JPS60179658A - Production of protein-bound filter paper - Google Patents

Abstract

PURPOSE:To manufacture protein-bound filter paper which has good sensitivity and accuracy and is adequate for a radioimmunoassay by bringing the carboxyl methyl group of the filter paper consisting of carboxymethyl cellulose and protein into reaction in the presence of a specific condensated matter. CONSTITUTION:Protein-bound filter paper is manufactured by bringing the -OCH2COOH of filter paper consisting of carboxymethyl cellulose and protein such as tumor marker protein 1-ethyl-alpha-fetoprotein or the like, specific antibody, etc. into reaction in the presence of water soluble carbodiimide such as 1-ethyl- 3-(3-dimethyl aminopropyl)-carbodiimide hydrochloride, etc. as a condensating agent. The filter paper obtd. in such a way has high sensitivity and good accuracy and low-non-specific reaction in a radioimmunoassay of alpha-fetoprotein, specific antibody, etc. and is therefore adequate for diagnosing pregnancy and diseases. The condensating agent has no toxicity. The protein-bound filter paper is thus safely obtd.

Description

[Detailed description of the invention] The present invention relates to a method for producing a novel protein-binding filter paper.

Many useful radioimmunoassay solid phase supports are in use today. For example, Sephadex, Sepharose 4B, F paper, polystyrene tubes, glass beads, etc. are used. Among these, Sephadex, Sepharose, and filter paper are all made by bonding bromocyanide and then protein, and the manufacturing process can be completed in a short time using this method. In addition, bromic cyanide has drawbacks such as toxicity.

The present inventors used F paper made of calhoki polymethylcellulose, which had not been used conventionally, as a support for solid phase method for radioimmunoassay, and obtained the protein by reacting H3 on it in the presence of a condensing agent. The present inventors have completed the present invention by discovering that the filter paper has a sensitivity and accuracy comparable to that of conventional protein-binding filter paper produced by the Bromsian method.

That is, the present invention consists of a method for producing F paper bound to flea white matter by subjecting the carboxymethyl groups of a filter paper made of carboxymethyl cellulose and proteins to anti-F5 in the presence of a condensing agent, and this production method is easy to operate. In addition, the reagents used during production are low in toxicity and have extremely high sensitivity and accuracy for use in radioimmunoassays.

The F paper used in the present invention may contain carboxymethyl cellulose and other components such as a binder. As a commercially available product, Toyo F Paper Lot No. 2
The O protein, exemplified by 1260121, may be any protein having an amino group that can react with a carboxymethyl group in the presence of a condensing agent, and those used as marker proteins in radioimmunoassay are usually used. Examples of proteins include tumor marker proteins such as α-fetoprotein, calcitene, and gliotinoxactigen, and specific antibodies.

The reaction between CMF paper and protein is carried out in the presence of a condensing agent.
As the condensing agent, l-ethyl-3-(3-dimethylaminoprobyl)-carbodiimide, Hct@olyosol,
Examples include Karl diimides.

In this reaction, the condensing agent and the protein may be added to the CMF paper at the same time, but it is preferable to first add the combination agent to the CMF paper and then add the protein. The carboxyl group of carboxymethyl cellulose is activated, and the amine group of the protein reacts with it to form an acid amide bond.

pHI at the time of reaction/'i. Usually pH is around 4-5,
Carboxymethyl cellulose and condensing agent are first adjusted to pH 4-5.
It is preferable to activate the acid amide bond at a pH of about 7.5 to 9, especially about pH 8 after activation at a pH of about 5, especially at a pH of about 5.
When an acid amide bond is formed, the bond between proteins is suppressed, and the carboxyl group of CMF paper and the amine 7 group of the protein react with each other. Such adjustment of p) to l is carried out using a phosphate buffer or the like.

The reaction time is usually about 4 to 6 hours, preferably 10 to 1
Although the activation time is about 5 hours, the activation of carboxyl groups is about 1 to 4 minutes, preferably lj: about 2 minutes.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 CMF paper with a diameter of 5 mm [Toyo F Paper Lot NO-21
260121) 100 discs 0.5 NHC46
0rnt for 45 minutes to form an H-type, then washed with water, and immersed in 6 mt of phosphate buffer (pH 5) to form a l-
Add 240 tnf of ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. At this time,
The final concentration of phosphate buffer is 12.57 nM. After gently shaking the reaction lacquerware for 2 minutes, 4.8 mV of anti-α-fetoprotein antibody was added to phosphate buffer (pH
8) Add the dissolved material to 8mt. At this time, the final concentration of the phosphate buffer should be 0.2M. The F paper was gently shaken at room temperature for 12 hours and washed with physiological saline to obtain anti-α-fetoprotein antibody-bound CMFM.

Example 2 Anti-alpha
- Fetoprotein antibody-bound CMF paper was obtained.

Example 3 In Example 1, the anti-α-fetoprotein antibody was
) Anti-α-fetoprotein antibody-conjugated CMF paper was obtained in the same manner as in Example 1, except that the time for the reaction was shortened to 6 hours.

Examples 4 to 6 Anti-α-fetoprotein antibody-bound CMF paper was obtained in the same manner as in Example 1 except for #1 in which the amounts of antibody and carbodiimide in Example 1 were set as shown below.

