JPS60127462A - Enzymatic immune measuring method - Google Patents

Abstract

PURPOSE:To prevent the blocking of a light path due to solid phase particles in the spectroscopic measurement of a specimen, in an enzymatic immune measuring method, by making the specific gravity of an enzymatic reaction solution lower than that of a solid phase system containing a carrier while making the specific gravity of said solution higher than that of the solid phase system after enzymatic reaction is stopped. CONSTITUTION:A reagent such as an antibody (antigen) is supported by emulsion polymerization latex of a polymerizable monomer, gell particles formed by suspension polymerization or a solid phase particulate carrier such as glass or alumina and a specimen containing an antigen (antibody) is reacted with a definite amount of an antigen (antibody) labeled with enzyme to allow the enzyme substrate to react with the reaction product of a substrate while a reagent emitting fluorescence or colored light is added to measure fluorescence or absorbancy. In this case, a substance selected from inorg. salts, an acid, a base, polyhydric alcohol or sugars is added to the reaction system so as to make the specific gravity of the solution, after reaction is finished, higher than that of the solid phase carrier and solid phase particles are allowed to float to prevent the optical measurement of the liquid phase from receiving hindrance. By this method, the accurate measurement of an antigen (antibody) is enabled.

Description

[Detailed description of the invention] Technical field The present invention relates to an enzyme immunoassay using an in-phase method.

Prior Art For the detection, identification, and quantification of biologically related substances such as amino acid-related substances such as peptide hormones, carbohydrates and their metabolites; lipids; and nucleotides, enzyme immunoassay using a solid-phase method using antigen-antibody reactions is used. used. The Sanderch method is known as one of the methods for quantifying a substance to be measured using this in-phase method. For example, when quantifying insulin using this sandwich method, first prepare beads made of ABS, polystyrene, etc. as a carrier, and have immunoglobulin G (IgG), which is an antibody for insulin, supported on the surface of the beads. A homophasized antibody is prepared. This in-phase antibody is added to an insulin solution of known concentration and immobilized on a carrier using insulin as an antigen to prepare a first solid phase thread. On the other hand.

IgG, which serves as an insulin antibody, is labeled with an enzyme such as HRP (peroxidase) in advance as a labeled antibody. When this is added to the first solid phase system, this labeled antibody binds to insulin, and the second (
The speed is 1 and it is completed. Insulin, which is a substance to be measured, is sandwiched between IgG, which is an antibody. ABTS (2・2′-azinorbis-(
When α is added to enzyme 8 such as 3-ethylbenzothiazoline-6-phonic acid), the substrate undergoes an enzymatic reaction. By measuring the oD value of the reaction solution of the resulting reaction product, the insulin concentration in the substance to be measured can be determined in advance from a calibration curve showing the relationship between the OD value and the insulin concentration. In this way, the specific substance contained in the unknown sample can be quantified. To measure the oD value, it is necessary to transfer a portion of the enzyme reaction completed solution to a cuvette (Se/L/). The carrier easily enters the cuvette when This mixed carrier blocks the optical path, causing measurement errors and inability to measure. Removing contaminant carriers from cuvettes is tedious. To this end, it is conceivable to keep the amount of enzyme-labeled antibody constant and measure the amount of enzyme that is not bound to the carrier, but this requires extra effort and is inefficient. In addition to the Sand-Deutsch method, a competitive reaction method is also used. In this case as well, interference with the optical path by the solid phase during measurement poses a problem, as described above. In carrying out the solid-phase method, it would be convenient if the diameter of the carrier could be reduced to increase the contact area with the reaction solution and increase the reaction rate in order to shorten and simplify the reaction time.

However, carriers with a small particle size can more easily enter the cuvette than carriers of normal size.

Therefore, fine particles must be removed using a centrifuge, which is inconvenient. In order to minimize interference with the optical path,
It may also be considered to use Acrylamide Ge M, which has excellent transparency, as a carrier. but.

Because a chemical bonding method must be used to immobilize the antigen or antibody, the operation is extremely inconvenient.Furthermore, since acrylamide is used as the material only from the viewpoint of transparency, it is not suitable as a material for the carrier. Not necessarily appropriate.

OBJECTS OF THE INVENTION An object of the present invention is to provide an enzyme immunoassay method using a solid phase method in which a carrier does not enter a cuvette and obstruct the optical path during spectroscopic measurement of a sample, thereby preventing the measured values from being distorted. The object of the present invention is to provide an enzyme immunoassay method that uses Ia small particles as a carrier to increase reaction efficiency and enables the quantification of a substance to be measured by a spectroscopic method without being affected by the in-phase system. Particularly.

