JPH03130664A - Modified amino-acid sensitive immobilized erythrocyte, manufacture thereof and detecting method for antigen or antibody using this erythrocyte - Google Patents

Abstract

PURPOSE:To establish a determing method which indicates high potency simply and is suitable as a screening method by adding modified amino acid to an erythroycte suspension, thereafter adding alhehyde, and performing the immobilization of the erythrocytes and the sensitization of the modified amino acid on an erythrocyte film at the same time. CONSTITUTION:Modified amino acid is added into the erythrocyte suspension. Then aldehyde is added. The immobilization of the erythrocytes and the sensitization of the modified amino acid to an erythrocyte film are performed at the same time. As the aldehydes, glutaraldehyde, formaldehyde and the like can be listed. The glutaraldehydfe is preferable since it indcates excellet reactivity. As the modified amino acid, the amino acids which are modified by phosphorylation, sulfonation, acylation and the like, e.g. tyrosine phosphate and the like, are used. The tyrosine phosphate is especially important in the sensor for detecting an anti-tyrosine phosphate antibody that is most effective for observing the kinetics of tyrosine phosphorylation. Thus, the immobiliztion and the sensitization of the erythrocyle which must have been individually conducted can performed at the same time. Therefore, the sensitive fixed erythrocyte indicating excellent activity can be simply obtained.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to modified amino acid-sensitized fixed red blood cells used for detecting or quantifying modified amino acids, specific antibodies thereof, proteins containing the same, methods for producing the same, and The present invention relates to a method for detecting antigens or antibodies using the same.

The present invention provides, for example, sensitized fixed red blood cells used for the detection or quantification of products of tyrosine phosphorylation reactions known as cancer-related protein modification reactions in vivo or in vitro, specific antibodies thereof, methods for producing the same, and the like. The present invention relates to a method for detecting an antigen or antibody using an antigen or antibody.

(Prior art) As methods for detecting or quantifying hapten antigens and their specific antibodies, enzyme immunoassays and radioimmunoassays using conjugates of haptens and carrier proteins (for example, bovine serum albumin or KLH) are often used. has been done. However, these methods do not use sedimentation and flocculation as the reaction principle, so
Antibodies that show high titers using these methods do not necessarily show correlated high titers when using other immunological methods, especially the immunoagglutination reaction method (precipitation reaction), the immunodouble diffusion method, and the immunoelectrophoresis method. do not have.

In addition, there is a method using a hapten antigen mediated by a carrier protein to measure the anti-hapten antibody titer of an antiserum obtained by immunizing an animal with a carrier protein.

This can only be done after absorption and removal of antibodies against the carrier protein.

In other words, conventional methods such as enzyme immunoassay and radioimmunoassay are used to screen for the production of monoclonal antibodies with high immunoprecipitation activity and to assay animal antisera raised with hapten carrier protein antigens. was not suitable.

(Problems to be Solved by the Invention) It is generally said that antibodies suitable for nine different immunochemical detection methods differ depending on the detection method. The antibodies against hapten antigens such as modified amino acids include hapten-antigens such as modified amino acids.
It is desired to recognize the antigen structure centered on the hapten moiety (or the modified moiety in the case of modified amino acids) of a carrier protein, and to have activities useful for antigen separation 1 analysis, such as antigen precipitation ability. . Appropriate detection methods must be employed to screen for the production of monoclonal antibodies and animal immune serum antibodies that serve this purpose.

Therefore, the present invention can be used for hemagglutination reaction, which is simple, shows high titer, and is suitable as a screening method when the Habten antigen is a modified amino acid.

Sensitized fixed red blood cells, a method for producing the same, modified amino acids using the same, and specific antibodies thereof.

The present invention provides a method for detecting or quantifying proteins containing the same.

(Means for Solving the Problems) That is, the present invention involves adding modified amino acids to a red blood cell suspension, and then adding aldehydes to simultaneously immobilize red blood cells and sensitize red blood cell membranes to the modified amino acids. The present invention relates to a method for producing fixed red blood cells sensitized with modified amino acids, which is characterized by: a fixed red blood cell sensitized with modified amino acids obtained by the method, and a method for detecting antigens or antibodies using the same.

