JPS60147650A - Formation of supernatant liquid from specimen - Google Patents

Abstract

PURPOSE:To remove easily and quickly unnecessary cells, etc. from the inside of a specimen contg. cells without the need for intricate machines and instruments by bringing a carrier fixed with cell agglutinin and said specimen into contact with each other. CONSTITUTION:Cell agglutinum such as concanavalin A, leukoavalutinin or the like is fixed to a carrier of a water soluble high polymer material such as polystyrene, agar or the like or an inorg. material such as glass or the like which can fix stably the cell agglutinin without spoiling the activity thereof. The cell agglutinin fixed to the carrier is brought into contact with a specimen contg. cells such as blood, lymph fluid, bodily vaginal fluid, urine or the like drawn from a patient. The supernatant from which the unnecessary cells, etc. removed is thus formed in short time without requiring intricate machines and instruments.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to a method for removing unnecessary cell tubes from a specimen to produce a supernatant.

[Technical background of the invention and its problems]

When performing biochemical analysis or immunological analysis as part of a clinical test,
The test sample is often a supernatant fluid of a specimen, such as blood, body cavity fluid, urine, etc., collected from a patient and containing various cells. In addition, in the field of serology, non-specific antibodies in the specimen are removed by adding red blood cell ghost membranes or fixed red blood cells to the specimen as a pretreatment for the immune reaction, and the resulting clear liquid is used for the immune reaction. There is b. (41 Physical Chemistry Experimental Methods Vol. 15 P, 112 Gakkai Publishing Center (1981))
In either case, it is necessary to generate and collect a clear liquid from the specimen. The general method for producing the supernatant liquid is the 1j method involving centrifugation or filtration using a membrane filter or the like.

However, although the method for producing supernatant liquid as described above is reliable and effective, it requires special machines and equipment, and is laborious and time-consuming. When using membrane filters, clogging with cells or cell debris is an important issue.
This is a problem and costs a lot of history.

On the other hand, there are many substances that naturally cause strong agglutination of G, L, cells, especially red blood cells. These substances are collectively called lectins. Lectin itself is a glycoprotein that recognizes various sugar chains on the cell surface and adsorbs there, which is thought to cause cells to aggregate. As a technique that utilizes such properties of lectins, there is a cell fractionation method using affinity i chromatography. In this method, for example, various lectins are immobilized on the surface of a dextran piece, and then a sample containing various cells is allowed to flow down an IR column filled with dextran beads. J due to differences in affinity
Then, the various ff1l cells in trial r1 are fractionated to obtain the desired specific cells. Dextran pieces on which various lectins are immobilized are commercially available, for example, from Pharmacia Shoninori of Sueden and commercially available as C1.

In addition to the lectins, there are antibodies that recognize the cells as antigens, which are called cell aggregins. Techniques using this antibody include blood type determination using 14F blood coagulation reaction.

Therefore, after diluting 15 l of supernatant from the specimen,
One possible method is to agglutinate, for example, red blood cells in a specimen by adding a lectin or a specific antibody as a single substance.

However, excess lectin antibodies may remain in the supernatant, and if lectin caps remain in the supernatant, the next J3 test will be adversely affected. !
For example, in the case of hemolysin measurement, the reaction system includes sheep red blood cells, hemolysin (anti-sheep red blood cell antibody), and complement. Complement in this case refers to guinea pig serum. Mol Nanatsu 1
~Serum originally contains heterophilic antibodies, such as Forsmann antibodies, and if used as is, it will hemolyze sheep red blood cells regardless of the presence or absence of hemolysin! I end up. Therefore, in advance, guinea pig serum and fixed sheep red blood cells (sheep red blood cell membranes treated with glutaraldehyde etc. to prevent hemolysis) or ghost membranes of sheep red blood cells, etc. Work to adsorb antibodies is performed.

At this time, lectins can be applied to separate fixed red blood cells and ghost membranes, but if excess lectin remains in the supernatant, red blood cells will agglutinate during the next reaction, that is, the lysis reaction. This will impede the reaction efficiency and suspension.

[Purpose of the invention]

This invention was made based on the above circumstances,
The object of the present invention is to provide a method for simply and quickly removing unnecessary cysts III from a specimen without using complicated machines or instruments.

(Summary of the Invention) The outline of the present invention for achieving the above object is characterized in that a sample containing cells is brought into contact with a cell agglutinin that is fixed alone.

[Embodiments of the invention]

This invention prevents aggregation of cells by bringing a specimen containing cells into contact with a cell agglutinin fixed on the body.
Obtain the supernatant.

Examples of specimens containing cells include blood, lymph, body fluid, urine, etc. collected from a single person. , including cells such as cell membranes.

Cell agglutinants include, for example, concavalin A10 icobartinin, wheat germ lektin, soybean lectin, peanut lectin, pine mushroom lectin, lima bean lectin, lentil lectin, pea lectin, fava bean lectin, di-11 gyomolectin, himalectin,
Snail lectin, galactose-binding protein isolated from the liver, protein A1, and other lipoproteins derived from the cell walls of various bacteria)
Examples include lectins and antibodies directed against the cells to be removed, such as anti-sheep red blood cell antibodies when the cells are sheep red blood cells.

The carrier used in the present invention may be any carrier that can impair the activity of the cell agglutinin (for example, poly-Frene, Dextran, cellulose, polyacrylamide, nylon
In, agar, starch, geratin, -" lagen, -1
-1~Water-insoluble polymeric substances on paper, water-soluble polymeric substances such as polyvinylalnylene, polyvinylpyrrolidone, soluble starch, polyamino acids, polyethylene glycol, glass, and inorganic substances on stainless steel hats. Can be mentioned. If glass is selected as the carrier, for example, a glass zamble cup can be used as the carrier, and the sample can be collected by simply placing the sample into the glass rimple cup that stably binds the cell agglutinin to the inner wall surface. Since the cells in the sample aggregate and settle, the process for obtaining the liquid can be easily replaced with step 2.In addition, if Suanless Steel is selected as the carrier, the 1J sample cup containing the sample is exposed to the magnetic field. By directing inwards, cells can be rapidly agglomerated and sedimented.Furthermore, water-insoluble polymers can also be good carriers for lectins, as mentioned above.However, when these insoluble carriers are used, The contact of lectins floating in the test solution is limited. Therefore, from the viewpoint of cell removal, it is not necessarily efficient to cover these insoluble cells. - When a water-soluble hemomolecule is used as a carrier for lectin, since the carrier itself is water-soluble, it is immobilized and there are more opportunities for contact between the lectin and the cells, and the carrier molecule itself becomes flexible. To have,
The cells can be adsorbed more efficiently. Furthermore, the cells that have reacted with the lectin aggregate and precipitate, but at this time, they also take in the lectin-immobilized water-soluble polymer that binds to the surrounding cells in a small number of cells, and become a huge clump and precipitate.

Therefore, by using lectin 11 and a τ water-soluble polymer, the cells can be efficiently removed and a clean supernatant containing no excess lectin can be obtained.

As a method for immobilizing cell agglutinin onto the carrier, conventionally known methods applied to immobilization of enzymes can be adopted.

Contact between the specimen containing cells and the cell agglutinin immobilized on the carrier can be carried out, for example, by adding a carrier that immobilizes the cell agglutinin to the specimen. Then, when the added liquid is cooled to 10° C. or higher, preferably around 4° C., the cells can be aggregated and sedimented in about 5 to 10 minutes, depending on the case.

As a result, a precipitate settles in the lower part of the added liquid, and a clear liquid forms in the upper part, so that this supernatant liquid can be separated by a sampling means such as a pipette or a suction nozzle.

Next, the present invention will be further embodied by accepting experimental examples of the present invention.

<Experimental Example> When performing an immunoassay, if a heterophilic antibody of A. fulsun antibody is present in the test serum as a specimen, the immunoassay may not be performed accurately. For example, when measuring hemolysin, a typical example is the absorption treatment of heterophilic antibodies for complement surface clean guinea pigs 1 to 2 (T'i).
For example, detyped sheep red blood cells may be added to the test serum for incubation.

The method of this invention is used to remove this heterophilic antibody.

Concabuvaline was used as a lectom, and it was immobilized on polyvinylnofluor into which an amino group had been introduced using water-soluble carbodiimide. Therefore, a red blood cell ghost membrane was added to the collected test serum, and after the heterophilic antibodies were absorbed by the ghost membrane, 1 d of test serum containing S]-Roma (5 my protein) was obtained. Next, add 0.2 ml of physiological saline in which polyvinyl alcohol carrying capvalin A is dissolved in the test serum 1d. After stirring, the mixture was cooled to 4° C. and allowed to stand for 5 minutes. As a result, the ghost film was completely precipitated, and a clear liquid containing no ghost film could be obtained. It was also confirmed that the supernatant liquid did not contain 11-linkerovaline A.

<Comparative Example> Serum obtained in the same manner as in the above experimental example was mixed with 1.0% of physiological saline.
211117 was added and left to stand after stirring, but the -1-strike crotch did not settle even after 30 minutes.

〔Effect of the invention〕

Claims (1)

[Claims] 1. A method for producing a supernatant from a specimen, which comprises bringing a specimen containing cells into contact with a cell agglutinin immobilized on a carrier.