DE3205849C2 - Reagent for immunological determination - Google Patents

Abstract

A reagent for immunological determination is described which comprises an insolubilized antibody and a labeled antibody each prepared from monoclonal antibodies. With the aid of the reagent according to the invention, a significant shortening of the reaction time is achieved without any adverse effect on the sensitivity and accuracy being observed. In addition, an improved sandwich method is provided using the above reagent, wherein insolubilized antibody and labeled antibody are reacted simultaneously with the antigen to be determined.

Description

The enzyme immunoassay (EIA) is a very sensitive and quantitative immunological determination method.

In particular, the sandwich method based on this determination is preferred due to the simplicity of the Procedure and high sensitivity performed frequently. However, since the sandwich method 1 to 4 days requires reaction, it is desirable to keep the required reaction time to a minimum. Also, due to the recent advances in clinical medicine, rapid evaluation of the Test results required in order to be able to subject the patient to treatment immediately. From From this point of view, a shortening of the reaction time is highly desirable. With the conventional one Sandwich method, however, leads to a reduction in sensitivity and a reduction in response time Accuracy of the determination, so that a significant reduction in the response time cannot be achieved can.

The conventional sandwich method is described in detail below. Although for the sake of Simplicity the method is described throughout the specification as being based on EI, the Declaration can also be applied to an FIA-based method (fluoroimmunoassay).

The conventional sandwich method is shown in the sequence shown in the schematic diagram in FIG. 1 is shown, performed.

i) Insolubilized antibody 2 ', which by binding an antibody for an antigen to be determined 3 is obtained on an insoluble carrier (solid phase) 4! is reacted with the antigen 3 in order to Antigen 3 to bind to antibody 2 'on the solid phase (first reaction).

ii) The solid phase 4 is washed to remove unreacted substances.

iii) The solid phase 4 washed in this way is labeled with antibody 1 ', which by binding of an enzyme 5 to an antibody for the antigen to be determined 3 was obtained, converted to the labeled antibody 1 'to an unreacted antigenic site of antigen 3, which is attached to the solid phase 4 was fixed to bind (second reaction).

iv) The solid phase 4 is washed in order to remove an excess of labeled antibody Γ; a substrate for the enzyme is then added and subjected to an enzymatic reaction.

Since antigen 3 and labeled antibody 1 'are bound to solid phase 4, the enzyme reaction takes place in Proportion to the amount of enzyme present. The amount of the antigen 3 to be determined is from the Determined amount of the reaction product obtained.

Since the sandwich method is generally classified as a non-competitive determination, could it can be assumed that there is no competition reaction. However, this classification does not necessarily exist the exact reaction states again. Indeed, the sandwich method can be a state of equilibrium present, which is shown schematically as follows.

First reaction

+ [Ag]

Ab-Ag a> a '

Second reaction

- Ag - Ab * l

- AgJ + [Ab *] Γ j Ab I j + [Ag - Ab-]

Γ I Ab J Ί + [Ag] + [Ab *]

ύί

z | (In the above scheme, | Ab | represents the insolubilized antibody; Ag means that

| L containing antigen; Ab * represents the marked antibody; a, a ', b, b'. c. c ', d, d', e and e 'mean equilibrium

Ij constants; and - means that the antigen and the antibody are linked.)

% If the complex of the insolubilized antibody and the antigen to be determined with the marked

I'J antibodies are converted in the second reaction, so bind the insolubilized antibody and the

; labeled antibodies compete with the antigen to be determined according to their affinity levels for the antigen

r (since the properties of the insolubilized antibody and the labeled antibody are almost the same,

ΐ% , their affinity for the antigen to be determined is assumed to be almost equivalent), and because of the

} v! Competition dissociates »the complex of insolubilized antibodies / antigen formed in the first reaction,

l ' ^: ' where complexes of various combinations as shown above are formed.

This is proven by the following experiment:

A plastic test tube with an anti-HCG - ^ - subunit antibody attached is mixed with HCG- / ?, which is labeled with peroxidase, reacted and then washed. After implementation with an anti-HCG - /? - Antibody gradually dissociates the enzyme-labeled HCG- /? Which has been fixed to the solid phase (from the solid phase and migrates into the liquid phase. Fig. 2 shows this state.

