JPS63313068A - 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 guinea pig. 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, for example, a guinea pig dosed with the antigen and is adsorbed to the solid phase is used as the solid phase antibody. As to the species of the animals for the labeled antibody and the solid phase antibody, the guinea pig is used for at least either thereof.

Description

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

B1 Summary of the Invention The present invention is a measurement reagent used in the Sand-Deutsche method, which is an immunoassay method, in which a labeled antibody is produced by binding a labeled substance to the remaining antigen-binding site after cutting off the crystallization site of an immunoglobulin. At the same time, by preparing at least one of the labeled antibody or the solid-phase antibody based on guinea pig serum, the amount of nonspecific adsorption of the labeled antibody to the solid-phase antibody is suppressed,
This improves measurement sensitivity and allows good reproducibility to be obtained.

C3 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 trace amounts 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 is prepared by binding a labeled substance, such as glucose oxidase (hereinafter referred to as rGODj), to the immunoglobulin. 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 solid-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 such immunoassays include rabbit-labeled antibody-rabbit solid-phase antibody, both of which are prepared based on rabbit serum, or rabbit-labeled antibody-rabbit solid-phase antibody, both of which are prepared based on goat serum. Goat-labeled antibodies--goat solid-phase antibodies, etc. have been conventionally used.

D9 Problems to be solved by the invention By the way, according to the measurement principle of the sandwich method, it is ideal that the number of directly adsorbed labeled antibodies to solid-phase antibodies is 0, but in reality, some of the labeled antibodies 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 adsorption amount as low as possible.

Looking at the nonspecific adsorption rate of conventional measurement reagents, in the case of rabbits it is approximately 4.8Xl (I' ~ 36.8X10
-”%, for goats approximately 7.lXl0-'~16
The value is quite high at ゜0XIO-'%. 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 combinations of animal species and found that if at least one of the labeled antibody and the solid-phase antibody was prepared using a reagent prepared based on guinea pig serum, the nonspecific adsorption rate could be reduced to 1.8 x 10-'' to 2.
．． It has been found that it can be suppressed to about 3×10-”%.

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 E2 Problems In the present invention, first, immunoglobulin (hereinafter referred to as rIgGj) is obtained from, for example, guinea pig serum to which an antigen has been introduced. As shown in Figure 1, this IgG has two antigen-binding sites (hereinafter referred to as rFcJ) at the crystallization site (hereinafter referred to as rFcJ).
a b 'j) are combined. Here, Fc is a region that has no binding strength with antigens and has the property of crystallizing at low temperatures, and F a b' is a region where antigen is
l! This part has a part at the tip where the target can be attracted. Then, Fc is removed by cutting the IgG as shown by the dashed line ``4'' to obtain Pab', and this F a b
For example, use COD (glucose okindase) bound to ' as the labeled antibody. As the solid-phase antibody, use one in which IgG obtained from, for example, guinea pig serum to which the antigen has been introduced is adsorbed to a solid phase. As for the animal species for the labeled antibody and the solid-phase antibody, a guinea pig is used for at least one of them.

F. Example (1) Example 1 The antigen α-fetoprotein (hereinafter referred to as FAI"Pj) was regularly subcutaneously injected into a guinea pig, and Pc was cleaved from IgG prepared based on the serum of the guinea pig. Obtain Fab' and use the Fab' bound to GOD as a labeled antibody, and the 1gG adsorbed to a polystyrene ball 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 Collect 21 guinea pig serum and add 2 mQ of saturated ammonium sulfate to it.
was added drop by drop and slowly stirred at a temperature of 4°C for 2 to 3 hours. The resulting self-localized reaction solution was placed in a centrifuge tube and centrifuged at a rotation speed of 3000 rpm for 15 minutes.
Next, discard the supernatant and add Q, OO5moQ/Q to the precipitate.
of 0.1 mo(1/(l) of phosphate buffered t
Add L (pI (6,0) 2xσ and dissolve. Then, the dissolved solution was transferred to a column (IX90 cm, manufactured by Pharmacia) under the trade name Sephacryl 300 equilibrated with the above mixture at a speed of 6 m (1/hour). eluted through the tube and aliquoted into 11 tubes.Subsequently, I
A portion with absorbance 2B0nx corresponding to the absorbance of gG is collected to obtain IgG.