The method for producing fixed red blood cells sensitized with modified amino acids of the present invention allows fixation and sensitization of red blood cells, which conventionally had to be carried out separately, to be carried out simultaneously, thereby obtaining sensitized fixed red blood cells that are simple and exhibit good activity. It is something that can be done.

Modified amino acids include phosphorylation and sulfonation.

Amino acids modified by acylation etc. are used.

For example, phosphorylated tyrosine, phosphorylated serine, phosphorylated threonine, sulfonated tyrosine, acylated tyrosine,
Further examples include synthetic peptides synthesized as a result of antigen site prediction.

Among these, phosphorylated tyrosine has been shown to exhibit the ability to phosphorylate protein tyrosine as an enzymatic activity of many cell membrane receptors for growth factors and oncogene products (R.A., Bradshaw, S., Prentes, eds.; Growth Factors, Published by El Sepia (1987)).

Since tyrosine phosphorylation is involved in a part of the intracellular transmission mechanism, it is particularly important in terms of detecting the most useful anti-phosphotyrosine antibodies to observe the dynamics of tyrosine phosphorylation. be.

To obtain highly sensitive sensitized fixed red blood cells using these modified amino acids, approximately 1 red blood cell per red blood cell.

It is preferable to carry out the reaction at a concentration of the modified amino acid equivalent to or higher than the IQIO molecule. For example, for 5x107 red blood cells, 200μ9 to 2ff! It is preferred to mix g modified amino acids. Here, if it is less than 200μ9, the sensitivity tends to decrease, and if it exceeds 2μ, it is saturated and there is no point in adding it.

After adding the modified amino acid to the red blood cell suspension, aldehydes are gradually added and reacted to simultaneously fix and sensitize the red blood cells.

The aldehydes include glutaraldehyde.

Examples include formaldehyde, but glutaraldehyde is preferred because highly purified products are easily available at low cost and exhibit good reactivity.

The amount of aldehydes to be used is preferably in the final concentration range of 0.1 to 1.0% (W/Vl) to ensure good sensitization and fixation.

Furthermore, the red blood cells used in the present invention are preferably human type 0 red blood cells or sheep red blood cells. Human type O red blood cells are susceptible to non-specific reactions - and high quality and stable sheep red blood cells are readily available, and non-specific reactions are well understood. The final concentration of red blood cells depends on the operability,
In terms of the sensitivity of the obtained sensitized fixed red blood cells, it is 5 to 10%.
(Y/V) is preferable.

Fixation and sensitization are performed for a long time (preferably 16 hours or more)
It is preferable to carry out this procedure from the viewpoint of obtaining sensitized fixed red blood cells with good sensitivity. If the fixation is too rapid, the shape of the red blood cells becomes abnormal (spike-shaped).

In addition, excess aldehydes are Tris-HCl.

It can be blocked by storing at 4°C in 0.14M NaCl (pH 7,4).

The modified amino acid-sensitized fixed red blood cells obtained as described above are used in the antigen or antibody detection method of the present invention.

Detection can be carried out using, for example, a 96-well microtiter plate. For antibody detection, an equal amount of sensitized red blood cells (preferably added to a final concentration in the range of 5 to 10% (v/v)) is usually added to a diluted sample, shaken and stirred, and then used in the apparatus. After 1 hour of Vi for human type O red blood cells and 2 hours after Vi for sheep red blood cells, the agglutination image is observed, and the highest dilution point at which agglutination is determined is determined as the titer of the sample. In addition, for antigen quantification, a known amount (often about 24 to 25) of antibodies is reacted in advance with a sample diluted several times (usually 3 times).
7 °C 1 hr), then final concentration 5-10% (v/v
) Add sensitized red blood cells and shake, then observe the agglutination image in the same way as for antibody measurement. The final concentration that does not cause aggregation is defined as the antigen titer. However, in order to compare titers between experiments, it is necessary to always keep the antibody concentration K constant. For this purpose, it is preferable to use the same sensitized red blood cells, measure the antibody before detecting the antigen, and dilute the antibody solution to the same titer.

(Example) In this example, phosphorylated tyrosine was used as a modified amino acid.

(1) Preparation of phosphorylated tyrosine-sensitized fixed red blood cells Commercially available stored sheep red blood cell blood (30% v/vl 25ml; mixed with 100 μg of Darbetz choline buffer and washed by centrifugation. .

