JPH0466872A - Specimen measuring method using affinity-chromatography-carrier and its measuring kit - Google Patents

Abstract

PURPOSE:To make a solidifying antibody disused by finding a specimen quantity using a radiometric immunoassay after making the analyzed specimen adsorbed and combined with an affinity-chromatography carrier and blocking the residual active radical. CONSTITUTION:The specimen quantity is found using a known radiometric immunoassay or nonradiometric immunoassay after the analyzed specimen is made adsorbed and combined with an affinity-chromatography carrier and the residual active radical is blocked. The measuring operation is simplified and a reduction of the measuring cost becomes possible because a solidified antibody in the sandwich method becomes unnecessary. Further, the measurement can readily be made by the sandwich method even if the specimen is a hapten.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for measuring an analyte using an affinity chromatography carrier and a kit for measuring the same.

Prior Art 2 J A radioimmunoassay method was devised by Bcrson et al. in 1959, and because of its high detection sensitivity, specificity, and simple measurement operation, it has been applied to the measurement of trace amounts of biological substances in blood and urine. It is becoming widely used for diagnosis and testing in the medical field. However, these methods using radioisotopes have the following problems: (1) The labeling reagent has an expiration date (half-life).

(2) It cannot be handled without a laboratory facility that meets certain standards.

(3) Radioactive waste cannot be easily disposed of.

Due to these problems, it cannot be used at all testing facilities.

The radioimmunoassay method is the RIA method (RIA method) using a labeled antigen.
adioimmunoassay method) and IRMA method (immunoradiometric method) using labeled antibodies.
As5ay method). The RIA method requires: (1) highly purified labeled antigen;

(2) Labeling with radioactive isotopes may denature antigens, which may reduce reactivity with antibodies.

However, it is widely used to measure many biological trace substances such as haptens, which are difficult to measure by the IRMA method described below. I RMA method includes competitive method and 2.
-si There are two types of IRMA methods. 2-site
The IRMA method involves sandwiching the antigen between a solid-phase antibody and a labeled antibody, and is therefore called the sandwich method. In this method, one antigen is sandwiched between two antibodies, so multiple antibody recognition sites must exist on the antigen. Therefore, it is impossible to measure haptens with small molecular weights. The I RMA method has the advantage of being able to perform measurements over 3 to 4 times, but when the antigen concentration becomes high, the amount of solid-phase antibody becomes relatively insufficient, and the amount of antigen binding to the same-phase antibody reaches a plateau. Therefore, it is necessary to select equipment and immobilization methods that can immobilize a large amount of solid-phase antibodies.

Enzyme immunoassays were developed as an alternative to radioimmunoassays. Among the measurement principles of enzyme immunoassay, the Sand-Deutsch method (non-competitive method) using two types of antibodies is used to measure macromolecules that have two antigen-determining sites in one molecule; , a heterogeneous competitive method is used in which the antibody or antigen is immobilized. Additionally, for some haptens, such as cortisol, a homogeneous competition method is used that does not require separation of antigen-antibody complexes and free antigen. Methods devised for the measurement of haptens that are difficult to easily measure by enzyme immunoassay, for example (1) A fixed amount of antibody is reacted with an enzyme-labeled hapten, and the resulting antigen-antibody complex is collected using the two-antibody method. Alternatively, the activity of the labeled enzyme is measured after separation using a solid-phase antibody method.

(2) A method in which a conjugate of a hapten and a protein is used as a solid-phase antigen, and an antibody bound to the antigen is detected using an enzyme-labeled second antibody.

Although it is possible to measure haptens using this method, the measurement operation becomes complicated because it avoids the use of radioactive isotopes, making it difficult to be widely used.

[Means to solve problems]

The gist of the present invention is as follows: (1) After adsorbing and binding the analyte to the affinity chromatography carrier and blocking the remaining active groups,
A method for determining the amount of specimen using a known radioimmunoassay method or non-radioimmunoassay method.

(2) After simultaneously adsorbing the analyte (test antigen) and a certain amount of labeled analyte (labeled antigen) onto the affinity chromatography carrier, the amount of analyte How to find out.