(b) Preparation of Fab' The IgG prepared in (a) above was added at 0.05to17/
Dialyze with 0.07 moQ/Q acetate buffer (pH 4.0) containing f2 sodium chloride at a temperature of 4°C for 18 hours, and add pevenone solution to the obtained IgG so that the weight ratio of IgG to pepsin is 100 + 3. Add 0.1 spots of Q and leave in a constant temperature bath at 37°C for 8 hours. This solution was then converted to 0, l, woO,/l).

Sephacryl 200 column (l x 90 cm, '7
(manufactured by Almasia) at a speed of 6 m (/hour) for elution, and the eluate was fractionated into test tubes for each IRQ.

Next, a portion of the fractionated liquid with an absorbance of 280nx corresponding to the absorbance of F(ab')2 is collected and F(ab')
get t. This F(ab')t is a conjugate of two Fab's. Furthermore, the obtained F (ab') 2 is 0
, 1 rttoQ/ (l mercaptoethylamine to 0
,05xQ was added and reacted for 90 minutes at a temperature of 37°C.
Break the bond between a and b'. After that, this reaction solution was transferred to a column (1X30c) of Sephadex G25 (trade name).
The eluate was desalted by passing it through a tube (manufactured by Pharmacia) at a rate of 6 mQ/hour, and the eluate was poured into a test tube.
A concentrated solution of Fab' can be obtained by fractionating each mQ.

(c) Preparation of maleimide COD Prepare 4 test tubes containing 0.3 ml of phosphate buffer of 0 and 12#, add about 3 q of COD to each, and add succinimidyl-4-maleimidobu. Dilate (GMB)
S) is added such that the molar ratio of COD to CMBS is 1:50. The solution thus obtained was heated at 0 and 1 t.
The sample was desalted by passing it through a Sephadex G25 column (lx90cm, manufactured by Pharmacia) equilibrated with a phosphate buffer solution of ttoQ/12 at a rate of l2i12/hour, and the eluate was poured into a test tube. The maleimide COD is obtained by fractionating 1 shoulder Q at a time.

(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. This solution was then mixed with O, l containing 0,005 moQ/Q of EDTA.
Sephacryl 300 column (1 x 90 cm,
(manufactured by Pharmacia) at a rate of 6 mQ/hour, and the eluate is aliquoted in 1 jIQ portions into test tubes to obtain a labeled antibody in which COD is bound to Fab'. Then, add 0.1% NaN3, O, 1% to this labeled antibody.
of bovine serum albumin (USA),
Store at a temperature of 4°C.

(e) Preparation of solid-phase antibody Based on guinea pig serum, IgG was obtained in the same manner as in (a) above. 0.1+o0. containing IgG of lrxg/rnQ. /Q phosphate buffer (pH
A polystyrene ball (hereinafter referred to as rPBj) having a diameter of 6.5 u to serve as a solid phase was immersed in 7.5) and allowed to stand overnight at a temperature of 4°C. Then the polystyrene ball is O, l then oQ/
Wash 3 times with Q phosphate buffer (pH 7,5) and remove
, 1 tttoQ/ Q of NaC, (2,0,1%l3
0.01 butt oQ/Q including SA and 0.1% NaN3
After washing three times with phosphate buffer (pi(7,0)), PB
is immersed in the mixture and stored at a temperature of 4°C.

PB thus coated with IgG.

That is, a homologous antibody is obtained.

(2) Example 2 A combination of labeled antibody and solid-phase antibody was obtained in exactly the same manner as in Example 1, except that the in-phase antibody was prepared based on rabbit serum.

(3) Example 3 A labeled antibody-solid phase antibody combination was obtained in exactly the same manner as in Example 1, except that the solid phase antibody was prepared based on goat serum.

(4) Example 4 A labeled antibody-solid phase antibody combination was obtained in exactly the same manner as in Example 1, except that the labeled antibody was prepared based on rabbit serum.

(5) Example 5 A labeled antibody-solid phase antibody combination was obtained in exactly the same manner as in Example 1, except that the labeled antibody was prepared based on goat serum.

(6) Examples 6 to 1O For Examples 1 to 5, CE was used instead of AFP as the antigen.
Combinations of labeled antibody-solid-phase antibody using A are referred to as Examples 6 to 10, respectively. Note that CEA is used for diagnosis of colon cancer.

G. Comparative Example (1) Comparative Example 1 AFP as an antigen was periodically injected subcutaneously into a guinea pig, and IgG obtained from the guinea pig serum was injected with CO.
D-bound was used as the labeled antibody, and as the solid phase antibody, the same one as in Example 1 was used.