However, if the time of the first reaction is long enough, the antigen and antibody binding will be | _t partially irreversible, and the antigen-antibody complex does not readily dissociate, even when labeled

^f antibody is added to it. On the other hand, it is said that when the time for the second response

is long enough for the reaction to proceed in one direction, after which it eliminates a complex 1 insolubilized antibody, the antigen to be examined and the labeled antibody «. the end

For these reasons, it is absolutely necessary in the conventional sandwich method to carry out the reaction for a sufficiently long time. Therefore, if the sandwich method is carried out with no initial reaction

or the duration of the first reaction is extremely shortened (for example, if the insolubilized antibody and the labeled antibody are reacted simultaneously with the antigen to be determined), the second reaction must be carried out for a longer time and nevertheless the sensitivity and accuracy of the determination are reduced. If the duration of the second reaction is shortened, the effect on the sensitivity and accuracy is even more disadvantageous. For these reasons, it was with the conventional method impossible to shorten the reaction time i without causing a reduction in sensitivity and accuracy.

With this in mind, Ichihara et al. (The Japanese Journal of Clinical Pathology,

26,1027 [1978]) reports that such a competitive reaction occurs, and that only if the reaction is carried out over a sufficiently long period of time, the reaction becomes partially irreversible.

The reaction system of the conventional method is therefore complicated, inefficient, and requires 1 long response time.

In the older, prior art patent application P 31 30 834.1 is an immunological Determination by the sandwich method using an insolubilized monoclonal antibody and labeled monoclonal antibody. The antibodies are able to differentiate between different To distinguish antigen determinants of the antigen to be determined. Similar prior art 'is also described in the earlier European patent applications EP 48 357 and EP42 755.

The object of the invention is to provide a reagent for immunological determination by the sandwich method ':; In which one takes the time required for the determination without reducing the sensitivity and can significantly reduce accuracy. This object is achieved by a reagent according to claim 1 solved.

I Fig.] Is a schematic diagram showing the manner of reaction according to the conventional sandwich method.

F i g. 2 and 3 are diagrams showing the presence and absence of competition between Reproduce antibodies,

F i g. Fig. 4 is a schematic diagram showing the manner of reaction with a reagent according to the invention shows

F i g. Fig. 5 is a graph showing a comparison between the use of the reagent of the present invention and reproduces the conventional method,

F i g. 6 is a graph showing the results of Example 1;
F i g. 7 is a graph showing the results of Example 2. FIG.

In the renal of the present invention, the insolubilized antibody and the labeled antibody are each able to perceive different antigenic determinants of the same antigen to be determined and to bind to them; or they include antibodies that intervene between different antigenic determinants of the antigen, so that it is possible to use the two antibodies to different antigens Sites that are located on the same antigen are sufficiently spaced apart to bind to the Binding of each of the antibodies does not interfere.

The monoclonal antibodies are produced by cell fusion between myeloma cells and antibody-producing Cells according to the method of Milstein et al. (Nature, 256, 495 [1975]). Because these antibodies are made from Antibody-producing cells of a single clone are formed, they are completely homogeneous and theirs Specificity for antigenic determinants is completely identical. As the antigens usually to be determined Having multiple antigenic determinants occurs when using monoclonal antibodies as insolubilized Antibodies and labeled antibodies that are capable of different antigenic determinants of the antigen to perceive and bind, there is no competition between these antibodies, and it becomes a highly specific one Determination of antigen achieved. It can also be a mixture of two or more monoclonals Antibodies that bind to various antigenic determinants, as insolubilized antibodies and / or labeled antibodies can be used provided that these antibodies do not cause competition.

The determination according to the invention can be carried out in the same way as is known in the prior art is to be carried out. However, the reaction mechanism compared to the conventional method is simplified, since the insolubilized antibody does not compete with the labeled antibody, as follows will be shown.

