JPS6018010B2 - Application of reagents to the medium and equipment therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to analytical devices or tools and methods of using the same, and more particularly to measuring amounts of water-soluble or Apparatus and method for applying water-dispersible reagents to water-containing solid media.

The various molecular types present in a sample can be analyzed to separate and identify them by applying the sample to a solid medium containing water and causing molecular diffusion of the sample into the medium. A method has been developed.

In particular, chromatography and fly aerophoresis, including immunoelectrophoresis, have been used - all of which effect the separation of different molecular types by differential diffusion of a sample through a water-containing solid medium. In such methods, ``various reagents that interact with one or more types of molecules in a sample are also introduced before, during, or during the separation process to aid in the separation or identification of the types of molecules in the sample. These reagents can then be applied to the medium.These reagents have traditionally been introduced into the medium in a variety of ways.

In some cases they are introduced into the medium when it is manufactured, but in many cases they are applied to the surface of the water-containing solid medium when needed, as a liquid solution or dispersion of the reagent. It has been applied by applying and traversing the solid medium to the surface of the medium or by dipping the solid medium into a solution or dispersion of the reagent in a suitable liquid vehicle. In either case, it is difficult and uncertain to accurately measure and control the amount and location of the medium in which the reagents are dispersed.
s) dispersing such reagents in a solid water-resistant binder as in U.S. Pat. No. 3,694,163 to Verbeck; It has also been proposed to keep the adhesive binder in contact with a moisture-containing or moisture-absorbing medium, such as paper.

However, reagents in water-resistant or water-tight binders cannot easily be completely extracted and diffused into a water-containing solid medium, making it difficult to control and measure the amount introduced into the medium. The present invention provides devices and methods that facilitate accurate and quantitative introduction of reagents into solid, water-containing media for use in molecular diffusion or affinity separation methods.

The present invention is particularly useful in conjunction with water-containing solid media in the form of thin layer media, ie, 0.1 to 2 layers thick. The present invention provides for use in molecular diffusion or affinity separation methods.
An apparatus is provided for applying a metered amount of a hydrophobic or water-dispersible reagent to a water-containing solid medium, the apparatus comprising a film-forming solid organic polymer that is soluble in water to an extent of at least 1% by weight at 20 qo. a solid film consisting essentially of a combined binder and a metered amount of the reagent incorporated into the binder film; It is characterized by having a size and shape suitable for being brought into contact with the shell.

The present invention also provides a method for analyzing a sample by subjecting the sample to molecular diffusion separation in a water-containing solid medium and reacting the components of the sample with reagents in the medium, the method being One side of the nuclear medium is placed in contact with the nuclear medium, and this is maintained for a sufficient period of time to allow complete diffusion of the drug and the binding agent into the medium.

Binders that are soluble in water are intended to include those substances which form colloidal solutions or dispersions as well as those which form true solutions.

Binders that can be used in the present invention include various polymeric substances such as dextran, water-soluble polyacrylamide, polyacrylic acid and its water-soluble metal salts, and water-soluble polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyvinyl Pyrrolidone "clarified guar gum ~ water soluble carboxymethyl cellulose water soluble hydroxyethyl cellulose ~ water soluble methyl cellulose" also algin "laginan" Water-soluble copolymers with various pinyl monomers to copolymers of maleic anhydride with vinyl ethers or vinyl esters or their corresponding salts are included. Dispersants may also be present to maintain the flexibility of the binder and to facilitate or accelerate its dispersion or dissolution in water.

The polymeric binder film may be of any desired thickness, but is preferably 0.01 to 2 thick.

