JPH03144366A - Immune measurement - Google Patents

Abstract

PURPOSE:To facilitate measurement with an avoidance of a delay phenomenon when an object to be measured exists in plenty by adding one from among an unbonded antibody identical to an antibody in a reagent and an unbonded antibody having a recognition section the same as the antibody. CONSTITUTION:At least one is added to a reaction mixed liquid from among an unbonded antibody identical to an antibody in a reagent for immune measurement and an unbonded antibody having a recognition part the same as the antibody. For example, a simultaneous reaction is caused between a solid bonded antibody with an antibody against an object to be measured immobilized to a solid phase, a labeled antibody having a label bonded to the antibody and an object to be measured in a sample and then, the antibody-bonded label bonded to a solid phase is measured to determine an amount of the object to be measured in the sample.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to an immunoassay method using a solid phase, in which an unbound antibody that is the same as an antibody in an immunoassay reagent and a recognition site that is the same as this antibody is used. It relates to an immunoassay method in which at least one of the unbound antibodies is added.

(Prior art) In immunoassays, enzyme immunoassays (hereinafter referred to as EIA
method) and radioimmunoassay method (hereinafter referred to as RIA method)
is widely used as a highly sensitive measurement method in the field of clinical testing. It is known that a phenomenon (hereinafter referred to as a "delay phenomenon") occurs that is only observed to a small extent in appearance.

Therefore, as a solution, a two-step sand-inch method that includes a washing operation in the middle, or diluting the sample containing the object to be measured and measuring it have been adopted. When the target substance to be measured is a sample that exists in a range of concentrations from low to high, it is difficult to use the sample as it is without pretreatment and obtain results.

(Problems to be Solved by the Invention) Increasing the number of solid-phase antibodies as a means of avoiding erroneous measurements due to the delay phenomenon has a limit in terms of the ability of the solid phase to bind antibodies, and increasing the number of labeled antibodies is This leads to increased non-specific adsorption to the solid phase and increases the blank value of the measurement. In addition, methods that measure at two points, the sample and the diluted sample, and the two-step sandwich method, which includes a washing operation in between, have been adopted, but these methods take longer to measure and require more effort such as washing. It is unavoidable that operations become complicated. Therefore, there has been a need for a method that can obtain accurate measurement results in a short time with simple processing operations.

Usually, the addition of antibodies as in the method of the present invention naturally causes a decrease in measurement sensitivity, but the EIA method and R
The IA method has high sensitivity, so even if it causes a slight decrease in sensitivity, it is a method that can be used to measure a very small amount of the object to be measured. Although this method lowers the measurement sensitivity for very small amounts of the measurement target, it is possible to measure the measurement target over a wide range of concentrations, from low to high concentrations, and the conventional complicated processing operations can be avoided.

(Means for Solving the Problems) The present inventors have discovered that in immunoassay methods using solid phases, especially p:IA method and RIA method, even when a large amount of the target substance is present in the immunoassay reagent, We have discovered that by adding at least one of an unbound antibody that is the same as the antibody and an unbound antibody that has the same recognition site as this antibody, the delay phenomenon can be avoided and measurement can be easily performed, and the present invention completed.

In the EIA method and the RIA method, the one-step method includes, for example, a solid phase-bound antibody in which an antibody against the analyte is immobilized on a solid phase, a labeled antibody in which the antibody and a label are bound, and the analyte in the sample. This method determines the amount of the target substance in the sample by simultaneously reacting and then measuring the antibody-bound labeled substance bound to the solid phase.

In the EIA method or RIA method, the present invention further adds to the reaction mixture at least one of an unbound antibody that is the same as the antibody in the immunoassay reagent and an unbound antibody that has the same recognition site as this antibody. There is a particular thing.

The antibodies used in the present invention can be those obtained according to known methods. For example, rabbits, goats,
The antigen or its derivative to be measured is administered to warm-blooded animals such as horses, guinea pigs, and chickens at 0.3 to 1 kg per 1 kg of body weight.
Antibodies can be formed in the animal's body by subcutaneously injecting it with an adjuvant into the back, foot pad, thigh muscle, etc. once to several times. The obtained antibodies are digested with proteolytic enzymes such as pepsin to F (ab')t, Fab', F
It can also be used after being decomposed into ab and the like.