(3) Adsorb and bind antibodies against the test specimen in advance,
A method of determining the amount of a specimen using an affinity chromatography carrier with residual active groups blocked, using a known radioimmunoassay method or a non-radioimmunoassay method.

(4) A measurement kit configured to carry out claims 1, 2, and 3.

(5) An affinity chromatography carrier used for carrying out claims 1, 2 and 3, which is immobilized on a laboratory instrument.

This compensates for the drawbacks of the known radioimmunoassay methods and non-radioimmunoassay methods mentioned above. That is, if the method of claim 1 is used, the immobilized antibody in the Sand-Deutsch method becomes unnecessary, so the measurement operation is simplified and the measurement cost can be reduced.

Furthermore, even if the sample is a hapten, it can be easily measured using the Sanderch method. According to the method of claim 2, B/F separation can be performed immediately. In the method of claim 3, the immobilization of the antibody against the test specimen can be shortened, and a sufficient amount of the antibody can be immobilized.

Although the present invention was originally devised to simplify measurement operations for non-radioimmunoassays, all of its techniques can be applied to radioimmunoassays. Furthermore, in addition to the method of using a commercially available affinity chromatography carrier as it is, the present invention also includes a method of using a solid-phase affinity chromatography carrier (
It also includes claim 5). Solid-phase affinity chromatography supports make the invention even more convenient.

That is, by immobilizing an affinity chromatography carrier on the surface of magnetic beads, magnetic particles, microparticles, etc., it is possible to apply it to a fully automated analyzer. Furthermore, by immobilizing an affinity chromatography carrier on a test tube, microplate, bead, strip, etc., it becomes possible to use it manually.

The present invention will be explained in more detail with reference to Examples below.

Example 1. Measurement of HBs antigen HBs antigen measurement kit A, kit contents (1) CNBr-activated Sepharose solid-phase microplate (2) peroxidase-labeled anti-HBζ monoclonal antibody (3) buffer solution (4) OPD substrate tablet (5) substrate Lysis solution (6) Reaction stop solution (7) Washing solution (8) HBs antigen positive control serum (inactivated) (9) HBs antigen negative control serum 00) Blocking reagent B, measurement procedure (1) CNBr-activation Dispense 50 ul of sample into each well of a Sepharose-immobilized microplate, mix,
Stir and incubate for 30 minutes at room temperature to remove H in the sample.
Bs antigen was adsorbed and bound to CNBr-activated Sepharose.

(2) The microplate subjected to the operation in (1) was washed three times with a washing solution.

(3) 0.
A 2M glycine solution (pH 8,0) was added, mixed, and incubated at room temperature for 1 hour.

(4) Perform the operation in (3) and wash the two microplates three times with the washing solution.

(5) Add 50 μl of the enzyme-labeled antibody solution to the microplate prepared in step (4), mix, stir, and incubate at room temperature for 45 minutes. formed.

(6) The microplate subjected to the operation in (5) was washed three times with a washing solution.

(7) O
100 μl of each PD substrate solution was dispensed and incubated at room temperature for 45 minutes.

(8) After dispensing 100 u/each of the reaction stop solution into the microplate that had been subjected to the operation in (7), 492 nm
The absorbance was measured. The same procedure was performed using HBs antigen positive control serum and HBs antigen negative control serum instead of the test specimen, and CUT OF
The F Index was calculated to determine whether it was positive or negative.

Example 2 Measurement of serum ferritin Serum ferritin measurement kit A, kit contents (1) CNBr-activated Sepharose-immobilized beads (2)
Peroxidase-labeled anti-human ferritin rabbit antibody 3) Buffer solution 4) OPD substrate tablet 5) Substrate solution 6) Reaction stop solution 7) Washing solution 8) Standard ferritin for creating a calibration curve 9) Reagent B for pro-reagent B Measurement procedure (1) ) 1 at of buffer and 50 B of sample in each small test tube.
, 1 were added and mixed. Next, CNB with the preservation solution well drained.
One total rNBr-activated Sepharose homophasing bead was mixed, vortexed, and incubated for 30 minutes at room temperature.

