JPS63117264A - Measurement of antigen - Google Patents

Abstract

PURPOSE:To enable highly sensitive measurement, by a method wherein an antigen (Ag) to be measured is sandwiched in a liquid phase with an antibody (Ab1) against the antigen and an labeled antibody (Ab2) different from the antibody to form an Ab1-Ag-Ab2 compound body and then, the Ab2 is separated using a second antibody of the Ab1. CONSTITUTION:First, an immunological reaction is caused among an object to be inspected (sample to be inspected) containing an antigen (Ag) to be measured, an antibody (Ab1) and an antibody (Ab2) so that the antigen (Ag) is sandwiched between both the antibodies to form an immunological compound body (Ab2-Ag-Ab2 compound body). Then, a second antibody of the antibody (Ab1) undergoes an immunological reaction with the compound body likewise to turn the immunological compound body to a sediment. The antibody (Ab2) existing isolated in a reaction system, namely, a free F is separated from the resulting bound (B) to measure the label activity of the B or F. The object being inspected herein used is a clinical sample such as blood in the form of serum or plasma. Thus, a trace of a substance to be inspected (antigen) contained in said clinical sample can be measured at a high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunoassay using the Sandwich method, and more specifically to a novel antigen measurement method that retains the advantages of the Sandwich method and enables more sensitive measurement. .

The Sanderutsch method, which is commonly used in immunoassays such as radioimmunoassay (RI△) and enzyme immunoassay (EIA), is a heterogeneous measurement method using immobilized antibodies and labeled antibodies. Compared to the reaction method, this method has the advantage of not requiring a labeled antigen. This is a particularly significant advantage in fields where antigens are difficult to obtain or where antigens are unstable and difficult to label.

However, the detection sensitivity of the Sand Inch method is not sufficient, and it is a major drawback, especially in the field of clinical chemistry, where components present in body fluids at extremely low concentrations are pathologically important. Therefore, it is desired to develop a new measurement method that takes advantage of these characteristics. for example,
Cytokinins, such as lymphokines and monokines, whose measurement in clinical samples is attracting attention for research on various immune deficiency diseases and abnormal immune responses, as well as for clinical diagnosis, particularly meet such requirements. This is one of the strong fields, and the Sandermanch method mentioned above cannot be used due to sensitivity.

Although such measurements are inferior in operability and accuracy, and it is necessary to always consider the presence of components that may interfere with the measured values, the current situation is that we have no choice but to rely on bioassays (biological quantitative methods).

Problems to be Solved by the Invention The purpose of the present invention is to retain the advantages of the existing Sand Deutsch method described above, eliminate its disadvantages, and easily and conveniently obtain a target sample with high sensitivity and precision. The object of the present invention is to provide a measurement method using a new immunoassay method that can perform measurements.

Means for Solving the Problems According to the present invention, in an immunoassay method, an antigen (Ag) to be measured is mixed with an antibody against the antigen (Ab+>) and a labeled same antibody (Ab2) of a different species than the antibody. ) in a liquid phase to form an Ab+-Ag Ab2 complex, and then a second Ab+ antibody is used to separate the complex from At)2 present in a free state. A method for measuring an antigen is provided.

In the present invention, the antigen to be measured (Ac+> is not limited in any way as long as it has reactivity with antibodies, and refers to antigens in a broad sense including incomplete antigens such as Habten. In particular, it is difficult to label them. Antigens of associated species and antigens of species that require extremely sensitive and highly accurate measurement, such as interferon, interleukin 1.2.3 or 4, lymphokines such as tumor necrosis factor, colony stimulating factor, monokines, etc. Preferred examples include various cytokines.

The antibody (Ab1) against the above antigen is not particularly limited as long as it recognizes the antigen (shows binding properties), and may be any of polyclonal antibodies, monoclonal antibodies, and purified products according to conventional methods. Such antibodies and their preparation methods are known per se, or can be carried out according to known methods.

The same labeled antibody (A
b2) is labeled and an antibody (Ab+
) is not particularly limited as long as it is an antibody from a different species of animal that has a different antigenicity;
It may have the same definition as .

Labeling to obtain the antibody (Ab2) can be carried out according to a conventional method [for example, Ann.

CIin, 3 iochem, , 2 u, 310
(1984): J. Biol. Chem., 254.9.
349-9351 (1979): Nature, 19
4,495<1962); Fluorescent antibody method, medical chemistry experiment course No.