Summary of the Invention In the enzyme immunoassay method using the in-phase method of the present invention, the specific gravity of the enzyme reaction solution is lower than the specific gravity of the in-phase system containing the carrier, and the specific gravity of the solution after the enzyme reaction has stopped is also greater than the specific gravity of the in-phase system. Thus, the above objective is achieved.

The method of the present invention can be applied to enzyme immunoassays using solid-phase methods such as the sandwich method and competitive reaction method. In the Sand-Deutsch method, an antibody or antigen is supported on a carrier, and a substance to be measured, which is an antigen or antibody that specifically reacts with the antibody or antigen, is detected and identified.

Or an in-phase method for quantitative determination, which includes (1) binding the analyte to the antibody or antigen supported on the carrier to form a first solid phase system; (3) forming a second solid phase system by binding an antibody or antigen labeled with a reactive enzyme to a substance to be measured on a first solid phase thread; (3) attaching a substrate to be subjected to an enzymatic reaction by the enzyme to a second solid phase system; The process of reacting with

and (4) spectroscopically detecting, identifying, or quantifying the analyte through the enzymatic reaction product.

The present invention will be explained below using the Sanderuch method as an example.

The phase used in the present invention may be any material as long as it can be formed into any shape, such as a bead shape, and can support the antigen or antibody by physical adsorption or the like.

Not particularly limited. An example of this is ABS.

There are thermoplastic resins such as folystyrene. This carrier is 1
It is usually used in the form of beads with a diameter of 6MNFli degrees. The diameter of the carrier may be even smaller.

Examples include latex obtained by emulsion polymerization of styrene and gel obtained by suspension polymerization.

When using imitation fine particles such as , the reaction form during the reaction approaches a liquid phase-liquid phase reaction, which increases the reaction rate. As a result, one reaction time can be shortened. If these carriers have a specific gravity appropriate for the reaction, they can be used as is in the reaction system, but it is also possible to increase their specific gravity by adding inorganic salts such as barium sulfate or calcium carbonate as fillers. be. In the case of latex or gel, if a filler is added to the monomer and polymerized, it becomes a carrier containing the filler. Furthermore, the specific gravity can also be changed by the polymerization method.

An antibody or antigen for the substance to be measured is supported on such a carrier, for example, by physical adsorption. For example, when insulin is the substance to be measured, Jk
IgG is supported on lll- as an insulin antibody. Insulin is bound to this to form a first-homeophase system. It was then labeled with an enzyme. A second solid phase system is formed by binding an antibody or antigen that specifically reacts with the substance to be measured. If the carrier is A, the substance to be measured is B, the antibody or antigen of the substance to be measured is C1, and the enzyme-labeled antibody or antigen is C3, then the solid phase threads are A-C, B.

The second solid phase system is represented by A-C1-B-C. cl and C9 may be the same substance. As for c and l.

For example, IgQ to which HRP (peroxidase) is bound as a molecule is used. Note that the labeled antibody or antigen may be used, for example, by J. Histochem, Cytoc.
hem.

22.1084 (1974) by the method of Nakane et al.

Next, an enzyme substrate solution is added to the second solid phase system to perform an enzyme reaction. At this time, it is necessary that the second solid phase system is completely immersed in the reaction solution. Therefore, it is desirable that the specific gravity of the second in-phase system is greater than the specific gravity of the reaction solution. A specific gravity adjusting substance is added to the reaction solution as necessary. Specific gravity adjusting substances include sodium chloride, magnesium chloride, and other alcohols, glycerol and other alcohols, glucone and other sugars, urine, acids, and bases.

These ratios [R adjustment product color↓ are added as appropriate within a range that does not interfere with the enzyme reaction. If a specific gravity adjusting substance is added to the substrate solution beforehand, then fP is I+i'i rli.

After the enzymatic reaction is completed, reaction products and enzymes appear on one reaction surface.

Contains solid phase threads. Reaction 1 to C? Liquid stop = /J11
to completely stop the reaction. To stop the reaction 6f, mix with 1 reaction solution 9. A12ffff agents, acids, and alkalis can be used. Reaction 1': The specific gravity has been adjusted in advance using the same specific gravity adjusting substance as used for the above reaction solution, and by adding it to the reaction solution, one reaction solution can be prepared. The solid phase fibers present in the liquid float on top of the liquid. It is important that the specific gravity-adjusted reaction stop solution is capable of stopping the enzymatic reaction and does not interfere with spectroscopic measurements.

After the reaction has stopped, the enzyme reaction solution is evaporated to zero by spectroscopic techniques.
0 measurement is good. From this OD measurement value.

Insulin resistance can be determined using a calibration curve prepared in advance. The specific gravity of the reaction solution after the reaction has been stopped has been adjusted, so when a portion of it is directly transferred to a cuvette, it will not sink to the bottom even if some solid chloride migrates into the cuvette. therefore.

