JPS6071954A - Reaction method by immobilizing solid layer - Google Patents

Abstract

PURPOSE:To simplify an operation for analysis by fractionating and measuring the resultant product of reaction by the respective superposed medium layers contg. reactive reagents. CONSTITUTION:An antibody 7 is coated on the 1st layer 11, from above, an enzyme immobilizing antibody 8 on the 2nd layer and an enzyme reaction substrate 9 on the 3rd layer, respectively. Gelatin, ''Ficoll'', etc. are used for the media to be coated on the respective layers. The 1st layer 11 is adjusted to the coefft. of buoyant density of the antibody 7, the 2nd layer 2 to the coefft. of buoyant density of the antibody 8 and the 3rd layer 13 to the coefft. slightly higher than the coefft. of the buoyant density of the combined matter 10 of an antigen 6 such as virus, the antibody 7 and the antibody 8. If the antigen 6 is contained in a specimen (for example, serum), the antigen 6 increases molecular weight by reacting and combining with the antibody 7 and settles quickly in the 2nd layer 12. The antigen increases further its molecular weight by reacting and combining with the antibody 8 in the 2nd layer 12 and settles in the 3rd layer 13. The antigen causes an enzyme-substrate reaction and the ELISA method is thus automatically executed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a solid phase immobilization reaction method in which media containing reaction reagents added sequentially are layered, and reaction products are separated and measured using the media.

In various qualitative and quantitative measurements, reaction reagents must be added sequentially in order to cause the reaction to proceed stepwise. In addition, if there are many reaction steps, it may be necessary to temporarily stop the reaction or remove the reaction solution depending on the complexity of the order of addition and the reaction system, so it is important to understand and familiarize yourself with the reaction system. requires an operator who is familiar with the

This generally poses a major obstacle when utilizing the reaction system and providing versatility.

For example, enzyme immunoassay (Enzyn+e L 1n
kedI mmunosorbent A 5saV
: ELISA), when measuring antigen, addition of antibody to the solid phase and washing after coating;
A total of 47 operations are required: addition of a sample containing an antigen and washing after the reaction, addition of an enzyme-immobilized antibody and washing after the reaction, addition of a substrate solution, 4 addition operations, and 3 washing operations. be done.

In addition, when measuring chelate compounds by adding a chelating agent, for example, the hardness of water can be measured using ethylenediaminetetraacetic acid (ED
To titrate TA), you must add potassium cyanide solution (10%) to the sample water, add a buffer solution, add Eliofrom Black (EB) T indicator solution, and titrate with EDTA. , requires a four-step operation.

The inventor of the present invention has conducted extensive research with the aim of eliminating the complexity of operations caused by proceeding the reaction stepwise as described above, and as a result of extensive research, the inventor has developed a method of coating or layering the reaction reagent in several layers. The inventors have completed the present invention by discovering that the reactions can proceed sequentially, and that the reactions can proceed sequentially by fractionating the reactants by utilizing changes in the molecular weight of the reactants caused by the reaction.

That is, the basis of the present invention is to coat or stratify a medium in which reagents are dispersed in a medium for each successive reaction according to the reaction order as shown in the following, and to allow the reaction to proceed over time.
Also, by taking advantage of the fact that the molecular weight of the reaction product changes due to the reaction, the medium is formulated so that the change in molecular weight becomes the rate-determining reaction rate within a certain period of time when moving on to the next reaction. There is a particular thing. More specifically, when the intermolecular distance between the primary, secondary, and tertiary products increases, in the reaction process, the primary, secondary, and tertiary products become Taking advantage of the fact that sedimentation constants, buoyant density coefficients, etc. change, and that it is possible to differentiate by sedimentation rate when using a substance with a molecular sieve effect as an immobilization medium, or by the difference in buoyant density coefficients, By controlling the rate of sequential reactions within the reaction system, most of the reaction system is reduced to a solid-phase immobilized reaction system.

For example, as shown in Figure 1, a method of fractionation using the difference in buoyant density coefficients uses a medium such as gelatin or Ficoll that can have an arbitrary buoyant density by adjusting its concentration. The buoyant density of one layer is the same as the buoyant density coefficient of the reactant (e.g. antibody) 3, and the second layer 2 of the lower layer is
The buoyant density coefficient is set to be larger than that of the reactant 3 and smaller than that of the bond 5 of the reactant 3 and the reactant (eg, antigen) 4. The reactant 3 is added to the first layer 1 in advance, but since the buoyant density coefficient of the reactant 3 is the same as that of the first layer 1 and smaller than that of the second layer 2, the reactant 3 is added to the second layer 2. It stays in the first layer 1 without spreading. When the reactant 4 is added to this, the reactants 3 and 4 react to form a bond 5, which settles because the buoyant density coefficient becomes larger than that of the first layer 1 and the second layer 2 due to the increase in molecular weight. The bond 5 transfers to the second layer 2.