Temporary removable supports in the form of paper or water-insoluble plastic films or imprints may also be provided.
Such supports or supports are only important for very weak or brittle films and can be omitted in the case of self-supporting films. If a support or backing is used, it is desirable that it be transparent. Suitable bushings include those made of plastics such as polyester, polystyrene, cellulose acetate, polyamide, etc. in various thicknesses. The thickness of the dowel is usually kept to a minimum to minimize cost, but at the same time to provide the desired mechanical reinforcement or strength. The degree of binding of the reagent-binder film to the diamond film is not critical. Sufficient bonding is usually obtained if the binder film is formed in situ from a solution or melt on the surface of the backing film. The reagents that can be incorporated into the polymeric binder film can be any water-soluble or water-dispersible substances, many of which are commonly used in analytical methods, such as antibodies, Antigens, enzymes, enzyme substrates, precipitin brighteners, colorants, precipitants, microorganisms and/or their nutrients, buffers, salts, etc., as well as radioactively labeled or fluorescent reagents of the above types.

The relative proportions of reagents and water-soluble polymer binder in the apparatus can vary widely, depending on size or metering as desired and a matter of choice or convenience. Typically, the amount of water-soluble polymeric binder is approximately 1% of the film.
It is most convenient to use devices in which the proportion is 0 to 95% by weight, with the reagent accounting for the remainder. It is also possible to use two or more different reagents mixed with each other.

Mixtures of two or more different polymeric binders can also be used, although there is usually no advantage in using such mixtures. It is noted that although the reagents can be incorporated into the polymeric binder film in a variety of ways, it is usually important to distribute the reagents as uniformly as possible throughout the polymeric binder material.

The reagents can be mixed with the polymeric binder while the polymeric binder is in molten form or in the form of a solution in an effervescent solvent, after which the mixture is formed into a film of desired thickness. and dry or cool to solidify the film. Films of water-droppable polymeric binders can also be formed separately from solutions or melts of the binder; a solution or dispersion of the reagent in a suitable liquid vehicle is then applied to the surface of the film, and the film is and allow the film to dry. In some cases, the reagent in dry, fine particulate form can be spread onto the surface of a film of water-soluble polymeric binder, and the film is then melted and resolidified. Forced air drying can usually be used to incorporate the reagents into the film, but vacuum- or freeze-drying can also be used in the case of heat-sensitive materials.The dimensions of the bag of the present invention are selective. This will depend on the nature of the analytical method being performed.

In general, it is not convenient to use foils with film dimensions smaller than about 5 mm square, but smaller foils may be used in special cases where it is desired to retain the reagent in a small local area of the medium. Larger containers can also be used, including those large enough to cover the entire surface of the medium in which the reagents are applied, even on the order of 16 square inches or more. The film can be of any desired shape, including annular or perforated.The device of the invention can be used for molecular diffusion or affinity separation methods such as chromatography and tortoise sardine, especially gels, membranes. For use in applying reagents to any water-containing solid medium used in the form of thin layers (i.e., maximum thickness of 2 layers), whether in the form of cellular products or tissues. I can do it.

Of particular interest are hydrated gels made from agarose-agar, polyacrylamide (cross-linked to prevent dissolution), or cellulose acetate, and also membranes of paper and cellulose acetate. When using the apparatus of the present invention, the side of the reagent-containing film is simply placed in contact with the moisture-containing solid medium, and complete dissolution or diffusion of the reagent and polymeric binder occurs and the reagent and binder remain in the medium. keep it in that state until it is dispersed. This step is usually carried out at room temperature, but other temperatures from 1°C to 6000°C can also be used. If a water-resistant backing or support is applied to the film as part of the device, it can be tacked onto the surface of the water-containing medium, or if desired it can be used as a reagent and binder. It can also be removed when the diffusion is complete. The book can also be used to introduce reagents into the medium used for bacterial culture, and can also be used for tissue staining by bringing a dye-containing device into contact with the tissue. The following examples are merely illustrative of the essence of the invention and do not in any way serve to limit the scope of the invention.

Example 1 A solution of lactate dehydrogenase (LD) identification reagent was prepared from a commercially available substrate-dye and buffer mixture.

To this solution, a 0.5% aqueous solution of polyethylene glycol 4000 binder (9 parts) was added, and the mixture was stirred. Next, 31〆 Umbrella of Mizufuko Bonito and hydrophilic polyethylene terephthalate sold under "This mixed solution 5 I also r Cronal (Cronate)"
The mixture was uniformly introduced onto a 4-inch-sized blade or support buoy and rapidly dried in a forced-air oven at 48-fins.
A H film was formed. A conventional hydrated agarose electrophoresis sardine medium (a layer of hydrated agarose 1 to 8 thick on a support of Clonal film) was equilibrated with a standard aqueous buffer solution, inoculated with human serum, and exposed to air. I was good at swimming.

