JPS5832166A - Analytical elements for immunoassay - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoassay methods using chemiluminescent substances, bioluminescent substances, etc.
and seventh, it relates to analytical elements used in the in-phase method to improve the sensitivity and ease of handling of this type of analysis.

Although the history of immunoassay using luonescent is relatively new, several reports have been made so far. for example,
h) A report by Hayyu (L, S, Hergh) et al. who measured human IgG by labeling IgG with luminol (Anal
, 13iochem, , L4J267 (1979
)), Simpson (J-S, A, S), who measured rabbit IgG by labeling rabbit IgG antibody with luminol.
jmpSon) et al. (Nature, 279.
646 (1979)) - Testosterone Al, Labeling Pumin with Luminol and Measuring Testosterone Pratt (J, J.

Pratt et al. (J, Irrmunol, Met
Hods, 21° 179 (1978), ), Schröder (H), who labeled T with isoluminol and measured T4.
, R, 3 chroeder) et al. (J, imm
unol 0Methods, 25.275 (197
9)) is said. These reports are still basic, and the results obtained cannot be said to be particularly sensitive compared to other immunoassay methods.

An invention for carrying out chemiluminescent immunoassay using an in-phase method, particularly a method in which immunoreaction and luminescence are caused between positive and negative electrode plates, was made by Bruce G. A. Oubanault et al. ).

However, this patent lacks specific figures, and the effects of implementation are unclear.

Immunoassay is used to determine specific ultralow-tone biochemical substances in a multicomponent mixture system, and must have high sensitivity and a high S/N ratio. Furthermore, in order to apply this to clinical tests, etc., it must be easy to operate. The present invention provides a chemiluminescent immunological lid that satisfies the above requirements.

or generally relates to a luminescent immune dose.

In the anti-inflammatory method, in which the antigen or antibody is injected with a chemical substance that has been passed through some kind of measuring means, after the antigen-antibody reaction is completed, the unreacted labeled antigen or labeled antibody is It is necessary to separate the antigen-antibody complex from the antigen-antibody complex. Solid phase methods have been devised to facilitate this separation and simplify operations. In the solid-phase method 2, antibodies are usually immobilized on insoluble polymers, glass, etc. by VIJ embedding adsorption, chemical bonding, etc., and this can be realized by known methods for inoculation.

Now, the light generated by chemiluminescence has no directionality and spreads between all walls, so even if the luminescent particles are large, it is difficult to detect them effectively. The present invention uses a surface phase method that is easy to operate, and allows the generated light to be efficiently collected on a photodetector.

Although various shapes of antibody-mouse carriers have been used in the past, carriers suitable for the present invention are preferably plate-shaped carriers that can transmit generated light. This carrier may be flat or curved. As carrier materials, transparent glass, insoluble polymers such as cellulose and polystyrene, and derivatives of these molecules are used. It goes without saying that there are many other light-transmissive insoluble materials that are suitable for the purpose of the present invention.

Analysis 1 of the present invention! The material is the above antibody immobilization carrier j-
The basic design is to provide a light-reflecting light on the opposite side of the solid surface. The light that has passed through the carrier 11 is also reversed by this reflective layer, and is incident on and detected by, for example, a detector placed in front of this analytical element. A vaporized aluminum film is suitable as the reflective layer, but of course this is not to be relied upon.

FIG. 1 is a diagram showing the basic melting of the analytical element of the present invention.

In the figure, 1 is a photomultiplier tube for measuring chemiluminescence, 2 is a transparent immobilization carrier that supports the antibody, 3 is a reflective layer that reflects light, and 7 is the antibody immobilized on the carrier 2. FIG. 1 shows the basics of the analysis element, and if this is shown in more detail, it will take the form as shown in FIGS. 2a to 2C. These are only the characteristic points shown, and combinations of these or forms with further improvements can similarly produce the effects of the present invention. In the figure, reference numeral 4 denotes a monomolecular L- or multi-molecular active layer, or an active layer made of an active substance, which has been chemically modified to suit the immobilization of antibodies on the surface of a transparent immobilization carrier.