(2) Place each small test tube after performing the operation in (1) on the washing liquid table 2.
Washed 3 times with m and l.

(3) Add 0.2 M to each small test tube after performing the operation in (2).
Add glycine solution (pH 8,0), mix and incubate at room temperature.
Incubated for hours.

(4) Each small test tube subjected to the operation in (3) was washed three times with a washing solution.

(5) Add 2 ml of each buffer solution and 50 μl each of enzyme-labeled antibodies to each small test tube in which step (4) was performed, mix them, incubate at room temperature for 45 minutes, and combine CNBr-activated Sepharose, ferritin, and enzyme. A labeled antibody complex was formed.

(6) Each small test tube subjected to the operation in (5) was washed three times with 2 ml of washing solution.

2 ml of each OPD substrate was dispensed into each small test tube subjected to the operations (7)+6), and incubated at room temperature for 45 minutes.

(8) In each small test tube in which the operation in (7) was performed, a wave valley was applied to stop the reaction.1. After dispensing ml, the absorbance at 492 nm was measured. The same operation was performed using standard ferritin for preparing a calibration curve at each concentration instead of the test sample, and the amount of ferritin in the test sample was determined from the prepared calibration curve.

Example 3 Measurement of thromboxane B2 A, kit contents l Non-immobilized epoxy activated Sepharose 6B2) C
``I] Labeled thromboxane B2 antibody 3) Standard thromboxane for creating a calibration curve 4) Control serum 5 Buffer solution 6) Washing solution 7) Blocking reagent B, measurement procedure (1) Non-immobilized epoxy activity suspended in buffer solution Aliquots of 50 µl of epoxy-activated Sepharose 6B were placed in small test tubes.Next, 50 µl of prostaglandin crude extract solution extracted by the PO-Vell method was added and mixed with non-immobilized epoxy-activated Sepharose 6B. Stir and incubate at room temperature for 1 hour to adsorb and bind thromboxane B2 in the solution to the non-immobilized epoxy-activated Sepharose.

(2) Perform the operation in (1) -) Add 500 u/washing solution to thromboxane B2-bonded non-solid-phase activated Sepharose 6B in each test tube, mix, and then add 2.000
Thromboxane B2 binding,
The non-solid phased epoxy activated Sephas 6B was washed.

(3) The remaining active groups of non-immobilized epoxy-activated Sepharose 6B were blocked with 1M ethanolamine.

CI2a engineering 1 was added to each small test tube that underwent the operation (4H3).
Add 200 μl of each labeled thromboxane Bt antibody, mix, and incubate for 1 hour in a water bath at 37°C to prepare a non-immobilized epoxy-activated Sepharose 6B/thromboxane B2/Cl25I] labeled thromboxane B2 antibody complex. did.

Wash wave trough 5 to each small test tube that underwent the operation of (5) + 4).
After adding 00 μl and mixing, incubate at 4°C, 3,50 rpm.
Free 25.1-labeled thromboxane B2 was separated by centrifuging the mixture for 30 minutes and aspirating and removing the supernatant using an aspirator.

(6) Using a well-type notillation counter,
The radioactivity of non-immobilized epoxy-activated Sephas 6B, thromboxane B2, and (''I)-labeled thromboxane B2 was measured.Instead of the test sample, standard thromboxane B2 for preparing a calibration curve at each concentration was used. Perform the same operation using
Thromboxane B2 in the test sample was prepared from the calibration curve.
I asked for the quantity.

Example 4 Folic Acid Measurement Folic Acid Measurement [3H] Kit A, Kit Contents (1) EAH-Sepharose 4B homophasing small test tube (2
) [″H] labeled folate antibody (3) Standard folate for preparing a calibration curve (4) Control serum (5) Buffer solution (6) Washing solution B Measurement procedure (1) Transfer 1 rnL of each buffer to EAH-Sepharose 4B solid phase Next, 50 μl of each test sample was added and mixed, and incubated at room temperature for 30 minutes to remove folic acid in the sample using EAH-Sepharose 4B.
adsorbed and bonded to.

(2) After removing the buffer solution from each small test tube in which the operation in (1) was performed, add 1 mL of washing solution to each tube and mix.
The washing solution was removed again.