After the aerophoresis was completed, the 18H-binder film was laid out on the agarose medium, lined side out, and the sandwich assembly was placed in a 37G salt chamber for 1 hour. If a visible purple line appears in the medium, this is also LQH.
indicates the position of the serum component that reacted. The backing film was then removed from the media surface and the media was briefly washed under running water and then soaked in a 5% by weight aqueous acetic acid solution to fix the colored lines and dried to form a permanent record as desired. Then, the reagent binder film after preparation can be stored in a bran eye before use by keeping it in a 3 mil polyethylene bag with a capacity of 4 to 800 mm.

Example 2 A reagent-binder film of the same type as in Example 1 was prepared, but in this case polyethylene glycol 400
A solution of 0 was spread separately onto a backing film and dried.

The LDH visualization solution (1 layer) was then evenly distributed over the surface of polyethylene glycol 4000 and dried. The device thus made was used in the same manner as described in Example 1. Example: To 10 pieces of a 0.5% aqueous solution of a polyvinylpyrrolidone binder, 0.4' of anti-human serum produced in goats was added as a reagent. Distribute onto a 4 x 4 inch kojichi film as in step 1 and heat in a forced air oven for 25 minutes.
A film of the reagent mixture with a thickness of approximately Q1 was formed on the backing film. It was used for electrical immunodiffusion by bringing the surface of a planar hydrated agarose film medium into contact with a reagent mixture film.

After 3 cycles at room temperature, the koji-uchi film was removed, the residual liquid was washed from the sample well, and the serum sample was placed in the well. After electrophoresis between 4 junctions at 100 volts, The desired visible r-rocket precipitate pattern appeared at the locations where interaction between the reagent and serum components occurred.

EXAMPLE 4 A reagent-binder film prepared as described in Example 1 was prepared using sodium chloride Q.

85% sodium azide and 0.05% sodium azide into which two wells of two columns in diameter were holed for radial immunodiffusion. I tried to make it come in contact with something.

After 30 minutes at room temperature, the backing film was removed and residual liquid was removed from the wells. Five human serum samples were placed in a well and the gel medium was placed in a humid chamber. After 8 hours, the gel showed the desired precipitin ring around the well, which was removed after diffusion. It shows the level of the reaction product between the reagent and serum. EXAMPLE 5 A reagent-binder film useful for the methods described in Examples 3 and 4 consists of ~ antiserum 0.4 side Z and polyvinylpyrrolidone binder Q. A 5% aqueous solution is mixed with 10' of a 5% aqueous solution. This mixed solution is lyophilized and the lyophilized solid is placed on a plastic backing of either a wet polyvinylpyrrolidone film layer or other contact adhesive. It was also possible to make ~ by expanding it.

EXAMPLES The same procedure as in Examples 3 and 5 was followed, but in this case normal human serum with no antigen was used as the reagent in the binder film and antibodies were used as the test sample.

Example 7 The same procedure as in Example 3 was used, but in this case the polyvinylpyrrolidone binder was replaced with polyvinyl alcohol, polyethylene oxide, polyethylene glycol 20,000 polyacrylamide (in water), sodium alginate, methylcellulose, clarified guar gum, respectively. , water-soluble hydroxyethyl cellulose, xanthan gum, soluble starch and dextran were used as binders.

Additionally, instead of backing films, films of polyvinylidene chloride (Saran-wrap), polycarbonate, polymethyl methacrylate, and cellulose acetate were used, all of which were water-impregnable. A supporting film was prepared from the above mixture by using a polytetrafluoroethylene film (Teflon) as a backing and then removing the dried reagent-binder film from the hair band before use.Example 8 Dyes for Lipoproteins Fatt.