4 4, 8 mW 240r1 5 4, 8 mlF ctotn9 6 2, 4 mW 6 0 mW Reference Example 1 The same procedure as Example IK was carried out in Example 1 except that carbodiimide was not added and the amide bond formation time was changed to 6 hours. went.

Reference example 2 Toyo pl#. tit diameter 5 mm. No. 51 using Ceska5 (Immunochem, 9.10
21. (1972)), anti-α-fetoprotein antibody-bound CMF paper was obtained by the Bromsian method.

Reference Example 8 In Reference Example 2, the same operation as in Reference Example 2 was performed except that bromcyan was not used.

The following experiments were conducted on the protein-binding disks obtained in Examples and Reference Examples.

In addition, in the following experiments, radioimmunoassay was performed by the following method.

(Radioimmunoassay) The anti-α-fetoprotein antibody binding disk of Example 1 (or Example 2.8.
4, 5.6, the disk of Reference Example 1.2.8) and then add α-fetoproin of various concentrations (1 mt prepared with normal horse serum or pregnant women's serum).
After shaking at room temperature for 8 hours, vacuum the well.
Remove the liquid inside. Add 250μ of physiological saline and shake for 1 minute to wash the filter paper (this is done 3 times). Iodolabeled anti-α-fetoprotein antibody 0.1 mt (number of counts =
T) and shake gently at room temperature for about 24 hours. Add 250 μt of physiological saline solution, shake for 100 minutes, and then wash by suction (do this 8 times). Transfer the filter paper to a test tube and measure the count (count number - B. B/TX
IOO=B/T%).

Experimental Example 1 The sensitivities of the radioimmunoassays obtained in Example 1 and Example 2 were compared, and the results are shown as a graft in FIG. In FIG. 1, Δ is that of Example 1,
The opening is similar to that of Example 2.

As is clear from this result, in Example 1, 10n
It is possible to measure concentrations up to around r.

Experimental Example 2 The calibration curves of the radioimmunoassays obtained in Example 3 and Reference Example 2 were compared and shown as a graph in FIG. In FIG. 2, △ indicates that of Example 3, M indicates that of Reference Example, ○ indicates that of Reference Example 2, and . indicates that of Reference Example 8.

As is clear from this result, the Bromsian method of Reference Example 2 has a high B/T (ch) Specific reactions are also low.

Experimental Example 3 The calibration curve of the radioimmunoassay obtained in Examples 4 to 6 is shown as a graph in FIG. In Fig. 8, △ is for Example 4, ○ is for Example 5, ×
shows that of Example 6.

As is clear from this result, in the method of the present invention, even if relatively small amounts of protein and carbodiimide are used, a product that can be used sufficiently can be obtained.

Experimental Example 4 The α-fetoprotein concentration obtained in Example 6 was O-10 Ont/ml, 100-20
The serum was diluted into pregnant women's serum kX4, X8, and X16 corresponding to Qnff/mL and 200-800 nl/mt, and the radioimmunoassay standard curve was plotted and shown as a graph in FIG. In Figure 4, Δ is 200-300nr/mtk, O is 10O-200nf
/mt f, X is O-10On? /mL. As is clear from this result, even in the dilution test,
It showed satisfactory linearity.

Experimental Example 5 Calibration curve of α-fetbrodi/ according to Reference Example 2 and α-7 ethobrodi / according to Example 6 for 11 pregnant women's serum
Inspection: it! The correlation between α-Fue I and protein content is calculated from each of them: V = 0.97° Y = 0.92X
+7.79, indicating a significant correlation (see Figure 5).

[Brief explanation of the drawing]

The first factor is a radioimmunoassay diagram using the protein-bound P# of the present invention, Figure 2 is a comparison diagram of a radioimmunoassay between the protein-bound filter paper of the present invention and a conventional filter paper, and Figure 3 is a diagram showing the amount of protein and the combination agent. Figure 4 shows the radioimmunoassay test i1 and m for serially diluted samples using the filter paper of the present invention, and Figure 5 shows the correlation between the filter paper of the present invention and the conventional filter paper. This is the diagram I investigated. Fig. 1 AFP nq/m+ Fig. 5 Mold formation procedures -? cni official document (spontaneous) August M, 1988 1, Indication of the case 1988 Patent Application No. 093186 2, Name of the invention Process for manufacturing protein-bonded 5-sheet paper 3, Person making the amendment - Relationship to the case Patent Applicant Name Hide Hirai Matsu 4, Agent ■541 Address 406 New Life Hirano-cho, 4-53-3 Hirano-cho, Higashi-ku, Osaka Telephone: (06) 227-1156 Document proving power of attorney (1) ) IJ1 thin! I have corrected the ``Calcitsuen, Prionik Actigun Jt ``Karunno Nigurionikkua/Chige 7'' on page 3, line 1z. (2) [double] on page 7 of the specification, 4th line from the bottom
Jpr double J corrected T/). (3) On page 8, line 10 of the specification, there is a correction to "as a craft" and "grafted" the hair. (4) Submit power of attorney T/).

Claims (3)

[Claims] (1) A method for producing a protein-bound filter paper, which comprises reacting carboxymethyl groups of a filter paper made of carboxymethyl cellulose with a protein in the presence of a condensing agent. (2) The manufacturing method according to claim 0, wherein the condensing agent is a water-soluble carbosiimide reagent. (3) The production method according to claim (1) or (2), wherein the protein is a tumor marker protein.