(3) [3H] for each small test tube that was subjected to the operation in (2).
Add 500 μl of each labeled folic acid antibody solution, mix, and incubate at 37°
The mixture was incubated in a water bath of C for 1 hour to prepare an EAH-Sepharose 4B/folic acid/[3H]-labeled folic acid antibody complex.

(4) Wash each small test tube that was subjected to the operation in (3),
Free [°H]-labeled folic acid antibody solution was removed.

(5) Using a well-type scintillation counter,
The radioactivity of the EAH Sepharose 4B/folic acid/(SH)-labeled folic acid antibody complex was measured. The same operation was performed using standard folic acid for preparing a calibration curve at each concentration instead of the test sample, and the amount of folic acid in the test sample was determined from the prepared calibration curve.

Example 5 Determination of digoxin (, +25'f, '1 Kit A, kit contents (1) Epoxy-activated Sepharose 6B (2) ("
I) Labeled digoxin antibody 3) Standard digoxin for preparing a calibration curve 4) Buffer solution 5) Control serum 6) Washing solution 7) Blocking reagent B, measurement procedure (1) Epoxy-activated Sepharose 6 suspended in buffer solution
50 ul of B was aliquoted into each small test tube. Next, 50 μl of each test sample was added and mixed, and incubated at room temperature for 30 minutes to adsorb and bind OH-containing substances such as digoxin in the sample to the epoxy-activated Sepharose.

(2) Add 0.5 ml of washing solution to each small test tube in which the operation in (1) was performed, mix, and then add 2.00 O
Centrifuge for 5 minutes at rpm and use an aspirator to
Epoxy-activated Sepharose 6B was washed by removing the supernatant by suction.

(3) Add 2 ml of the blocking reagent pot to each small test tube in which the operation in (2) was performed, and after reacting at room temperature for 4 hours, remove the blocking reagent.

(4) For each small test tube (125
I) Add 200 μl each of labeled digoxin antibodies, mix,
Incubate for 1 hour in a 370G water bath and add epoxy-activated Sepharose 6B Digoxin (+2sJ).
A labeled digoxin antibody complex was prepared.

(5) Place each small test tube after the operation in (4) at 4°C.
, centrifuged at 500 rPm for 30 minutes, and aspirated.
The free (+
25 steps] The labeled digoxin antibody was removed.

(6) Using a well type scintillation counter,
The radioactivity of the epoxy-activated Sepharose 6B phased beads, digoxin, and (: +251) labeled digoxin complex was measured.Similar operations were performed using standard digoxin for preparing a calibration curve at each concentration instead of the test sample. The amount of digoxin in the test sample was determined from the prepared calibration curve.

Example 6 Quantification of human IgE Human IgE measurement [3H] Kit A, kit contents (1) CNBr-activated Sephas homogeneous filter paper (2) (
”H) Labeled anti-human IgE antibody (3 Standard human IgE for creating a calibration curve) (4) Control serum (5 Buffer solution (6) Washing solution (7 Bronok reagent B, measurement procedure (1) 1 mL of buffer solution in each small test tube Next, add 50 ul of each test sample and mix.Next, CNB
rActivated Sepharose immobilized filter paper (0.3cm x 03m
), one plate of each was added, stirred gently, and incubated at room temperature for 30 minutes.

(2) The buffer solution and the test sample mixture in each small test tube in which the operation in (1) was performed were removed. Next, the CNBr-activated Sepharose-immobilized filter paper was washed by adding 1 ml of the washing solution to each small test tube, stirring gently, and then removing the solid solution.

(3) 2 ml of each blocking reagent was added to each small test tube in which the operation in (2) was performed, and after reacting at room temperature for 2 hours, the blocking reagent was removed.

(4) Add 1 ml of [3H]-labeled human anti-IgE antibody solution to each small test tube in which step (3) was performed, stir, and then incubate for 1 hour in a water bath at 37°C.
Activated Sepha0-Sll human IgE, (''H) labeled human IgE antibody complex was prepared.

(5) Free [3H]-labeled anti-human engineered gE antibody was removed by suction from each small test tube subjected to the operation in (4).