Dissolve 0.2 parts of Red 7B (Sigma) in 0.2 parts of polyethylene glycol 400 and mix the mixture thoroughly with melted polyethylene glycol 40.
The mixture was then rapidly and uniformly spread onto a 31'4 x 4 inch backing film of hydrophilic polyethylene terephthalate and cured.
The tofilm was used without any other changes by placing the reagent-binding agent surface in contact with the surface of a hydrated agarose gel medium in which serum proteins had previously been subjected to electrophoresis. After 10 minutes of contact at room temperature, the shot film was removed and bands of reacted poproteins were visible in the medium. In another embodiment ~ the reagent-binder mixture contains a nonionic surfactant, namely Trito.
n) Octylphenoxyboethoxy (9-10) ethanol sold under the trade name x-100~00
12 was added.

Reagent-binder films made in this manner exhibited more rapid diffusion into the agarose gel medium. Example 9 0. Mix equal volumes of each of a 1% aqueous solution of ionic strength bait 8.2 barbital buffer and polyethylene glycol 400M compound and spread this mixture 5' onto the surface of a 4 mil hydrophilic polyethylene terephthalate backing film. Pour and dry in a forced air oven at 40 qo.

The resulting reagent-binder film, about 0.5 mm thick, is placed in contact with the surface of the hydrated agarose gel film medium, about 2 mm thick, and the reagent-binder film is separated from the sinter body. I left a film of the shot by my side. After 10 minutes at room temperature,
The cast film was removed, and the gel medium was now ready to receive the test sample prior to the incubation process. Example 10 Dextran Sulfate A specific precipitant for lipoproteins can be used when incorporated into a reagent-binder film.

Approximately 0.5 ml of dextran sulfate reagent was dissolved in 1 ml of water and uniformly spread on the surface of a pre-prepared and dried polyethylene glycol 400 thickening film with a thickness of approximately 0.25 ml, and then forcedly evaporated. Dry in oven at 40 qo. Human serum samples were electrophoresed in a conventional hydrated agarose gel medium approximately one cell thick supported on a hydrophilic polyethylene terephthalate film backing.

After the pneumophoresis is completed, the surface of the reagent-binder film is placed in contact with the gel medium and left in this state for 1 hour at room temperature.
After holding for 5 minutes, the surface of the medium was washed with water. A white band of the precipitated reaction product of riboblotin and dextran sulfate was visible in the medium, the measurement of which allowed the determination of the amount of lipoprotein. Example 11 1:5 alpha 0 antitrypsin in 0.85% saline solution.

Claims (1)

[Claims] 1. A method of analyzing a sample by subjecting the sample to a molecular diffusion separation operation in a water-containing solid medium and reacting components in the sample with reagents in the medium, the method comprising: % of a film-forming solid organic polymer binder mixed with a measured amount of a water-soluble or water-dispersible reagent is prepared, and one side of the film is covered with the water-containing material. A method for analyzing a sample, characterized in that it is placed in contact with a solid medium and maintained in contact for a sufficient period of time to ensure complete diffusion of the reagent and binding agent into the medium. 2. The method of claim 1, wherein the film has a supporting backing of water-insoluble synthetic plastic bonded to the other side. 3. The method of claim 1, further comprising the step of removing the supporting backing after diffusion is complete. 4. A solid film consisting essentially of a film-forming solid organic polymeric binder that is soluble in water at 20°C to the extent of at least 1% by weight, and a measured amount of a reagent mixed into the binder film. for molecular diffusion or affinity separation operations, characterized in that the film has dimensions and shapes adapted to be placed in contact with the medium for complete diffusion of the reagents and binders into the medium. A device for applying a metered amount of a water-soluble or water-dispersible reagent to a water-containing solid medium for use. 5. The device of claim 4 including a supporting backing of water-insoluble synthetic plastic bonded to one side of the film. 6 The binder is dextran, polyacrylamide, polyacrylic acid, polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, guaya gum, carboxymethylcellulose, hydroxyethylcellulose, methylcellulose, algin,
5. The device of claim 4, wherein the device is selected from the group consisting of carrageenan, xanthan gum, starch, and a copolymer of maleic anhydride and vinyl monomer.