal dilution ratio.
．． The washed solid-phase antibody is immersed in the mixture with 2xσ and left overnight at room temperature. Then, wash the solid-phase antibody three times with solution A, transfer it to a new test tube, and add 0.5 m
0,01 moQ/Q with 12/12 glucose
0.3 mQ of acetate buffer (pH 5, 1) was added, and the mixture was left standing at a temperature of 37°C for 2 hours.

Thereafter, 0.1 mQ of this solution was sampled, 0.5 mQ of 0.2 moQ/ρ carbonate buffer solution (pH 9,8) containing 2x 10-7 moQ,/(! of luminol) was added, and a further 6 mQ was added. X 10-”yoQ/
Add 0.5x Q of potassium ferricyanide aqueous solution to 1
After waiting 5 seconds, the amount of light emitted for 30 seconds is accumulated. The amount of luminescence corresponds to the amount of labeled antibody adsorbed to AFP captured by the solid-phase antibody, that is, the amount of AFP.

The results of the measurement range thus obtained are shown in Table 3, and the results of the detection limit are shown in Table 4.

Table 3 Table 4 Table 39 As can be seen from Table 4, when the combination of labeled antibody and solid-phase antibody is rabbit-derived Fab'-goat-derived IgG, the nonspecific adsorption rate is low, so Ig
Both the measurement range and the detection limit are more permissible than when G is used, or when Fab' is used as the labeled antibody and the animal species are rabbit-rabbit or goat-goat.

(3) Reproducibility The simultaneous reproducibility and day-to-day reproducibility of the detection limit of Comparative Example 2 determined in (2) above were investigated for Example 1 and Comparative Examples 1 to 3, and the detection limit of Comparative Example 6 was The simultaneous reproducibility and day-to-day reproducibility of Example 2 and Comparative Examples 4 to 6 were investigated. Simultaneous reproducibility here refers to reproducibility when measurements are performed continuously, and day-to-day reproducibility refers to reproducibility when measurements are performed on different days. Reproducibility was evaluated using the standard deviation above the mean and the coefficient of variation. Example 1 and Comparative Example 1~
For the reproducibility test with 3, AFP was used as the antigen,
Furthermore, for the reproducibility tests of Example 2 and Comparative Examples 4 to 6, CEA was used as the antigen.

The results are shown in Table 51 and Table 6, respectively.

Table 5 Table 6 As can be seen from Table 51 and Table 6, rabbit-derived Fab'-goat-derived Ig
The combination of G is better than either the combination of rabbit-derived Pab'-rabbit-derived 1gG and goat-derived Fab'-goat-derived 1gG.

Although AFP or C,EA was used as the antigen in the above examples, similar results were obtained using other antigens such as human placental gonadotropin, insulin, IgE, α,-microglobulin, etc.

Furthermore, in the present invention, CO is used as a label that binds to Fab'.
Other enzymes instead of D, such as horseradish peroxidase, β-D-galactosidase.

Glucose-6-phosphate dehydrogenase, alkaline phosphatase, etc. may also be used. In addition, instead of using enzymes as labels, fluorescent substances (fluores-ceini
sothiocyanate, tetramethy
lrhodamine so-thiocyanate
etc.), chemiluminescent substances (aminoethyleL-hy
lisoluminol, aminobutylet
hylisoluminoLaminopentyle
thylisoluminol, aminohexyl
ethylisoluminol, etc.), or radioactive isotopes (3H.

14C, 3tp, +15I, +31f, etc.)
may also be used.

10. Effects of the Invention As described above, according to the present invention, a labeled antibody is used as a labeled antibody to the remaining Fab' from which Fc has been separated from IgG, and the animal species in the labeled antibody and the solid-phase antibody are Focusing on the fact that the combination of
- Since the labeled antibody is used as a labeled antibody, the nonspecific adsorption rate of the labeled antibody to the solid-phase antibody can be suppressed to a low level, as seen from the comparative test results of Examples and Comparative Examples. Enlargement and detection limits can be improved, and good reproducibility can be obtained.

[Brief explanation of the drawing]

Figure 1 is a structural diagram showing immunoglobulin. Figure 1 Reward Groff ηn0 lecture drawing

Claims (1)

[Scope of Claims] A solid-phase antibody is prepared by adsorbing immunoglobulin obtained from the serum of an animal injected with an antigen to a solid phase, and the antigen is specifically adsorbed to the solid-phase antibody. Immunization in which a labeled antibody formed by binding a labeled substance to globulin is specifically adsorbed to the antigen adsorbed on the solid-phase antibody, and the amount of the antigen adsorbed is indirectly determined by checking the amount of the labeled substance. In the antigen measurement reagent used in the chemical measurement method,
An immunoglobulin is obtained from the serum of a rabbit to which the antigen has been injected, and a labeled antibody is obtained by cutting off the crystallization site and binding a label to the remaining antigen-binding site, and a labeled antibody obtained from the serum of a goat to which the antigen has been injected. An antigen measurement reagent consisting of a combination of a solid-phase antibody made by adsorbing immunoglobulin on a solid phase.