Γ I from I - AgI

- Ag + [Ab *]

Γ I Ab j j + [Ag] + [Ab *] j j Ab | - Ag - Ab * l

v
Γ Ab 1 + [Ag - Ab *]

p> p ', q> q', r> r ', s> s'

(Ab, Ag, Ab * and - have the same meanings as given above, and p, p ', q, q'. R. R ', s, s'

each represent equilibrium constants.)

Since there is no competition between the insolubilized antibody and the labeled antibody, if these have different binding sites on an antigen, the reaction proceeds in the direction that the Components in the reaction solution include an "insolubilized antibody-antigen-labeled antibody" comp! Ex form.

This can be proven by the following experiment:

In the same reaction system as the conventional sandwich method, there were two different anti-HCG monoclonal antibodies used as insolubilized antibodies and labeled antibodies, the were able to sense various antigenic determinants of HCG and a similar experiment was carried out. The results show (Fig. 3) that as soon as the antigen reaches the insolubilized Antibody is bound, this does not dissociate from the insolubilized antibody (solid phase) if a Anti-HCG antibody corresponding to different antigen determinants is added. Consequently it is understandable that competition between antibodies for the same antigenic center does not occur. Accordingly, according to the invention, similar results are obtained when the insolubilized antibody, the antigen to be determined i.ind the labeled antibodies are converted at the same time, or the insolubilized antibodies and the antigen are first mixed and then reacted with the labeled antibody or the insolubilized antibody is reacted with the antigen and after a while the mixture is reacted with the labeled antibody. There is therefore no limitation in the sequence of reactions.

The use of the reagent according to the invention is shown in FIG. 4 shown. Since the insolubilized antibody 2 and the labeled antibody 1 do not compete with each other, a larger amount of labeled Antibody 1 will be bound to antigen 3 within a shorter period of time, and it is not only possible to shorten the time for the determination, but also the accuracy and sensitivity of the determination to increase.

Table 1 shows the advantages when using the reagent according to the invention compared to conventional method using polyclonal antibodies in the determination of A-fetoprotein (AFP) compiled. The table shows that according to the invention many advantages, such as. Legs significant shortening of the determination time, a simplification of the determination procedure and an increase can be achieved in the determination accuracy and sensitivity.

Table 1

Application of the conventional method according to the invention
reagent

Procedure Reaction— "Wash First Reaction—" Wash

—Enzyme reaction - »second reaction — washing

—►Enzyme reaction

Antibodies used Monoclonal antibodies Polyclonal antibodies

Required time 30 min 270 min

Sensitivity 3 ng / ml 10 ng / m

accuracy

Intra-experimental error, 1.2% 2.5%

C. V. for 10 repetitions

Intra-experimental error, 3.7% 5.6%

C. V. for 5 repetitions

The determination was made using a fluorophotometer on the serum of a pregnant woman as the sample to be determined, a polystyrene test tube as the solid phase, horseradish peroxidase as the labeling Enzyme and phloretinic acid carried out as substrate.

F i g. 5 shows standard curves in the determination of CEA (carcino-embryonic antigen) when the insolubilized Antibody and the labeled antibody can be combined as shown in Table 2. It shows clearly that the determination system based on the combination according to the invention to the best results.

Table 2

Insolubilized marked person

Antibodies antibodies

Reagent according to the invention monoclonal monoclonal

Comparative method 1 monoclonal polyclonal

Comparative method 2 polyclonal monoclonal

Conventional method polyclonal polyclonal

The monoclonal antibodies used were produced by cell fusion techniques using Mouse myeloma cells are obtained from and are derived from clones that are responsive to different antigenic sites. Polystyrene test tubes were used as the insoluble supports; Horseradish peroxidase was used as the enzyme and o-phenylenediamine was used as the substrate.

Any desired combination of myeloma cells and antibody-producing cells can be used Obtaining monoclonal antibodies can be used and there is no limitation on the animal from which the cells originate, as long as both cells in combination can form hybridomas and produce antibodies as they grow.

According to the invention, all insoluble carriers can be used as they are with conventional ones Immuno-determinations are used. Examples of such insoluble carriers are polystyrene, polyethylene, Acrylic acid resins, polytetrafluoroethylene. Paper, glass and agarose. The insoluble carrier can be called gear-like Form, bead, rod, disc or cuvette are present. He can z. B. an optical cell, a test tube be.