On the other hand, it can also be obtained as a monoclonal antibody. In that case, the above-mentioned object to be measured is injected into the mouse's peritoneal cavity several times along with an adjuvant. If the object to be measured has a low molecular weight, it can be combined with a suitable polymeric substance such as a protein to form an antigen. Thereafter, the enlarged lung cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like, and among the fused cells, those that produce the antibody are grown as monoclonal cells by cloning.

A monoclonal antibody obtained by growing the monoclonal cells thus obtained in the peritoneal cavity of a mouse can be used.

In the present invention, the amount of unbound antibody to be added can be appropriately determined depending on the expected amount of the target substance to be measured, and when using IgC as the unbound antibody, it should be in the range of 0.1 ng to 5 mg per 1 ml of the measurement sample. Can be done. For example, if the measurement target is AFP, 1 mff1 of measurement sample
ng~2.5mg, CEA 0.1ng~2+mg
, Q for CA 19-9, ranges from lng to 300 pg, etc. Furthermore, when using a fragment obtained by enzymatically decomposing IgG, the amount of the above-mentioned antibody to be added can be determined depending on the molecular weight.

Solid phases used in the present invention include polystyrene beads, nylon beads, microplates, and various magnetic particles. The solid phase-bound antibody can be physically or chemically bound to the solid phase by a known method ("Enzyme Immunoassay" 2nd edition, Igaku Shoin, (see 1987).

In the EIA method, an enzyme-labeled antibody can be produced by chemically bonding the antibody produced above with an enzyme. As the binding method, periodic acid method, maleimide method, etc. can be used (Proteins, Nucleic Acids, Enzymes, Separate Volume No.

31.37-45 (1985)). The enzyme used here can be selected as appropriate for the substrate, such as peroxidase, alkaline phosphatase, β
- Galactosidase etc. can be used.

For information on substrates on which enzymes act and measurement methods using these substrates, see "Enzyme immunoassay" Igakushoin (1987 edition)
It can be carried out according to the substrate and method described in .

In the RrA method, +251 is used instead of the enzyme in the EIA method.
This can be done by labeling with radioactive isotopes such as. For example, a commercially available Polton-Hunter reagent can be used to label antibodies. Furthermore, a scintillation counter is used for measurement.

Examples of objects to be measured in the present invention include drugs, hormones, and trace components derived from various diseases contained in serum or urine.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

1i! i, IL Anti-human α-fetoprotein (AF
P) Preparation of mouse antibody particles Magnetic particles with a particle size of 0.3 μm (manufactured by Nippon Paint ■)
10 mg was placed in a tube, 200 μl of ethylene glycol was added, and after stirring, ultrasonication was performed to disperse the particles. The particles were collected at one end of the tube using a magnet and the ethylene glycol in the supernatant was removed. Subsequently, 1 mj2 of distilled water was added, and after stirring, the supernatant distilled water was removed again using a magnet. This operation was repeated 7-8 times to wash the particles. An anti-human AFP mouse IgG solution (0.1M acetate buffer pH) prepared to IB/mf was added to the washed particles.
5,5) Add 200 μl and stir gently for 1 night at 4°C. This was then washed with distilled water using the method described above, and
% bovine serum albumin (BSA) solution, 0.15 M
It was suspended in 50mM 1-Lis buffer pH 7.2 containing NaCl and gently stirred at room temperature for 2 hours. Next, this was washed four times with 2% BSA solution using the above method, and 2% BSA solution was used.
SA, 0.15M NaC1! ,,1mM MgC
50 mjl containing 1,z, Q, 1% NaN! Suspended in Tris buffer pH 7.2,
)) Mouse antibody particles were prepared.

im Preparation of anti-human AFP mouse Fab'-ALP conjugate Dissolve 10 mg of anti-human AFP IgC in 3 ml of 0.1 MM phosphate buffer pH 5.5 and give 0.067 mg (7)
Hepsin was added and stirred at 37°C for 1 hour. 0.1
After adjusting the pH to 7.0 with N NaOH aqueous solution, Im
0.1 M phosphate buffer pH 6.3 containing MEDTA
anti-human AFP mouse Ig by gel filtration using the above buffer.
3B of G F (ab')z was obtained.