(6) Using a well-type scintillation counter,
The radioactivity of the CNBr-activated Sepharose/human IgE/r″H]-labeled anti-human IgE antibody complex was measured.Similar operations were performed using standard human IgE for preparing a calibration curve at each concentration instead of the test sample. The amount of human IgE in the test sample was determined from the prepared calibration curve.

Example 7 Measurement of plastaglandin E2 Prostaglandin E2 measurement kit A, kit contents (1) Epoxy-activated Sepharose 6B solid-phase magnetic particle suspension (2) Peroxidase-labeled prostaglandin E2 (
(3) Buffer solution (40PD substrate tablets (5) Substrate solution (6) Reaction stop solution (7) Washing solution (8) Standard prostaglandin E2 for creating a calibration curve (methyloximeated) (9) Block Reagent B, measurement procedure (1) Transfer, mix, and stir 50 μl of the crude extracted solution extracted by the method of Powell I into a test tube containing a certain amount of epoxy-activated Sepharose 6B in-phase magnetic particle suspension. Immediately, 50 μl of each peroxidase-labeled prostaglandin E2 solution was added, mixed, and stirred. After incubation at room temperature for 30 minutes, peroxidase-labeled prostaglandin E2 and peroxidase-labeled prostaglandin E2 were immobilized on epoxy-activated Sepharose 6B. Adsorbed and bonded to magnetic particles.

(2) Each test tube subjected to the operation in (1) was washed twice with a washing solution.

(3) One liter of each OPD substrate solution was added to each test tube subjected to the operation in (2), and incubated at room temperature for 30 minutes.

(4) 1 ml of reaction stopper was added to each test tube subjected to the operation in (3), mixed, and the absorbance at 492 nm was measured. A similar operation was performed using standard prostaglandin E2 for preparing a calibration curve, and the amount of prostaglandin E2 in the sample was determined from the prepared calibration curve.

Example 8 Interferon γ ff1U constant interferon γ measurement kit A, kit contents (1) B lur St: pbar++s+・CL −
6B immobilized beads (2) Peroxidase-labeled interferon γ antibody (Fah') 3) Buffer solution 4) OPD substrate tablet 5 Substrate solution 6) Reaction stop solution 7 Washing solution 8 Standard interferon rB for creating a calibration curve, measurement procedure (1) ) 1 ml of buffer wave trough and 50 μl of sample were added to each small test tube and mixed. Next, remove the storage solution from BlueSe.
Take one each of Pharose C1 and 6B solid-phase beads, mix and stir, and incubate at room temperature for 30 minutes.

(2) Wash each small test tube after performing the operation in (1) with Wave Valley 2.
Washed 3 times with ml.

(3) Add peroxidase-labeled interferon r antibody (Fab') to each small test tube in which step (2) was performed, mix, and incubate at room temperature for 1 hour.
S (l city arusr CL-68/interferon r
A II enzyme-labeled antibody complex was formed.

(4) Pour each small test tube in which the operation in (3) was performed into two washes.
ml twice to separate free enzyme-labeled antibodies.

(5) 2 mL of OPD substrate pot was added to each small test tube subjected to the operation in (4), and incubated at room temperature for 1 hour.

(6) After adding and mixing 1 ml of the reaction stop solution to each small test tube in which the operation in (5) was performed, the absorbance at 492 nm was measured.

The same operation was performed using interferon γ for preparing a calibration curve at each concentration instead of the test sample, and the amount of interferon γ in the test sample was determined from the prepared calibration curve.

Example 9 Measurement of soluble liver-specific lipoprotein (LSP) antibody LSP antibody measurement kit A, kit contents (1) CNBr@Sepharose-immobilized test tube (2
) Peroxidase-labeled protein A3) Buffer solution 4) OPD substrate tablet 5) Substrate solution solution 6) Reaction stop solution 7) Washing solution 8) Standard LSP antibody for creating a calibration curve 9) Blocking reagent B, measurement procedure mcNgr activation Sepharose immobilization 1. each buffer solution in test tubes. Add μl of each of aL and LSP solutions, mix, and incubate at room temperature for 1 hour to adsorb and bind LSP to CNBr-activated Sepharose.