4, 263-270: ACta, EndOcrin
ol.

5upp1. ．． 168.206 (1972); P
roc.

Nat, Acad, 3ci, USA, 57.71
3 (1967) etc.].

The labeling agent used in the above-mentioned labeling may be any of the various commonly used labeling agents, such as radioactive substances, enzyme labeling substances, fluorescent substances, and the like. The radioactive substance used as the labeling agent includes radioactive iodine such as 1251, and the fluorescent substance includes fluorescein isothiocyanate (
FITC'), tetramethylrhodamine isothiocyanate (TRITC>), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, dichlorotriazine fluorescein (DT
AF), and enzyme labeling substances such as peroxidase (POX), alkaline phosphatase,
Microperoxidase, β-galactosidase, urease, chymotrypsinogen, procarboxypeptidase, glyceraldehyde-3-phosphorus m1lJR hydrogenase, amylase, phosphorylase, D-nase, P
-nase, etc. can be exemplified.

The second antibody of the antibody (Ab+), that is, the anti-antibody, is known per se and may be appropriately selected depending on the animal species of the adopted antibody (Ab+). can be adopted successfully.

The basic operations of the assay method of the present invention can be carried out in accordance with ordinary immunoassay methods, and for example, the assay method described in detail below can be adopted.

That is, first, a sample (
Test sample), antibody (Abl), and antibody (Ab2) are immunoreacted in the liquid phase, and the antigen (Ag) is sandwiched between both antibodies to form an immune complex (AbIAg A2 complex).
to form. Next, a second antibody of the antibody (Abl) is immunoreacted in the same manner, and the above immune complex is precipitated, and the bound (B) and the antibody (Ab2) present in a free state in the reaction system, that is, free. The method of the present invention is carried out by separating (F) and measuring the labeling activity of either B or F.

In the above, various specimens can be used, such as blood in serum or plasma state, cell tissue fluid, lymph fluid, thymic fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, bone marrow fluid,
Examples include clinical samples such as various body fluids such as saliva, and according to the present invention, trace amounts of test substances (antigens) contained in such clinical samples can be measured.

As the solvent used in the measurement system in the above, any of the usual solvents that do not adversely affect the reaction can be used, such as citrate buffer, phosphate buffer, Tris-HCl buffer, acetate buffer, etc. It is preferable to use a buffer solution having a DH of about 5.0 to 9.0. In addition, in the present invention,
About 1 to 10 v/v% of serum (not containing the measurement target) and/or about 0.1 to 0.5 M
It is preferable to include a certain amount of NaGQ because it is more consistent with the purpose of the present invention.

In addition, in the method of the present invention, the reaction order of the sample containing the antigen (8g) and the antibody (Ab+) and antibody (Ab2) is as follows:
There is no particular limitation, and the reactions may be carried out simultaneously, but it is preferable to react the sample and the antibody (Ab2) and then react the antibody (Ab+), as this enables a more sensitive measurement.

The immunoreaction conditions during the measurement are not particularly limited and can be the same as those used in ordinary measurement methods of this type. Generally, the reaction may be carried out at a temperature of 45°C or less, preferably about 4 to 40°C, and at a rate of about 1 to 10 v/v degrees.

In addition, B/F using the second antibody of the above antibody (Ab+)
When separating, it is more preferable to have polyethylene glycol present in the reaction system, and the separation means can be conventional methods such as centrifugation, filtration, and washing.

Furthermore, the measurement of labeling activity can be carried out according to known methods depending on the type of labeling substance used.

Thus, according to the method of the present invention, even when a sample containing a trace amount of a test substance such as a clinical sample is used as a sample, the test substance in the sample can be quantified with high accuracy and sensitivity, and with a simple operation. be able to.

A particularly convenient method for carrying out the method of the present invention is a method that utilizes a kit for the above-mentioned measurements. The kit includes the antibody (Ab2), the second antibody thereof, and the antibody (Ab2).
It is important to contain. The antibody reagent may also contain stabilizers and/or stabilizers such as glycerol and live serum proteins.
Or a preservative can be added. This antibody reagent is
Freeze-drying may be performed, and the kit may contain a water-soluble or water-miscible solvent. Furthermore, the antibody reagent may contain a buffer solution to maintain a constant II of the reconstituted reagent system and/or a preservative and/or stabilizer to prevent the sample from deteriorating. Although the buffer is not an essential component of the kit reagent, it is preferable to use one that gives a p day of about 5.0 to 9.0 when carrying out the assay method of the present invention. The reconstitution agent preferably contains water, but part or all of the water may be replaced by a water-miscible solvent. Examples of the water-miscible solvent include well-known examples such as glycerin, alcohols, and glycol ethers.