Here, we will explain the preparation of labeled antibodies, and we will explain the preparation of labeled antibodies (,
The IgG obtained in section a) was added to 0. O05moQ/QED
0.1 oQ/Q phosphate buffer (pH 6,
0) In addition to 2xC, add this solution and S-acetylmercaptosuccinic acid to S-acetylmercaptosuccinic acid and IgG
D M F so that the molar ratio of and is 300 + 1
(N-dixetylroriamide) to dissolve and stir at room temperature for 30 minutes. This solution was then diluted with 0,1 *oQ/(l of Tris-HCl (p) (7,0
)0,1x(1,0,1xoQ/Q's EDTA
(pH 7,0) 0.0'2xQ and l oQ/Q hydroxyamine aqueous solution (pH7,0) 0,1xC were added to a mixture of 0.1xC and reacted at a temperature of 30°C for 4 minutes, and then this reaction solution was 0 including 0.005xof2/f2EDTA,
1 jIo (1/ f2 phosphate buffer (pH 6,0)
Sephadex G25 column (I
x30cm, manufactured by Pharmacia) 12xi2/hour
By passing the eluate through the tube at a speed of
gG is obtained, which is collected and concentrated in a collodion bag in water cooling. The thus obtained SH-1xC and the above (c)
SII-IgG
A labeled antibody to which maleimide COD is bound is obtained.

(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 of the labeled antibody and solid phase antibody was rabbit-rabbit.

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

(4) Comparative Examples 4 to 6 Regarding Comparative Examples 1 to 3, C was used instead of AFP as the antigen.
Comparative Examples 4 to 6 are the combinations of a labeled antibody using EA and a solid-phase antibody.

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

Table 10, Comparative Test (1) Non-Specific Adsorption Examples 1-10 and Comparative Examples 1-6 were compared regarding non-specific adsorption of the labeled antibody to the solid-phase antibody. The operating procedure is as follows.

First, the labeled antibody was diluted with 0,111o(1/(l) of NaCl
2 parts, 0.1% l3SA and 0.1% NaN
0 including 3. O1 shoulder oQ/Q phosphate buffer (pH 7,0
) C or below, this mixture is called r AljLj) is 100
Collect O, l x Q of the diluted solution, and add 0 of solution A to this.
．． The solid-phase antibody is immersed in a solution containing 2xσ and left overnight at room temperature.

After that, remove the solid phase antibody, wash it three times with Solution A, and transfer it to a new test tube. Then add 0, 5 to this test tube.
0, 01 no(1) containing glucose of txoQ/Q
/Q acetate buffer (p■■5,1) to 0,3y
(l is added and left to stand at a temperature of 37°C for 2 hours. Then, 0.1 J!+2 of this solution is sampled and 2 x
0.2mof containing luminol of l O-7xoQ/Q
Add 0.!5 mQ of a mixture of 2/(2 and carbonate buffer (p I(9,8)), and add 6
Add a potassium ferricyanide aqueous solution of 0, 5 y, (l), wait 15 seconds, and then integrate the luminescence amount for 30 seconds. This luminescence amount is equal to the amount of labeled antibody nonspecifically adsorbed to the solid-phase antibody On the other hand, since the total ff1xz of labeled antibodies is known in advance, the nonspecific adsorption rate is (x l/X 2)
It is calculated as X lo o. The experimental results thus obtained are shown in Table 2.

Table 2 As can be seen from Table 2, when Fab' was used as the labeled antibody and at least one of the labeled antibody or the solid-phase antibody was obtained based on guinea pig serum (Example 1-1
0), the nonspecific adsorption rate is lower than when IgG is used as the labeled antibody and the animal species combination of labeled antibody and solid-phase antibody is guinea pig-guinea pig (Comparative Examples 1 and 4). small. In addition, as a labeled antibody, Pab'
Even if the combination of labeled antibody and solid-phase antibody is rabbit-rabbit (Comparative Example 2.5) or goat-goat (Comparative Examples 3 and 6), the nonspecific adsorption rate will be It is comparable to the combination of 1gc derived from guinea pig and 1gG derived from guinea pig. 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 Examples 1 to 10 and Comparative Examples 1 to 6 were compared in terms of antigen measurement range and detection limit. The detection limit was determined using the F test (n=
lo). The operating procedure is as follows. The same applies to the case of antigen or CEA.