Labeling substances which are commonly used include e.g. B. peroxidase, / 7-D-galactosidase and alkaline phosphates for EIA, and fluorescein isothiocyanate for FIA.

When the labeling agent is an enzyme, a substrate is required in order for it to be active determine. The substrate can be chosen so that, after reaction with the corresponding enzyme, the Amount of product obtained or the amount of substrate removed or remaining slightly is determined. For example, 5-aminosalicylic acid-H2O2, o-phenylene- ^ C ^. Phloretinic acid-H2O2, etc. as Substrates for peroxidase, and fluorescein-di - (/? - D-galactopyranoside), o-nitrophenol-yf-D-galactopyranoside, 4-methyl-umbelliferyI - /? - D-galactosid etc. can be used as substrates for ^ -D-galactosidase.

Substances which can be determined with the reagent according to the invention must have at least two antigenic determinants in the molecule. Since the substances which have been determined using the sandwich method have two or more antigenic determinants, essentially all substances which have been determined using the conventional method can be determined using the method according to the invention. Examples of such substances include enzymes such as γ-glutamyl transpeptidase ( γ-GTP), alkaline phosphatase and transglycosidase; Protein hormones such as thyroid stimulating hormone (TSH), luteinizing hormone (LH), human chorionic gonadotropin (HCG), insulin, secretin and growth hormone (GH); Plasma proteins such as fibrin degradation product (FDP), C-reactive protein (CRP), «i -acid glycoprotein (λτι-AG), Λι-antitrypsin (« i-AT), as-plasmin inhibitor (Λ2-ΡΙ), / Jrmicroglobulin (/ J ₂ -MG) and immunoglobulin; carcinoembryonic proteins such as Λ-fetoprotein (AFP), carcinoembryonic antigen (CEA) and embryonic ferritin; and lymphocytes and bacterial cells, cell surface antigens, cell fractones, etc.

In addition to the insolubilized antibody and the labeled antibody, the reagent according to FIG Invention contain various auxiliaries that increase its usefulness. These can e.g. B. a dissolving one Agent for the labeled antibody (when added as a lyophilized product), one Washing liquid for washing the solid phase after the reaction of the insolubilized antibody and the to determining antigen and after further reaction with the labeled antibody, a substrate for Determination of the enzyme activity of the labeled agent (if this is an enzyme) and a reaction stopper for the enzymatic reaction.

example 1

Reagent for the determination of carcino-embryonic antigen (CEA) a) Manufacture of purified CEA

40 g of cancerous tissue of the large intestine were removed by means of a homogenizer after adding 100 ml of distilled water homogenized. The same amount of 1.2 M perchloric acid was added to the homogenate and extracted for 30 minutes with stirring. The centrifuged supernatant was turned against distilled water dialyzed to obtain the crude CEA extract.

The crude extract was concentrated to 10 ml and gel filtration using Sepharose 4B. which was equilibrated with physiological saline, subjected and collected the first fraction. This was again subjected to a gel filtration on Sephadex G-200, which was also equilibrated, and the second fraction collected. This was concentrated to 2 ml to give 135 µg of purified CEA.

b) manufacture before. inonoclonal anti-CEA antibodies

Using the purified CEA prepared above, 8 clones of anti-CEA anti · ', per-producing hybridoma were obtained. Mice were immunized using 30 µg of the purified CEA for each immunization. 1 × 10 ⁶ hybridomas were inoculated intraperitoneally into a female BALB / c mouse to which 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristan) had been administered intraperitoneally. The ascites were collected two weeks later. These were chromatographed with the aid of DEAE cellulose equilibrated with 0.01 M phosphate buffer of pH 7.0, a non-adsorbed fraction being obtained as a monoclonal anti-CEA antibody.