Effects of the Invention According to the present invention, antigens can be measured simply and with extremely high sensitivity and accuracy while retaining the advantages of the existing sandwich method, and in particular can be used to measure antigens that are contained in trace amounts in clinical samples. It is slow and useful for measurements, etc.

EXAMPLES Reference examples and examples will be given below to explain the present invention in more detail.

Reference Example 1 Human IL produced according to genetic recombination technology used as standard for preparation of labeled antibody (Ab2)
-1β [Biochemistry, Yudan.

8, D840 (1986); EPO 187991] to BALB/C mice with complete Freund's adjuvant! ! ! ! Administered intracavitally. Immunization was performed by administering the same amount twice with incomplete adjuvant every 3 to 4 weeks. 3-4 weeks later, final immunization: 30μq
A saline solution of human IL-1β was administered intravenously. 3 to 4 days after the final immunization, the conventional method (Method in En
zymoloqy.

73, pp. 3 (1981), etc.], cell fusion was performed.

M-cell fusion is performed between the immunized tile cells and myeloma cells (P3
U1, Currnet Topics in M icr
obiology and ImmunoloclV
, 81, 1-7 (1978)) at a ratio of 10:1, polyethylene glycol (PEG-4000>
This was done using

After selecting hybridomas in HAT medium, the supernatant was transferred to a 96-well microplate coated with the human IL-1β and peroxidase-labeled goat anti-mouse globulin antibody [E, Ly. Love (E.

A strain producing antibodies against the target human l-1β was detected by an enzyme immunoassay method using the following enzyme-linked immunosorbent assay (manufactured by Y. Lab.).

Cloning was repeated using the limiting dilution method to obtain the desired antibody-producing clone.

This monoclonal hybridoma was cultured intraperitoneally in mice, 1q of ascites was collected, and IcxG purification kit (MOP
It was purified to iQG+ using S kit, Bio-Rat (manufactured by 3io-Rad) to obtain the desired antibody against human IL-1β.

Using 2μq of IQG) obtained above, the iodoken method (
Fraker, P. J., and 5peck, J.
,C.

(1978), Biochcm, 3iophys
, Res.

Commun., 80, 849-857), followed by 0.02M phosphoric acid, 0.02M phosphoric acid, IMNaCQ
, 5mM EDTA, 0.1% BSA and 0.01%
Gel filtration using Bio-Rat P-30 equilibrated with thimerosal buffer (pH 7.4) [Eluent: same buffer, flow rate: 0.5 min/min, column size: 1 cmx
30 cm) L/, the active part is 10,000 Cpm with the same buffer.
/200tlQI,l: was prepared to obtain the desired isotope-labeled antibody. This was stored at 4°C.

Reference Example 2 Preparation of Antibody (Ab+) 50 μq of human IL-1B solution 14 in PBS used as a standard was mixed with an equal amount of complete Freund's adjuvant, and rabbits were immunized at multiple points. Thereafter, the animals were immunized 10 times every 2 to 3 weeks, and whole blood was collected one week after the final immunization.
The desired antibody (antiserum) was obtained. In addition, an incomplete adjuvant was used for the second and subsequent immunizations.

Then, the obtained antibody was mixed with 0.02M phosphoric acid and 0.1M phosphoric acid.
NaCQ, 5mM EDTA, 0.1% BSA and 0.01% thimerosal buffer (pH 7.40
It was diluted 0 times and stored at 4°C.

Reference Example 3 Preparation of second antibody of antibody (Ab+) To a 1mM mf2 PBS solution of rabbit γ-globulin, an equal volume of Freund's complete adjuvant was added to make a suspension, and the suspension was given to a goat 2+1112/1 time. Subcutaneous immunization was performed every two weeks. After six immunizations, blood was collected to obtain the desired antibody (antiserum).

Then, the obtained antibody was mixed with 0.02M phosphoric acid and 0.1M phosphoric acid.
NaCQ, 5mM EDTA, 0.1% BSA and 0.01% thimerosal buffer (pH 7.40
It was diluted twice and stored at 4°C.

Reference Example 4 Preparation of human hlL-2 Human IL-2 was produced using Escherichia coli according to the methods (genetic recombination technology) shown in (a) to (c) below.