100 ml of a solution (buffer A) containing vanadylate and zinc chloride at a final concentration of 0.1 mM each! Further wash the sheep red blood cells. After repeating this process twice,
Sheep red blood cells are suspended in 100 bottles of buffer A to a concentration of 5% (v/v). The antigen phosphorylated tyrosine is 100■
4 volumes of 0.5 M phosphate buffer (p)i7.4),
After suspending in 0.14 M NaCl, it is added to the washed sheep red blood cell suspension with stirring. Stir to homogenize the red blood cell suspension using a stirrer, etc.

Glutaraldehyde (commercially available for electron microscopy research, freshly opened) diluted to 25% (v/v) with phosphate buffer is added dropwise over 5 hours.

Stirring is continued even after the dropwise addition is completed, and continues for 20 hours or more from the start of the first dropwise addition. After the reaction is completed, phosphorylated tyrosine-sensitized fixed red blood cells are obtained as a sediment by centrifugation. This was washed with Dulbets cholic acid buffer, and further washed with 20mM) Liss-HCl, 0.
Repeat washing three times with 14 M NaCl (pH 7,4). Until use, it is suspended in the above Tris buffer to a concentration of 10% (v/v) and stored at 4°C.

(2) Example of detection of anti-phosphotyrosine antibody using prepared phosphotyrosine-sensitized fixed red blood cells An example of detection is shown in FIG.

The dilution series is shown on the horizontal axis. The antibody to be tested is a standard product of each purification process of immunized rabbit serum prepared using bovine serum albumin-phosphorylated tyrosine conjugate as an immunization material.

Antibodies in each row are shown in Table 1.

Table 1 Each antibody was diluted multiple times to create a dilution series of up to 12 tubes.

Lines 1 to 4 are reaction systems that do not contain antagonists.

Antibody titer is shown. For lines 5 to 8, add 1 antibody in advance.
It was reacted with mM phosphorylated tyrosine, and then the residual antibody titer was measured. The antibody in the third row that showed the highest titer (titer 211) was also completely inhibited by this concentration of phosphorylated tyrosine (row 7). Ir1 (line i11) was not inhibited at all, demonstrating the high specificity of the single method.

As described above, compared to the method of the present invention, the enzyme antibody method using a conjugate of carrier protein and phosphorylated tyrosine does not reflect this agglutination force, resulting in high titer and high agglutination force. It was unsuitable for screening for the production of antibodies, etc.

(Effects of the Invention) The hemagglutination reaction using fixed red blood cells sensitized with modified amino acids of the present invention enables short-time detection and quantification of antibodies to the modified amino acids, proteins containing the same, or modified amino acids. be.

Furthermore, the present invention is useful for screening for the production of high agglutinating titer monoclonal antibodies or immune serum that exhibit high performance in various rice immunological identification methods based on a simple indirect hemagglutination reaction.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph of a microtiter plate showing the characteristics of anti-phosphotyrosine antibody detection of phosphotyrosine-sensitized fixed red blood cells in an example of the present invention. Attachment 2 Engraving dilution series of drawings → 123456789101112 1 book + 2,000 + + + 1 + + 3 +l + + l + +! -1 - 10 sho 14 sho 7 + 10 ± 9 + H - + 10 sho 10 + l + + l + + + l - s 11 +
＋l t＋＋ 廿廿廿＋± 12+Shishishi 1st figure

Claims (1)

[Claims] 1. Modified amino acid sensitivity characterized by adding modified amino acids to a red blood cell suspension and then adding aldehydes to simultaneously immobilize red blood cells and sensitize red blood cell membranes to the modified amino acids. Method for producing fixed red blood cells. 2. The method for producing modified amino acid-sensitized fixed red blood cells according to claim 1, wherein glutaraldehyde is used as the aldehyde. 3. The method for producing fixed red blood cells sensitized with modified amino acids according to claim 1 or 2, wherein phosphorylated tyrosine is used as the modified amino acid. 4. Modified amino acid-sensitized fixed red blood cells obtained by the production method according to claim 1, 2 or 3. 5. A method for detecting an antigen or antibody, which comprises using the modified amino acid-sensitized fixed red blood cells according to claim 4.