(2) The buffer solution in each test tube subjected to the operation in (1) was removed, and the test tubes were washed twice with 1 mL of washing solution.

(3) Add 1 ml of blocking reagent to each test tube treated in (2) and incubate at room temperature for 2 hours.
T-ko.

(4) Wash each test tube with 1 liter of washing solution after performing the operation in (3).
Washed twice with td.

(5) Add one dose of buffer to each test tube in which step (4) was performed.
ml and 50 μl of the test specimen were added and incubated at 37°C for 1 hour.

(6) 1 ml of peroxidase-labeled protein A solution was added to each test tube subjected to the operation in (5), mixed, and incubated at 37°C for 1 hour.

(7) Each ml of the OPD substrate solution was added to each test tube subjected to the operation in (6), mixed, and incubated at room temperature for 30 minutes.

(8) 1 ml of the reaction stop solution was added to each test tube subjected to the operation in (7), mixed, and the absorbance at 492 nm was measured. A similar operation was performed using a standard LSP antibody for preparing a calibration curve, and the amount of LSP antibody in the sample was determined from the prepared calibration curve.

Example 10 Preparation of solid-phase affinity carrier (A) CNBr-activated Sepharose solid-phase test tube A water-insoluble synthetic glue was thinly and uniformly applied to the inside of a small test tube, and dried CNBr-activated Sepharose was sprinkled. ,CNB r
A thin layer of activated Sepharose was formed. After drying for 24 hours at room temperature, the CNBr-activated Sepharose layer was shaped and subjected to each test.

(B) EAH-Sepharose 4B immobilized beads diameter 8
Apply a thin and uniform layer of synthetic glue insoluble in water and organic solvents to a polystyrene ball, place it on top of the dried EAH-Sepharose 4B powder, and place the EAH on the polystyrene ball.
- A thin layer of Sepharose 4B was formed. After drying at room temperature for 24 hours, 50 mM phosphate buffer (pH 7, 2
, 0.1% NaH.

(contains) and stored.

(C) Blue 5epharose CL-6B solid-phase plate After applying a thin and uniform synthetic glue insoluble in water and organic solvents to the inside of separate strip wells (8 wells) for ELISA, dry Blue 5ep
Fill the wells with harose CL-6B powder and incubate for 24 hours.
Let it dry for a while and add extra Blue Sepl+ar
ose CL-6B powder was removed and solidified Blu
e 5rpl+arose CL-6B layer was shaped and subjected to each test.

Q)) AG POLY (I), POLY (C)
T, YP (! 6 Solid phase strip 0.3 cm
I).

Sprinkle POLY(C) Type 6 powder and glue.
It was dried for 24 hours. The back side was also treated in the same way and subjected to each test.

(5) Hollow cylindrical EL solid-phased with CNBr-activated Sepharose
Synthetic glue insoluble in water and organic solvents is thinly and uniformly applied to the inner and outer surfaces of a hollow cylinder of a size that can be inserted into the wells of a microplate for ISA (6 diameters and 8 lengths).
They were buried in boxes filled with dry CNBr-activated Sepharose powder, removed after 24 hours, shaped into hollow cylinders, and subjected to each test.

(Margin below)

Claims (5)

[Claims] (1) After adsorbing and binding the analyte to the affinity chromatography carrier and blocking the remaining active groups,
A method for determining the amount of specimen using a known radioimmunoassay method or non-radioimmunoassay method. (2) After simultaneously adsorbing the analyte (test antigen) and a certain amount of labeled analyte (labeled antigen) onto the affinity chromatography carrier, the amount of the analyte is determined by a known radioimmunoassay method or non-radioimmunoassay method. How to ask. (3) Adsorb and bind antibodies against the test specimen in advance,
A method of determining the amount of analyte using a known radioimmunoassay method or non-radioimmunoassay method using an affinity chromatography carrier in which residual active groups are blocked. (4) A measurement kit configured to carry out claims 1, 2, and 3. (5) An affinity chromatography carrier solid-phased on a laboratory instrument used for carrying out claims 1, 2, and 3.