B. Plasmid containing DNA sequence encoding IL-2 1) Construction of tl'pI L-2D8 Human tonsil mRNA
Plasmid pHlG5-3 containing IL-2C DNA cloned from A.
t,, 3iochem.

Biophys, Res, Comm,, 115
．． 1040-1047 (1983), size approx. 4.4
kbp) with the restriction enzymes HindIII and pvul[
A DNA fragment of approximately 1.2 kilobase pairs (kbp) containing IL-2c DNA was isolated by agarose gel electrophoresis. The isolated DNA fragment was digested with the restriction enzyme HaiA I, and a DNA fragment (approximately 0.8 kbp) encoding IL-2 was isolated by agarose gel electrophoresis. Synthetic oligonucleotide [5'-TG
CCAT T AT -3' and 5' -CGA
TAA-GGCA-3' (only the former oligonucleotide has its 5' end phosphorylated in advance)] was converted into T4D
The DNA fragments obtained by ligation using NA ligase were purified by agarose gel electrophoresis.

The IL-2-encoding DNA fragment bound to this synthetic oligonucleotide was pre-cleaved with restriction enzyme C1aI into plasmid I) AT153 (Nature, 28
3.216 (1980), size approximately 3.7kbp)
was ligated to the CIaI cleavage site of D AT mI using D4DNΔ ligase, the resulting conjugate was used to transform E. coli strain H8101, and the desired plasmid D AT mI was transformed from the transformed strain.
A transformed E. coli HB101/p AT ml L2-2 harboring L2-2 (approximately 4.5 kbl in size) was obtained.

The obtained E. coli HB101/l) AT mI L2-
From 2, plasmid pΔTmIL2-2 was isolated, and after cutting it with restriction enzyme C1aI, a DNA fragment (approximately o, a kbp>) containing the region encoding the IL-2M gene was isolated by agarose gel electrophoresis. I let go.

On the other hand, vector plasmid t) TMl (Imamotobotono, Metabolism Vol. 22, p. 289 (1985), size approximately 4.7 kb
t)) was digested with the restriction enzyme ClaI Te, the DNA fragment encoding the IL-21 gene obtained above was ligated to this cleavage site using a T4 DNA ligator, and the ligated product was used to infect E. coli H.
B101 was transformed and spread on LB agar medium containing 50 μg/mQ ampicillin, and the ampicillin-resistant strains that appeared were tested using the boiling method (Engineering, Maniatis).
etal, Molecular Cloning,
p 366 (Cold Sprink + Harbo
A plasmid was isolated using the plasmid plasmid (E. coli H
B101/D trl) I L2D8) was obtained.

b. Human IL-2 expression plasmid ptrpIL2D8Δ
Construction of the plasmid ptrpIL from the transformed strain obtained in step (a) above.
2D8 was extracted and treated with restriction enzyme 3tB and Hind.
A DNA fragment obtained by cutting with III and removing the 3' end non-gene encoding portion was isolated by agarose gel electrophoresis.

Next, this DNA fragment was converted into DNA volimera=t”I(
Klenow fragment) and further T4DN
Self-conjugated with Aligapi, and the conjugate was used to bind E. coli HB1.
01 to transform the desired plasmid 1) trp
Transformed E. coli l-lB101/p containing IL2D8Δ
trp IL2D8Δ was obtained.

C. Production of human ■[-2] The thus obtained transformed strain was treated with ampicillin 5C)
After culturing overnight at 37°C in LB medium 20 OmQ containing zg/ma, the cells were inoculated into M9 medium 10Q containing 1% casamino acids and 50 μg/ITIG ampicillin, and incubated at 37°C for 8
．． After culturing for 5 hours, the bacterial cells were collected by centrifugation.

The cells were suspended in physiological saline, the supernatant was removed by centrifugation, and the precipitate was suspended in Tris-HCl buffer (pH 8.0>) containing 25% sucrose and 0.01% sodium azide. and add 50mM of 10mg/mi2 lysozyme to this.
It was dissolved in 10 mM Tris-HCl buffer containing EDTA and added.

The above solution was disrupted by ultrasonication on ice and further centrifuged to obtain a precipitate. Further, the precipitate was suspended in a Tris-HCl buffer containing 1M sodium chloride, disrupted by ultrasonication, and then centrifuged to obtain a precipitate.