With the help of a test for the identification of antigen-recognizing sites, the antibodies obtained in be divided into three groups, d. H. 5 lots, 2 lots and 1 lot, each as an anti-CEA antibody [A]. [B] and [C] have been designated.

c) (i) Preparation of anti-CEA antibody sensitized test tubes

Polystyrene test tubes were each washed with PBS and 2 ml of anti-CEA antibody [A] (1 mg / ml), which had been prepared according to b) were added to the same and reacted at 56 ° C. for 20 minutes. After the reaction, the test tube was washed with PBS, whereby an anti-CEA antibody [A] sensitized Test glass was obtained.

c) (ii) Production of enzyme-labeled anti-CEA antibodies

5 mg of horseradish peroxidase (hereinafter abbreviated as HRPO) was dissolved in 1.0 ml of 0.3 M sodium bicarbonate buffer solved; 0.1 ml of a 1% strength ethanol solution of 1-fluoro-2,4-dinitrobenzene was added to this solution and the mixture reacted for 1 hour.

Subsequently, 1.0 ml of 0.06 M sodium periodate solution was added to the mixture and reacted for 3 minutes. Then 1.0 ml of 0.16 M ethylene glycol solution was added and the reaction was carried out for 1 hour. The reaction mixture was dialyzed against 0.01 M sodium carbonate solution at pH 9.5. To the resulting solution, 2 batches of anti-CEA antibodies, which had been prepared under b), were each added in an amount of 5 mg and reacted at room temperature for 3 hours. Then 5 mg of sodium borohydride were added and allowed to react overnight. The reaction mixtures were each dialyzed against 0.01 M PBS at pH 7.2, whereby HRPO-labeled anti-CEA antibodies were obtained.

d) Preparation of a reagent for CEA determination

HRPO-labeled anti-CEA antibody obtained according to c) (ii) became 1:50 with physiological saline solution diluted; 0.2 ml of the diluted antibody was added to each of the (anti-CEA antibodies) sensitized Test tubes obtained under c) (i) above are added and lyophilized, a reagent for CEA determination was obtained.

e) Determination of CEA

The purified CEA prepared as described above under a) became healthier with the serum Individuals diluted to a concentration of 100, 30, 10.3 and 1 ng / ml, respectively. Each 0.2 ml of the diluted CEA and 0.8 ml of distilled water were added simultaneously to the CEA determination reagent, which as above

hend described under d) was prepared, added and reacted with stirring for 20 minutes. To After the reaction, the test tube was washed with washing agent; then 3 ml of substrate solution, which 300 mg / dl of phloretinic acid and 0.01% hydrogen peroxide was added. The reaction was carried out for 10 minutes carried out; then 0.1 ml of 5% sodium sulfite was added to stop the enzyme reaction. in the The following was the fluorescence intensity at an extinction wavelength of 323 nm and a fluorescence 7.5 wavelength of 420 nm using a fluorophotometer. The standard curve obtained is in F i g. 6 reproduced.

Example 2

Reagent for HCG-ß- determination (chorionic gonadotropin determination)

a) Production of the HCG - /? - subunit

1 g HCG (2000 iu / mg) was dissolved in 2 ml 0.025 M phosphate buffer (pH 5.6); the solution was fractionated by means of chromatography on DEAE-Sephadex A-50 (3 g), which had previously been equilibrated with the same buffer. A fraction eluted with 0.05 M phosphate buffer (pH 5.6) was collected and dialyzed against distilled water to obtain a solution containing 308 mg of purified HCG, which was then lyophilized. 300 mg of the lyophilized HGC were dissolved in 10 ml of 10 M urea (pH 4.5) and ^{reacted at 40 °} C. for 1 hour. The reaction mixture was fractionated by means of chromatography on DEAE-Sephadex A-50 (2 g), which had previously been equilibrated with a solution containing 0.03 M glycine and 10 M urea. A fraction which was eluted with a solution of 0.2 M glycine, 1 M NaCl and 8 M urea was dialyzed against physiological saline, 147 mg of HCG - /? - subunit being obtained.