This solution was concentrated to 1 mf at P E 020000, 111 μl of an aqueous solution of 0.1 M 2-mercaptoethylamine hydrochloride was added, and the mixture was left at 37° C. for 3 hours. This solution was added to a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer pH 6.3, and 2-mercaptoethylamine was desalted to obtain Fab' 2■.

In addition, alkaline phosphatase (manufactured by Boehringer,
ALP) 5 mg to 0.1 M phosphoric acid pH 7.0 Ni? 5 μl of N-(r-maleimidophyloxy)succinimide (CMBS) dissolved in 100 μl of DMF was added, and the mixture was left at room temperature for 2 hours. This solution is 0.1
Add to a Sephadex G-25 column equilibrated with M phosphate buffer pH 7.0, remove unreacted CMBS,
4 pieces of ALP-GMB were obtained.

4 mg of this ALP-GMB and Fab'1.3 ■ obtained by the above method were mixed and allowed to stand at room temperature for 1 hour.

The obtained Fab'-ALP complex was added to a Mono S column equilibrated with 50 mM acetate buffer pH 5.0,
Apply a concentration gradient of O-IM NaCl F ab':A
LP=1:1 conjugate was eluted to obtain anti-human AFP mouse Fab'-ALP conjugate 2■.

Implementation ■Preparation of luminescent substrate 40■3-(2'-spiroadamancune)4-methoxy-4-(3"-phosphorinoxy)phenyl-1,
2-Dioxetane disodium (hereinafter referred to as AMPPD) in 0.1 containing 200ml of 1mM MgCft
M) Dissolved in Squirrel buffer pH 9.8 and stirred well. Furthermore, 200 μg of poly[vinylbenzyl (penzylmethyl-ammonium chloride)) (BDMQ)]
ynj! was dissolved in the above buffer solution and stirred well. These 2
00ml of 0.2mg/mj2AMPPD and 200ml
was mixed with 0.4 mg/mj2BDMQ and stirred well to prepare a luminescent substrate solution.

Measurement of human AFP Take 50 μl of human AFP sample at each concentration into a plastic tube and prepare anti-human AFP mouse Fab'-ALP conjugate solution (Example 2) at 50 μl/ml.
was added, and after mixing, the mixture was allowed to stand at room temperature for 30 minutes. Furthermore, this solution was added with anti-human AFP mouse IgG2.5 used for particle sensitization.
500 μl of 0.02% anti-human AFP mouse IgG sensitized particle suspension containing μg was added, and after mixing, the mixture was allowed to stand at room temperature for 15 minutes. The particles in this magnetic particle suspension were collected at one end of the tube using a magnet and the supernatant was removed. Subsequently, 1 mf of physiological saline was added, the suspension was resuspended, and the supernatant physiological saline was removed using a magnet again. This operation was repeated three more times to wash the particles. Add 300 μl of the gaseous solution to the particles, stir, and let stand at room temperature for 5 minutes.
Luminescence measurement was performed using C1inC11niLu manufactured by RThold. (5 seconds integration) The results are shown in Figure 1.

! Human AFP measurement by colorimetric method Using human AFP solutions of various concentrations, imuzine-AFP was measured.
A colorimetric measurement curve was obtained using a reagent (manufactured by Fujirebio). The results are shown in Figure 1.

(Effects of the Invention) The present invention provides an unbound antibody that is the same as an antibody in an immunoassay reagent and an unbound antibody that has the same recognition site as this antibody in immunoassay methods such as EIA and RIA using a solid phase. By adding at least one of the antibodies, it has a wide measurement range and enables measurement without delay even in the presence of a large amount of measurement target, making it an extremely useful measurement method.

[Brief explanation of the drawing]

Figure 1 shows the AFP measurement song for each concentration using the EIA colorimetric method! 191 (-·-) and each concentration AFP measurement curve (-0-) by the chemiluminescent EIA method of the present invention.

Claims (1)

[Claims] (1) In an immunoassay using a solid phase, at least one of an unbound antibody that is the same as the antibody in the immunoassay reagent and an unbound antibody that has the same recognition site as this antibody is added. The measuring method.