After repeating the above operation two more times, the resulting precipitate was subjected to gel chromatography and reverse phase chromatography using Ultrogel AcA34 (manufactured by LBK), and 5DS-P
Recombinant human IL-2 (
rhuman IL-2) was obtained.

Reference Example 5 Preparation of Antibody (Ab2) Using the human IL-2 obtained in Reference Example 4 above, a desired isotope-labeled mouse antibody against human IL-2 was obtained according to Reference Example 1.

Reference Example 6 Preparation of Antibody (Ab1) A desired goat antibody against human IL-2 was prepared according to Reference Example 2 using the human IL-2 obtained in Reference Example 4 above.

Example 1 Measurement of IL-1β Human IL-1β described in Reference Example 1 was used as a standard,
0.02M phosphoric acid, O,IM NaCQ, 5mM
Serial dilution series (200μQ/
Tube) was created.

Into this tube, add 20% of the isotope-labeled mouse antibody (Ab2) against human IL-1β obtained in Reference Example 1.
0 μQ/100,000 cpm was added and reacted at 37° C. for 2 hours. Subsequently, the human IL obtained in Reference Example 2 was added to the reaction mixture.
Rabbit antibody against -1β (Ab+ > (7) 1001
1Q was added and reacted at 37° cr for 1 hour. Finally, add 100 μQ of the A7 antibody [second antibody (Ab+ > second antibody)] against the rabbit antibody obtained in Reference Example 3 and 6% to the reaction mixture.
Polyethylene glycol (dissolved in PBS) 50
Add 0μQ and incubate for 30 minutes at room temperature.
Centrifugation was performed at 3000 rl) m for 15 minutes at 4°C to separate bound (B) and unbound labeled antibody (F) to obtain a precipitate (B).

To the obtained precipitate, 0.05% Tween-20 PBS solution was added and mixed, centrifuged under the same conditions, and the above precipitate was washed, and the radioactivity of the precipitate thus obtained was determined by - Measured with a counter.

The results are shown in Figure 1.

In Figure 1, the horizontal axis is IL-1β temperature (nMml2)
, the vertical axis indicates radioactivity (cpm).

Example 2 Measurement of 1-2 Human IL used as a standard in Example 1
The human IL-2 obtained in Reference Example 4 was used instead of -1β, the mouse antibody against the isotoped deceased IL-2 obtained in Reference Example 5 was used as the antibody (Ab2), and the antibody (Ab+) A standard curve was prepared in the same manner except that the rabbit antibody against human IL-2 obtained in Reference Example 6 was used.

The results are shown in FIG. 2 in the same manner as in FIG.

Comparative Example 1 Measurement of IL-1β by conventional sandwich radioimmunoassay method The same standard serial dilution series as in Example 1 was used.

One polystyrene bead (composite antibody) adsorbed with the same antibody (Ab+) as in Example 1 was added to this tube and reacted under the same conditions (37°C, 2 hours).

After removing the reaction solution and washing the beads three times with 0.05% Tween-20 in PBS solution 11TII2, the same antibody (Ab2) as in Example 1 was added and the beads were incubated under the same conditions (200tlQ/
The reaction was carried out at 100,000 C1)m, 37°C, 1 hour). After washing the beads in the same manner, bound radioactivity was measured in the same manner.

The obtained results are shown as line (1) in FIG. 3 in the same manner as in FIG. In addition, in FIG. 3, the results of the method of the present invention obtained in Example 1 are also shown as a line (2).

From FIG. 3, according to the method of the present invention, the variation (eV)
is smaller, and the measurement sensitivity is more than 10 times better.
It can be seen that highly sensitive and highly accurate measurements are possible.

[Brief explanation of the drawing]

FIG. 1 shows a standard curve for human IL-1β measurement according to Example 1 of the present invention. FIG. 2 shows a standard curve for human IL-2 measurement according to Example 2 of the present invention. FIG. 3 shows a comparison of standard curves between the method of the present invention and the conventional method. (That's all) ・Mi・ru′

Claims (1)

[Claims] (1) In the immunoassay method, the antigen to be measured (Ag
) is sandwiched in a liquid phase using an antibody (Ab_1) against the antigen and a labeled same antibody (Ab_2) of a different species than the antibody to form an Ab_1-Ag-Ab_2 complex, and then a second antibody of Ab_1 A method for measuring an antigen, which comprises separating the complex from Ab_2 existing in a free state using a method for measuring an antigen.