Of course, the optical path will not be obstructed.

If the method of the present invention is applied even when measuring by competitive reaction method, since the specific gravity of the carrier, enzyme reaction solution, and reaction terminator is adjusted, there will be no interference from the solid A'14 system during the measurement. Can perform old and accurate measurements.

Example The present invention will be described below with reference to examples.

Example 1 (A) Preparation of immobilized antibody: klz-shaped beads with a diameter of 6.8511JR were formed using vorinutylene as a carrier. These beads are 5% 5cat 2Q X-N
(manufactured by Hankai Kagaku Co., Ltd.), washed with water, and air-dried. The ratio 11d was 1.05. Separately, one guinea pig was given purified porcine insulin (Acrapid M manufactured by Noho).
C) was immunized to produce anti-insulin serum. This serum was subjected to ion exchange chromatography using f) Ii A E-cellulose to obtain an IgG fraction.

This was 0.1M p to be 10 lzg/wt.
Dissolved in a mild phosphoric acid shield of H7,0. Beads 10 above
Take 0 pieces in a beaker and add the above IgG phosphoric acid solution (
97a25rrl was added and incubated at 37°C for 1 hour. This was left in the oven at 4''C for 16 hours.

The produced immobilized 4[monoantibody] was removed and thoroughly washed with the above-mentioned phosphofI2 buffer. <1. ) was added to the immobilized antibody with bovine serum albumin in 0.1% phosphate buffer (B S A/F
H(ff, )25wi was added and left at 4°C for 16 hours.

(B) Enzyme-labeled antibody *IX4 ff+! ! The IgG fraction obtained in Section 4 was obtained from J, llistochem, Cy.
Peroxidase (H
orse radish peroxidase)
Labeled with. The obtained enzyme-labeled antibody was diluted 1200 times with a BSA solution.

C) Preparation of the 1st solid phase system: Sourium chloride 1-1JumO, 1M.

Magnesium chloride '0.01 M, BSA m14
pH containing 10.1% and 0.1% sodium azide
A 70.02M phosphate buffer (hereinafter referred to as 0.02M!J phosphate buffer) was prepared. Purified Quintin insulin (Novoi)
Earthenware, Actravid MC) above 0.02M! J
diluted with acid buffer to give concentrations o, lo, 2o, 4o, go.
, xeo, a2op unit/ @I! A dilution series was prepared. 100% insulin solution of each concentration in a test tube
0.5 ml of 0.02 M'J acid buffer was added to each μl portion. The in-phase antibodies prepared in section (A) were added one by one to this, and the mixture was incubated at 37°C for 1 hour. Incubation stage, suction ρ 12d 0.02M
! A first solid phase system was obtained by washing once with J acid buffer.

(b) Preparation of second solid phase system: Enzyme-labeled antibody solution 30011J obtained in section 0 was added to the solid phase thread obtained in section 0 and incubated at 37°C for 2 hours. Absorb reaction 11￥ and pass through δ-i, 0.02M phosphate buffer 2 tut
A second 1III phase filament was obtained by washing with suction twice.

(E) Enzyme reaction: Add 0.5 ml of a 0.1% phosphate buffered solution of O-nitrophenyl-β-lactopyranoside as a substrate to the second solid phase system obtained above.

Incubate for 1 hour at 37°C. Then add 0.1 rvx sodium carbonate solution containing 20% glycerol.
The enzymatic reaction was stopped by adding fil 2 wl.

(F') Spectroscopic measurement: The solution after the termination of the reaction in section (E) was poured into a cuvette, and its (monthly) value at 420 nm was measured. At this time, it does not exist in the measurement solution /: J solid A
The 'Ll' mark does not float on the upper layer of the liquid, so it does not interfere with the optical path during measurement. The 0D value for 1S (vs. insulin 1) was plotted to create the line shown in Figure 9. Using this, the amount of insulin contained in an unknown sample can be quantified.

Example 2 (~ Preparation of immobilized antibody: Same as Example 1 except that ABS was used as a carrier.

The specific gravity of the spherical beads serving as the carrier was 1.02.

(11) Preparation of tr¥-labeled antibody: I obtained in section (A)
N-(m-maleimidobencyμoxy)- in the gG fraction
The action site of succinimide is β-D-galactosidase (
Labeled with β-Gal). The obtained marker color chart is 0.
02M! It was diluted 500 times with J acid buffer.

0th - Preparation of solid phase thread: Same as Example 1.

p) Preparation of second solid phase system: Same as Example 1.

(E) Enzyme reaction: An enzyme substrate solution containing 2.5 mM of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and 5 m H of hydrogen peroxide at pH 7. .1M! J acid buffer was prepared.