Furthermore, as shown in Figure 2, a method for fractionating using the difference in molecular weight uses a cross-linked dextran, porous resin, etc., and the upper first layer 1' adsorbs the molecular weight of the reactant 3. The lower second layer 2' is a material capable of adsorbing or retaining a compound having a molecular weight of 5 of the reactant 3 and the reactant 4. The reactant 3 adsorbed or retained in the first layer 1' reacts with the added reactant 4 to form a bond 5 with a large molecular weight, which is then sedimented and retained in the second layer 2'.

To further explain the present invention by applying it to the above-mentioned ELISA method and chelate titration method, ELISA
For example, when measuring an antigen as described above, seven operations are normally required, but when the solid phase immobilization reaction method of the present invention is used, the complexity is reduced as follows. be able to.

That is, as shown in FIG. 3, the first layer 11 is coated with the antibody 7, the second layer 12 is coated with the enzyme-immobilized antibody 8, and the third layer 13 is coated with the enzyme reaction substrate 9. Coat. At this time, the coating medium for each layer is gelatin, Ficoll, etc., and the first layer 11 has a buoyant density coefficient of antibody 7, and the second layer 12 has an enzyme-immobilized antibody 8.
By adjusting the buoyant density coefficient of the third layer 13 to be slightly higher than the buoyant density coefficient of the conjugate 10 of the antigen 6 such as a virus, the antibody 7, and the enzyme-immobilized antibody 8, if the sample (
For example, if antigen 6 is contained in serum), first layer 11
The enzyme-immobilized antibody 8 reacts with the antibody 7, increases the FWi, and quickly precipitates in the second layer 12.
It reacts with the compound, increases its molecular weight, and precipitates in the third layer 13.
An enzyme-group 6-substance reaction is caused, and an ELISA method can be performed, and the number of operations can be reduced to one. Here, the ELISA method can be performed according to the same principle by using, for example, crosslinked dextran, which has a sieving effect depending on molecular weight, as a medium instead of gelatin or Ficoll, which utilizes a difference in buoyant density.

Next, in the case of chelate titration, for example, water hardness measurement, four operations are required as described above, but according to the present invention, the first layer is coated with EDTA and the second layer is coated with EDTA. This can be carried out by preparing a multilayer body in which a buffer solution composition and potassium cyanide are coated on the first layer, and a third layer is further coated with EBT. That is, when a certain amount of sample water is added to this multilayer body, Ca- ions in the sample and EDTA react in the first layer, forming a chelate compound and increasing the molecular weight, which advances to the second layer and then to the second layer. pH adjustment and the like are performed in the second layer, and the process progresses further to the third layer, where coloration by EBT is performed. In this case, unlike titration, the quantitative measurement requires reaction of the sample diluted in stages, but the operation is extremely easy as it only requires one addition.

It should be noted that the solid phase immobilization reaction method of the present invention is not limited to the above-mentioned embodiments, and as shown in FIG.
Antibody 16 and enzyme-immobilized antibody 17 are adsorbed to each medium 14.15 (for example, porous adsorption resin) of 1' and second layer 12', and antigen 18 and medium 14 of first layer 11' are adsorbed.
When the antibody 16 adsorbed on the medium 14 is reacted with the antibody 16, the antigen-antibody conjugate 19 is detached from the medium 14 due to an increase in molecular weight due to the antigen-antibody reaction, moves to the second layer 12', and is transferred to the second layer 12'.
It is also possible to detect it by reacting with the enzyme-immobilized antibody 15 adsorbed on the substrate, desorbing again due to an increase in molecular weight, moving to the third layer 13', reacting with the substrate 20, and causing coloration. Of course, various changes may be made without departing from the gist of the present invention.

As described above, according to the solid phase immobilization reaction method of the present invention, in sequential qualitative or quantitative reaction operations, the medium containing each reaction reagent is sequentially immobilized in a layered manner, and changes in molecular weight due to the reaction are controlled. Since the reaction is carried out in one step and the complicated reagent addition and washing operations required in the past are unnecessary, it is significantly simpler and has improved versatility. Demonstrates excellent cumulative effects.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of one embodiment of the present invention, FIG. 2 is an explanatory diagram of another embodiment of the present invention, FIG. 3 is an explanatory diagram of yet another embodiment of the present invention, and FIG. 4 is an explanatory diagram of another embodiment of the present invention. FIG. 2 is a detailed explanatory diagram of the invention. 1.1', 11 is the first layer, 2.2', 12 is the second layer, 3.4 is the reactant, 5.10 is the conjugate, 6 is the antigen, 7
8 indicates an antibody, and 8 indicates an enzyme-immobilized antibody. −〇−

Claims (1)

[Scope of Claims] 1) A solid-phase immobilization reaction method, characterized in that each medium containing a reaction reagent to be added sequentially is layered one after another, a sample is added, and the reaction is carried out in one reaction operation. 2) A solid phase immobilization reaction method characterized by fractionation and measurement using changes in the molecular weight of reactants due to reaction. 3) The method is characterized in that each medium containing a reaction medium to be added sequentially is layered one after another, a sample is added and reacted to cause a change in molecular weight, and the reaction product with a changed molecular weight is separated and measured. Solid phase immobilization reaction method.