5 is a protective layer, and 6 is a solution-permeable hydrophilic porous layer.

The porous layer 6 is easily peelable and can be removed before measuring chemiluminescence.

The present inventors used glass and a solid pot molecular film as transparent 1^1 carriers, deposited aluminum on one side of these carriers, immobilized antibodies on the other side, and developed the present invention. We investigated the effects of Aluminum can now be deposited on not only glass but also polymer membrane surfaces.

It has become a technology that can be carried out in general today. In addition, solidification of the antibody was carried out according to a known method (for example, Chibata, ed., Immobilized Enzymes, Koeisha, Tokyo, 1974).In the case of polymer carriers, if the treatment method is incorrect, they may become opaque. The effect of the present invention is reduced. Therefore, the method of manufacturing the active layer 4 is important. For example, active 1- having an aminophenyl group on the polystyrene surface was introduced as follows. (1)
Polystyrene bath liquid is cast onto a flat glass plate to a thickness of #
? After making a J200μ membrane and melting it at 120C to remove minute wall holes, a mixture of nitric acid and sulfuric acid (HNOs:
He S Oa:H, 0=26:57:17 (heavy ratio)
) for 2 hours at 20C. After washing this with water, a 4m solution of stannous chloride (HCt:S”Ctt ・2H1
0:8 n=130:67:3 (juice violet ratio)) to 60C
Soak for 5 hours. This allows the surface 1 of the polystyrene membrane to
Only So is aminated. (2) After nitration according to the conventional polystyrene method, the solution is bath-dissolved in dimethylformamide;
Create a molten layer by casting on a flat glass plate.

After evaporating the solvent, a polystyrene bath in toluene is cast onto the surface of the thin film to form a composite layer.

After heat-treating at 120C to remove any residual media,
The nitro group is reduced in the same manner as in 1).

Although the above is the rt+ when polystyrene is used, the transparent carrier and the active material can be formed using other polymer films according to the properties of each compound.

Immobilization of the antibody was carried out after the carrier was coated with an aluminum reflective layer, protective group, etc.

The multi-Ii# membrane containing the antibody t+6d, ie, the analytical element, was stored at 4C and removed for each experiment.

As mentioned above, immunoassays labeled with chemiluminescent substances have been attempted in various places. It is also known that bioluminescent storage can be used in a similar manner (H, R, 5chr
oeder, et al, ”Methods
1n) i:ngymOtOgy” vot, 57.
AcHdemic press.

NeW YOrk, N, Y, 11978) O Since the analytical element of the present invention does not depend on any type of luminescent substance, luminol, which is well known as chemiluminescence vIJ award, was selected.
This was labeled with a human IgG antibody to examine the effects of the invention.

Fig. 3 shows an example of the standard violet of IgG. Curve 11 is the result when using the analytical element of the present invention, curve 12 is the reflection;
It shows the result when there is no.

The Sanderutsch method was used as the method of determination. The experiment was conducted as follows. 20II solidified anti-human IgG
Human IgG standard serum i to analysis pg of II + × 10 wIm
The mixture was cooled to 10 μtm, left at 37C for 4 hours, and washed with phosphate buffer. Next, 0 μt of anti-Human IgGl labeled with luminol was added dropwise, and after being left at 37C for 4 hours, it was washed in the same manner. Anti-Human IgG labeled with luminol was further bound to the antigen (human IgG) bound to the anti-Human IgG thus immobilized. The luminol activity on the analytical cost was measured by binding hemin to hydrogen peroxide.

[Brief explanation of drawings]

Figure 1 is a diagram showing the basic formation of the analytical elements of the present invention, Figure 2a
20 are configuration diagrams of the analysis elements of the present invention, and FIG. 3 is a diagram showing experimental results of the present invention.

Claims (1)

[Claims] A transparent support layer that transmits light and a luminescent immunoassay analytical element that uses the transmitted light as a countersign.