JPH01274066A - Enzyme immunoassay method - Google Patents

Abstract

PURPOSE:To shorten operating time to a large extent and to make it possible to perform accurate measurement, by inserting a film wherein antigen or antibody is fixed and a central part is hollow into the well of a microplate. CONSTITUTION:Commercially available microplates can be used as it is. It is desirable that the shape of a film is the same as the shape of the bottom surface of the well of the microplate. The films are not especially limited when cavities are provided at the central part of the film and the shapes of the films do not hinder a light path in measurement. However, it is desirable to use films having a circular pattern. As an antigen or antibody which is to be fixed on the film, the antigen or the antibody to which allergen is bonded are desirable. As the measuring methods, enzyme immunoassay, light emitting immunoassay, fluorescent immunoassay and the like which are ordinarily used can be used. The measuring procedures are as follows; e.g., at first the film is inserted into the well of the microplate, then a specimen of body fluid incorporating the antigen or the antibody is injected, cleaning is performed, then a label antibody is inputted, cleaning is performed, and the measurement is performed with enzyme reaction and an optical device.

Description

Detailed Description of the Invention The present invention is an enzyme immunoassay method using a microplate, in which a membrane having an antigen or antibody immobilized thereon and having a hollow center is inserted into a well (hole) of a microplate. The present invention relates to a method for measuring the concentration of an analyte using an optical device without taking out the sample.

In immunological analysis methods such as enzyme immunoassay, fluorescence immunoassay, and luminescent immunoassay, antigens or antibodies are conjugated or non-chemically identified to insoluble carriers such as polystyrene balls, glass beads, filter paper, or nitrocellulose. There is a way to measure it.

If a microplate well is used as one reaction tank, after removing the carrier or transferring the measurement solution to another container, the color development caused by the enzyme reaction,
It is necessary to measure fluorescence etc. spectroscopically.

Another method that allows measurement without removing the insoluble carrier is to place it on the wall of the well of a microplate.

There is also a method of spectrophotometrically measuring the color, fluorescence, or luminescence generated by an enzymatic reaction without requiring any operations such as removing the carrier by directly immobilizing the antigen or antibody through a covalent bond or non-chemically. It is being

By the way, the surface area of microplate wells, polystyrene balls, and glass beads is very small, and the effective surface area is several to several times larger, so it is difficult to increase the sensitivity, which depends on the antigen or antibody binding. There was a problem.

On the other hand, filter paper, nitrocellulose, nylon fiber membranes, and fluorine fiber membranes, which have an effective surface area several hundred times larger, are difficult to remove and handle, and furthermore, these membrane materials are opaque and do not allow light to pass through, making them difficult to use with microplate absorbance reading devices. It is not possible to insert it as is and measure it. Another problem is that the membrane adheres to the wall surface during cleaning, making it difficult to clean the surface of the membrane. Is cleaning a blank reaction such as carry-on reaction? It is an important step to reduce Ifl, and the form of collection greatly affects the cleaning effect.

Therefore, we cut a membrane that has the function of covalently bonding proteins into a JFe similar to the bottom of the wells of a microplate without contaminating the surface, cut out the center part to make a cavity, and then applied antigens or antibodies to this membrane. After binding and reacting with the test serum, the enzyme! It was discovered that the conventional problems could be solved by injecting No. 32 antibody solution and performing an enzyme immunoreaction, and the present invention was accomplished.

That is, the present invention provides an enzyme immunoassay method using a microplate, in which anti-1g is added to the wells of the microplate.
Alternatively, the present invention relates to a method in which a membrane in which an antibody is immobilized and a hollow center is inserted and the concentration of a sample is measured using an optical device.

As the microplate used in the present invention, commercially available microplates may be used as they are.

However, the bottom surface should be transparent and preferably transparent and smooth.

The shape of the membrane is preferably the same as the shape of the bottom of the well of the microplate, and the size is 1. For example, if the well is cylindrical, the diameter of the membrane is 0.0 mm of the diameter of the well.
70 to 0.95 times is preferable. The material is not particularly limited, but examples include nylon membranes, fluororesin membranes, filter paper, nitrocellulose membranes, etc. In order to increase the surface area, fibrous membranes, such as nylon fiber membranes and fluorine fiber membranes, are more preferred. preferable. There is a cavity in the center of the above membrane, and the shape of the membrane is not particularly limited as long as it does not obstruct the optical path during measurement, but is preferably circular. For example, when the diameter of the well is 7 mm and the diameter of the crotch is 6 mm, the diameter of the cavity is preferably 2.5 to 3.5 mm.

Antigens or antibodies to be fixed on the membrane include 1. Allergens such as mites, house death 1-1 pollen, egg white, milk, α-α, mold, and animal hair; Antiserum; Hormones such as progesterone and insulin; A.I. DS, ΔT I, ,
Antibodies to viruses such as hepatitis and herpes; leukocyte surface antibodies such as HI and A; and enzymes such as prostatic acid phosphodase. Preferably, those bound to allergens are suitable.