A solid-phase antibody obtained based on antigen AFP was diluted with AFP standard solution 0.0. 0.21 of liquid A per l
The specimen is immersed in a solution containing the following ingredients and left to stand at room temperature for 6 hours. Thereafter, 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 an optimal dilution ratio of 0, I Jl (! and Solution A 0.2).
The washed solid-phase antibody is immersed in the mixture with xQ 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 0.5RQ/
0.0 containing Q glucose. Add 0.3 mQ of O1 shoulder oO,IQ acetate buffer (pHs, t) and incubate at 37°C for 2 hours.
Let stand for a while.

Thereafter, 0.1 mQ of this solution was sampled and 0.1 mQ containing 2x 10-7 moQ/Q of luminol was added.
, 0.5 mQ of carbonate buffer (pH 9, 8) of 2 tnoQ/Q was added, and further 6 x l 0-3yto
Add 0.5 mQ of potassium ferricyanide aqueous solution of Q/Q, wait 15 seconds, and then integrate the amount of light emitted for 30 seconds.

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 As can be seen from Table 31 and Table 4, when Fab' is used as the labeled antibody and at least one of the labeled antibody or the solid-phase antibody is obtained based on guinea pig serum, IgG is used as the labeled antibody. If Fab' is used as the labeled antibody and the animal species combination is rabbit-
Since the non-specific adsorption rate is lower than in the case of rabbits and goats, both the measurement range and the detection limit are improved.

(3) Reproducibility The simultaneous reproducibility and day-to-day reproducibility at the detection limit of Comparative Example 2 determined in (2) above were compared to Examples 1 to 5 and Comparative Examples 1 to 3.
In addition, we investigated the simultaneous reproducibility and daily difference reproducibility at the detection limit of Comparative Example 6 in Examples 6 to 10 and Comparative Example 4.
-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~
AFP was used as the antigen for the reproducibility test between Example 5 and Comparative Examples 1 to 3, and CEA was used as the antigen for the reproducibility test of Examples 6 to 1O and Comparative Examples 4 to 6. The results are shown in Table 51 and Table 6, respectively.

Table 5 Table 6 Table 59 As can be seen from Table 6, in terms of both simultaneous reproducibility and day-to-day reproducibility, when F a b' is used as the labeled antibody, at least On the other hand, the combination using guinea pigs is better than any combination of rabbit Fab'-rabbit IgGGTJt and goat Fab'-goat IgG.

Although AFP or CEA was used as the antigen in the above examples, similar results were obtained using other antigens such as human placental gonadotropin, insulin, IgE, α1-microglobulin, etc.

Furthermore, in the present invention, other enzymes, such as horseradish peroxidase and β-D-galactosidase, are used instead of COD as a label that binds to F a b '.

Glucose-6-phosphate dehydrogenase, alkaline phosphatase, etc. may also be used. In addition, instead of using enzymes as labels, fluorescent substances (rluores-ceini
sothiocyanatestetramethyl
rhodamineiso-thiocyanate, etc.), chemiluminescent substances (aminoethylet-hyl
isoluminollaminobutylethy
lisoluminol.

aminopentylethylisolumino
lSaminohexylethyl-isolumi
nol etc.) or radioactive isotopes (3H2+40.
32p, 1ff5, 1311, etc.) may also be used.

(1) Effects of the Invention As described above, according to the present invention, a labeled antibody is used as a labeled antibody by binding a label to the remaining Pab' from which Pc has been separated from IgG, and a label flow 0 and a solid-phase antibody are used. The combination of animal species in is also non-r.

Focusing on the fact that it has a large effect on heterogeneous adsorption, guinea pigs are used as at least one of the animal species for labeled antibodies or solid-phase antibodies, as can be seen from the comparative test results of Examples and Comparative Examples. In addition, the nonspecific adsorption rate of labeled antibodies to solid-phase antibodies can be kept low, thereby expanding the antibody measurement range and improving the detection limit, and achieving good reproducibility. can.

[Brief explanation of the drawing]

FIG. 1 is a structural diagram showing immunoglobulin.

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,
A labeled antibody was constructed by binding a labeled antibody to the antigen binding site remaining after the crystallization site of the immunoglobulin was cut off, and at least one of the labeled antibody or the solid-phase antibody was prepared based on guinea pig serum. A reagent for measuring an antigen, characterized in that: