KR100385713B1 - Immunostain buffer - Google Patents

Abstract

The present invention relates to a buffer for immunopathological staining, which can be stained according to the characteristics of a sample to be subjected to tissue staining with a buffer solution containing petal bovine serum to give specificity to the antigen-antibody reaction. Provide a buffer for staining. The present invention also provides the effect of cost savings as well as accurate diagnosis.

Description

Immunostain buffer

The present invention relates to a buffer for immunopathological staining, and more particularly, to a buffer for immunopathological staining, which enhances the binding ability of antigen antibodies and inhibits nonspecific antigen antibody responses during immunopathological examination.

A clinical test that determines the presence or absence of pathology by examining the exfoliated or collected cells from a part of the living body with an optical microscope is a cell morphological test that is effective for malignant tumors and various infectious diseases. After reacting the antibody specifically reacting to the substance to the cell, the dye composition is added to react only to the antigen antibody bound, and then the degree of staining, color, and distribution of the cell is determined through an optical microscope. Stained cells can be diagnosed simply because they have different staining types than normal cells depending on the presence of disease. As a result of diagnosis through this process, as soon as it is directly related to life, it is important to rapidly and accurately stain and staining method capable of reliably reproducing the characteristic of each component of the diagnostic cells. Accordingly, various staining solutions have been developed and commercialized, and medical institutions are diagnosed by performing a dyeing reaction in a short time using a large amount of samples using an autoimmunostiner.

Therefore, it is a matter of course that the dyeability and reactivity of the sample are most important, and dyeing methods have been developed for this purpose, but the dyeing buffer solution that can improve the dyeability and increase the specificity of the antigen antibody while focusing only on the dyeability of the sample Has been neglected.

Staining buffer stabilizes the reaction during staining through antigen antibody reaction, can control the degree of antigen antibody binding and can control the nonspecific binding of antigen antibody. However, currently used Tris buffer or PBS buffer only plays a role in stabilizing the antigen antibody response and does not play a role in controlling the non-specific reactivity of the more important antigen antibody. In addition, the buffer solution sold by the company that developed the autoimmune dye unit is being used at a high cost.

Therefore, the development of immunopathological staining buffer is required to increase the specificity of the antigen-antibody reaction so that a large amount of samples can be efficiently stained in a short time.

Accordingly, an object of the present invention is to provide a buffer for staining that can express the specificity of the antigen-antibody response in immunopathology.

It is another object of the present invention to provide a buffer for staining which can reduce the cost of immunopathological examination.

Figure 1 shows a tissue photograph carried out with an immunobuffer solution of the present invention,

Figure 2 shows a photograph of what was carried out with a conventional immune buffer solution.

In order to achieve the above object, the present invention is characterized by comprising tris buffer saline, Petal bovine serum (FBS), sodium azide, and Triton X-100 (X-100). It provides a buffer for immunopathological staining.

In another aspect, the present invention is a cell staining method comprising the step of reacting the antigen and the antibody of the test cell in the sequence, washing step, secondary antibody reaction step, washing step, and color development step,

(a) using the buffer solution of claim 2 in the step of reacting the antigen with the antibody,

(b) binding the secondary antibody to the antigen antibody reactant using the buffer of claim 3,

(c) using the buffer solution of claim 4 in the color development step, and

(d) In the washing step, a cell comprising the buffer solution containing 0.5 to 3 g of sodium azide, 0.01 to 5 mL of Triton X-100 and 500 mL of sterile water in 500 mL of the buffer solution to be used. Provide a dyeing method.

Hereinafter, the present invention will be described in detail.

The present invention provides a staining buffer for use in immunopathologic tests comprising FBS, sodium azide, and Triton X-10 in TBS solution.

The TBS contains tris buffer saline of 0.05 M, pH 7.6, trizma, sodium chloride, hydrochloric acid, and sterile water. TBS is prepared by adding sterile water to a mixture comprising 6.1 g of Trisma, 8.5 g of sodium chloride, and 3.75 mL of hydrochloric acid to prepare 1 L. The hydrochloric acid is added to titrate the pH of TBS to 7.6 and is not fixed to 3.75 mL indicated above. In addition, the composition of TBS may be adjusted to be used at a ratio that is commonly used among those skilled in the art.

The autoimmunostiner of the present invention or the buffer for immunostaining of cells consists of the following four kinds. _

(1) Buffer No. 1: A buffer used for binding an antigen to an antibody.

(2) Buffer No. 2: A buffer used for secondary antibody reaction.

(3) Buffer No. 3: A buffer used to induce a color reaction by adding a color substrate to the antigen antibody.

(4) Buffer No. 4: A buffer used as a wash solution at the end of each reaction.

The buffer No. 3 is used when reacting a substrate, an enzyme, or the like for the color reaction of a secondary antibody, and it is particularly preferable to use avidine, which is widely used, to the reaction product.

The buffer NO. 4 is a washing step after reacting the primary antibody reactant with the sample; In the washing step performed after binding the secondary antibody to the antibody bound to the antigen of the sample; It is used as a washing solution in the step of washing after inducing color development or fluorescence by adding color substrate to the secondary antibody.

Components and contents of the above buffers are shown in Table 1 below.

TABLE 1

Buffer Ingredient content Buffer No.1 FBS 5 to 25 mL Sodium azide 0.5 to 3 g Triton X-100 0.01 to 5 mL 0.05 M TBS 500 mL including above Buffer No. 2 FBS 0.01 to 5 mL Sodium azide 0.5 to 3 g Triton X-100 0.01 to 5 mL 0.05 M TBS 500 mL including above Buffer No. 3 Sodium azide 0.5 to 3 g Triton X-100 0.01 to 5 mL 0.05 M TBS 500 mL including above Buffer No. 4 Sodium azide 0.5 to 3 g Triton X-100 0.01 to 5 mL Sterile water 500 mL including above

The FBS of Table 1 of the present invention is a petal bovine serum (fetal bovine serum) in combination with sodium azide and Triton X-100 to inhibit non-specific antigen antibody binding when antigen-antibody binding.

The immunopathological staining buffer of the present invention exhibited the same stainability as that of the conventionally used expensive buffer solution. Therefore, the immunopathological staining buffer of the present invention can be used for autoimmune staining or cell immunostaining to express the specificity of the antigen-antibody reaction, thereby facilitating accurate diagnosis. In addition, the buffer of the present invention can reduce the test cost of immunostaining due to the cost-saving effect.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1

After dissolving by mixing 800 mL of sterile water in 6.1 g of Trisma (T-8524) and 8.5 g of sodium chloride (sigma; S-9888), the pH was adjusted to 7.6 with hydrochloric acid (NaOH), followed by In total, 1 L of 0.05 M TBS was prepared.

Preparation of Buffer No. 1

12.5 mL of Petabovine Serum (FBS, GIBCO; NO. 26140-079), 1.5 g of sodium azide (sigma; S-2002), 0.4 mL of Triton X-100 (sigma; X-100) Then add TBS to make a total of 500 mL of buffer.

Preparation of Buffer No. 2

1 mL Petalbovine Serum (FBS, GIBCO; NO.26140-079), 1.5 g sodium azide (sigma; S-2002), 0.2 mL Triton X-100 (sigma; X-100) Then add TBS to make a total of 500 mL of buffer.

Preparation of Buffer No. 3

Mix 1.5 g sodium azide (sigma; S-2002), 0.2 mL of Triton X-100 (sigma; X-100), and add TBS to make a total of 500 mL of buffer.

Preparation of Buffer No. 4

Mix 1.5 g of sodium azide (sigma; S-2002) and 0.2 mL of Triton X-100 (sigma; X-100) and add sterile water to make a total of 500 mL of buffer.

[Comparative Example]

A commercially available bufferkit from DaKo ChemMate was purchased.

Experimental Example

The cytoplasm was stained with the immunobuffer solution of Example 1 and Comparative Example. The buffer solution of Example 1 and the comparative example was composed of four types, respectively, and each was used in each of four stages. Human tissues were stained according to the manual of autoimmune staining machine and the results were compared. Figure 1 is a result of the immunobuffer solution of the present invention and Figure 2 is a photograph of what was carried out with the conventional buffer. Figure 1 and Figure 2 shows the same staining, the immunobuffer solution of the present invention expresses the specificity of the antigen antibody reaction as it facilitates the diagnosis of the test.

As mentioned above, the immunopathological staining buffer of the present invention can be used for autoimmune staining or immunostaining of cells to increase the specificity of the antigen-antibody reaction and facilitate the diagnosis by accurate staining. In addition, the buffer of the present invention can reduce the test cost of immunostaining due to the cost-saving effect.

Claims (5)

(delete) In the immunopathological staining buffer used for antigen and antibody binding, The buffer comprises 5 to 25 mL of Fetal Bovine Serum (FBS), 0.5 to 3 g of sodium azide, 0.01 to 5 mL of Triton X-100 and the balance of 0.05 M TBS to 500 mL Buffer for staining. In the immunopathological staining buffer used in the secondary antibody reaction, The buffer comprises 0.01 to 5 mL of fetal bovine serum (FBS), 0.5 to 3 g of sodium azide, 0.01 to 5 mL of Triton X-100 and the remaining 0.05 M TBS for 500 mL Buffer for staining. In the immunopathological staining buffer used to induce a color reaction by adding a color substrate to the antigen antibody reaction, Wherein said buffer comprises 0.5 to 3 g of sodium azide, 0.01 to 5 mL of Triton X-100 and the remaining 0.05 M TBS for 500 mL of the immunopathological staining buffer. In the cell staining method comprising the step of reacting the antigen and the antibody of the cell to be tested in a sequence, washing step, secondary antigen reaction step, washing step and color development step, In the step of reacting the antigen with the antibody using the buffer of claim 2, In the secondary antigen reaction step using the buffer of claim 3, In the color development step using the buffer of claim 4 and In the washing step, a cell staining method using a buffer containing 0.5 to 3 g of sodium azide, 0.01 to 5 mL of Triton X-100 and the remaining amount of sterile water for 500 mL.