Add the above substrate solution to the second solid phase yarn obtained in section σ〕) at 37°
Incubate for 1 hour at C. Then, 21 hours of 0.5M sulfuric acid was added to stop the enzyme reaction.

(Ladle spectroscopic measurement: 01 at 405 nm)
It is the same as in Example 1 except that the values were measured.

Effects of the Invention According to the method of the present invention, as described above, in the enzyme immunoassay using the in-phase method, the reaction solution 1, the reaction stop solution, and the carrier constituting the enzyme reaction system each have their specific gravity adjusted. At a fixed time, all solid systems in the cuvette float to the surface.

Therefore, the optical path is obstructed by the in-phase thread? 15 is absent, and a positive (t111) constant can be obtained.Furthermore, since the carrier floats to the layer j<11 in the reaction solution regardless of its particle size, it is necessary to use microparticles of the most waste material as the carrier in the 9 reaction system. Therefore, the magnitude of the 7 reaction increases and the Mlil constant range can also be expanded.

4. The ffi'i n' explanatory diagram of the drawing shows the insulin injection method in the method of the present invention! 1 degree and O
This is a graph showing an example of the relationship with the D value.

IJ　J：.

4 people: Bondsui Chemical Industry Co., Ltd. JP-A-Sho GO-127462 (6) (Issuance of procedural amendment) 1. Displaying incidents 1981 Patent Application No. 235613 2. Name of invention Enzyme immunoassay 3. Person who makes corrections Relationship to the incident: Patent applicant Postal code 530 Address: 2-4-4 Nishitenma, Kita-ku, Osaka 5 Contents of amendment +11 Line 5 from the bottom of page 11 of the specification [01% of]” Correct it to "contains 01%."

(2) From the third line from the bottom of page 11 to the third line of page 12 [(B) Preparation of enzyme-labeled antibodies:...
・(snip)・・・It was diluted. ” [(B) Preparation of enzyme-labeled antibody: The IgG fraction obtained in section (A) is treated with N-(m-maleimidobenzoyloxy)-zacdiimide, and β-D-galactosidase (hereinafter referred to as β
-Gal). The obtained labeled enzyme was diluted 500 times with 0.02M phosphate buffer to which 1fiM magnesium chloride was added. Correct it as j.

(3) On page 12, lines 5 to 6, the statement "Contains magnesium chloride O, 01M, BSA solution 01% and sodium azide O, i%" has been changed to (-s J containing 0.1% SA). I am corrected.

(4) On page 13, line 1 It says "filtration" [Corrected to remove J.

(5) On page 13, line M5, replace [-0.1% phosphate buffer solution] with [0.1% phosphate buffer solution].
Add 1mM magnesium chloride containing 90,02M
Phosphate buffer solution] Correct.

(6) On page 14, lines 1 to 5, the text ``(B) Preparation of enzyme-labeled antibody: diluted to... (omitted)...'' was replaced with ``(B) ) Ig production of enzyme-labeled antibody: (The IgG fraction obtained in section N was
, 22, 1084 (1974).
Peroxidase (Horse ra
dish peroxidase). The obtained enzyme-labeled antibody was diluted 1,111,200 times with a BSA solution. ” he corrected.

(7) Page 14, line 8 "202'-" “2. Correct it as z’-J.

(8) Page 14, line 10 It says [5 pain H] Correct it to "5mM".

that's all

Claims (1)

[Claims] 1. The specific gravity of the solution during the enzymatic reaction is smaller than the specific gravity of the phase filament containing the carrier, and the specific gravity of the solution after the enzyme reaction has stopped is set so that it is also larger than the specific gravity of the same phase system. Enzyme immunoassay using an in-phase method using 9 spectroscopic measurements including: 2. The measuring method according to claim 1, wherein the reaction system contains a specific gravity adjusting substance at the start of the enzyme reaction. 3. The measuring method according to claim 1, wherein the reaction terminator for stopping the enzyme reaction contains a specific gravity adjusting substance. 4. The measuring method according to claim 1, wherein the carrier is a polymer compound. 5. The measuring method according to claim 1, wherein the carrier is glass or alumina. 6. The measuring method according to claim 1, wherein the carrier contains a filler. 7. The measuring method according to claim 1, wherein the carrier is a latex obtained by emulsion polymerization of polymerizable monomers. 8. The measuring method according to claim 1, wherein the carrier is a gel obtained by suspension polymerizing a polymerizable monomer. 9. The specific gravity adjusting substance is an inorganic salt, an acid, or a base. The measuring method according to claim 2 or 3, which is at least one selected from the group consisting of polyhydric alcohols and saccharides. 10. The measuring method according to claim 6, wherein the filler is an inorganic salt.