As for the measurement method, commonly used enzyme immunoassays, luminescent immunoassays, fluorescence immunosinus chambers, etc. are used.

Furthermore, as for the optical device, a commercially available microplate reader or the like may be used.

For example, the order of subchambers'f when using the method of the present invention is as follows.

(1) Transfer the membrane to the wells of the microplate (2) Inject the sample such as body fluid containing antigen or antibody (3) Wash (4) Add the labeled antibody (5) Wash (6) Enzyme reaction (7) Optical Immobilization using a device The method of the present invention is suitable for detecting trace amounts of proteins in body fluids such as allergens, antiserum, hormones, enzymes, virus antibodies, and white blood cell surface antibodies by changing the type of antigen or antibody to be immobilized on the membrane. There is.

It can also be applied to screening monoclonal antibodies.

According to the method of the present invention, light can pass through the membrane by making a cavity in the center of the membrane, and even if there are marks on the bottom of the well, it can be directly inserted into the microplate absorbance reader for measurement. In addition, the membrane is lifted up by injection of cleaning solution, reaction solution, or sample, and the solution sufficiently spreads to the back side of the membrane, making it possible to perform measurements with less variation.In other words, the membrane can be removed during cleaning and measurement. Since there is no need for this, the operating time is greatly reduced, and there is no difference in measurement values regardless of the presence or absence of a membrane, and accurate measurements can be made, providing superior enzyme immunotherapy compared to conventional methods. It can be said that it is a measurement method.

In particular, it is a very useful method for automatic measurements because it does not require any special extraction operation.

Next, the present invention will be explained in more detail with reference to Examples.

<Example 1> Detection of allergens by enzyme immunoassay A membrane (Imopilone-Millipore Ltd. USA), which has the function of covalently bonding proteins, was placed in a similar shape to the bottom of a microplate well without contaminating the surface. Place it in place and cut out the center part to make a hollow.

The membrane, which has a hollow center, is immersed in an aqueous mite-extracted allergen solution to bind the allergen to the membrane.

Load this mite allergen-binding membrane into the wells of a microplate (the membrane is at the bottom of the well).

50 μm of serum from a patient suffering from mite allergy was placed in this well and allowed to stand at room temperature for 1 hour.

Next, the injected serum is aspirated, and the membrane and the inside of the well are washed several times with a washing buffer and drained.

100 μm of anti-human IgE antibody labeled with alkaline phosphatase (ALP) is injected into the well and left at room temperature for 1 hour.

21 Aspirate the antibody solution, collect with washing buffer, wash the inside of the well several times, and drain.

Next A1. Inject 100 μl of ditrophenol phosphoric acid solution, which is a substrate for P, and leave it at room temperature for exactly 30 minutes. Add 200 μl of a reaction stop solution and measure the absorbance using a microplate absorbance reader.

<Execution @ 2> Using plate readers from two companies, the influence of the presence or absence of a membrane carrier in the wells of the microplate on absorbance measurement was investigated, and the results are shown in Table 1.

<Table L> (405 nm○, D, (+IT) As seen from Table 1, there was no difference in the measured values depending on the presence or absence of the film (disk). There were no problems with interference.

<Example 3> Various allergen extracts were bound to the scars, and the effects of varying the dilution ratio were investigated, and the results are shown in FIG.

In FIG. 1, the horizontal axis represents the dilution ratio, and the vertical axis represents the absorbance. Egg white, candida, cedar pollen, mites, and house dust were used as allergen extracts.

respectively, −〇−1−x−5−・− One row, indicated by -+-.

As is clear from Ui4t, even if there is a membrane carrier in the allergen extract well at various dilution ratios, it can be accurately measured spectrophotometrically, and there is no problem with operations such as washing.
Furthermore, even if diluted with serum and applied to the reaction system, the dilution H (
f. Since the kneading property is maintained, the qualitative properties of the measurement are very excellent.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the effect on the subchamber when various allergen extracts are bound to the membrane and the dilution ratio is changed. The horizontal axis represents the dilution ratio, the vertical axis represents the absorbance, and -0-, -x-, -.-1-rho, and -+- represent the allergens of egg white, candida, cedar pollen, mites, and house dust 1-, respectively. Procedure Amendment 3 (Method) 1986
August 25th

Claims (2)

[Claims] (1) In enzyme immunoassay using a microplate, a membrane with an antigen or antibody immobilized thereon and a hollow center is placed in a well of a microplate, and the sample concentration is measured using an optical device. (2) The measuring method according to claim 1, characterized in that an allergen (allergy-causing substance) is immobilized on the membrane.