b) Production of anti-HCG - ^ - antibodies

11 lots of anti-HCG - ^ - antibodies were prepared according to the method of Example 2-b), with the Exception that rats from the Wistar strain were used as animals for the administration of antigen, instead of from BALB / c mice. The antigen-recognizing sites of these antibodies have been identified. The received Antibodies were divided into two groups, namely one group consisting of 8 batches of anti-HCG - /? - antibodies [A] and another group consisting of three lots of anti-HCGvi 'antibodies [B] duration.

c) Preparation of anti-HCG - /? - antibody sensitized beads

(insoluble carrier)

100 polyethylene balls were added to 100 ml of PBS containing 50 mg of anti-HCG - ^ - antibody [A1 prepared as described above under b), contains, added and reacted at 56 ° C. for 20 minutes. Then the Polyethylene spheres were washed to obtain anti-HCG - / # antibody [A] sensitized spheres.

d) Production of enzyme-labeled anti-HCG - /? - antibodies

The same procedure as described under 2-c- (ii) was repeated, using the anti-HCG - ^ - antibodies [B] prepared as described under b) above were used. After diluting the By volume with PBS, H RPO-labeled anti-HCG - /? - antibodies [B] were obtained.

e) Manufacture of the washing agent

9 g NaCl and 50 μ! Tween 20 were precisely weighed and placed in deionized water to give 100 ml of a solution dissolved. The solution was placed in a gas bottle to give a 10-fold concentrated Solution of the washing agent.

f) Production of the enzyme substrate

26.40 g of 5-aminosalicylic acid, 5434 g of monopotassium phosphate and 5.72 g of disodium phosphate were mixed and powdered in a mortar, 550 mg of the mixture was accurately weighed and placed in a glass bottle filled 1.5 ml of hydrogen peroxide (30% solution) was diluted with deionized water, i. H. to 100 ml filled up. 1 ml of the solution was filled into an ampoule which was then sealed by melting. on in this way the enzyme substrate was prepared

g) Production of a stopper for the enzyme reaction

Two grams of sodium azide was precisely weighed and dissolved in 100 ml of distilled water. 2 ml of the solution were filled into an ampoule which was then sealed by melting and a stopper for the Enzyme reaction formed.

h) Preparation of the reagent for the determination of HCG

A reagent for the determination of HCG was prepared by combining the materials as described under c] and d) were prepared in a vessel.

i) Determination of HCG

The following determination was made using the reagent for determination of HCG as above under h) described, was used and 0.4 ml of the HRPO-labeled anti-HCG - ^ - antibody [B.

ίο and a piece of an anti-HCG - /? - antibody [A] -sensitized ball in a test tube contained Danr 0.1 ml of the standard solution obtained by diluting HCG as described in the Japanese Pharmacopoeia with the serum of healthy people to a concentration of 10,000, 100, 10 and 1 miu / m, respectively was added and allowed to react for 15 minutes. After the reaction, the washing agent was used distilled water diluted tenfold, and the ball washed with the diluted washing agent

15 Thereupon 3 ml of the substrate solution, which was obtained by dissolving the entire enzyme substrate in 150 m distilled water was obtained, and the reaction was carried out for 30 minutes. Then became 0.025 ml of the stopper was added to stop the reaction. The absorbance of the reaction mixture was measured at 500 nm. The standard curve obtained is in FIG. 7 reproduced.

20 6 sheets of drawings

Claims (6)

Patent claims: 1. Reagent for immunological determination by the sandwich method, comprising insolubilized monoclonal antibodies and labeled monoclonal antibodies that are able to distinguish between different To distinguish antigen determinants of the antigen to be determined, characterized in that that the two antibodies are already in one before the determination is carried out single vessel. 2. Reagent according to claim 1, characterized in that balls or a vessel made of plastic or glass represent the carrier for the insolubilized antibody. 3. Reagent according to claim 1, characterized in that the labeling agent in the manufacture of labeled antibodies represents an enzyme or a fluorescent substance 4. Reagent according to claim 1, characterized in that the labeled antibody is in one Plastic vessel or glass vessel is located which is the carrier for the insolubilized antibody 5. Reagent according to claims 1 or 3, characterized in that the labeled antibody is lyophilized 6. Use of a reagent according to claims 1 to 5 for carrying out an immunological Determination by the sandwich method.