JPS6029665A - Quantitive determination of antigen - Google Patents

Abstract

PURPOSE:To enable easy and quick quantitative determination of an antigen with good sensitivity by adding an antibody for the antigen to a red blood cell suspension in which protein A is bonded with the red blood cells then adding further the antigen and complement thereto and bringing said liquid into reaction. CONSTITUTION:This method consists of bringing the antigen to be quantitatively determined and an antibody into reaction directly on the surface of red blood cells and determining quantatively and indirectly the antigen from the hemolysis of the red blood cells by the bonding reaction of the complement. The protein A is usually obtd. by refining the same from a yellowish staphylococcus. The sensitized red blood cells bond the protein A. The kind of the antigen is enumerated by, for example, alpha-fetoprotein, ferritin, C-reactive protein, HB, immune globulin G, immune globulin M, myoglobin, etc. It is preferable to adsorb chemically the protein A to the red blood cells in the case of bonding the protein A to the red blood cells. The antigen is determined quantitively with high sensitivity by increasing the amt. of the protein A to be sensitized.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for theorem of antigens.

In recent years, in the medical field, it has become an extremely difficult problem to easily and quickly determine the angle of antigens with high polarization for the purpose of diagnosing diseases, etc.

Conventional immunochemical assays for antigen determination include activation immunoassay (radioimmunoassay) (4), oxygen immunoassay (B), reverse passive hemagglutination (CJ), and radial immunodiffusion. law) etc. are being carried out,
Each of these constant K methods had the following runner-up points.

Method A: Requires cleaning operations, and requires special equipment to remove radioisotopes, resulting in huge equipment costs and time-consuming procedures.

Method B requires a cleaning operation depending on the age of the ship and takes a long time to make a determination.

Method C: We criticize the complicated 1,000-F method in order to make it difficult to understand the operation of stepwise diluting the test piece to 2 times with a dilator and the operation of dropping diluted liquid etc. once with a dropper. In addition, it takes a long time to make a determination, and since the antigen amount is determined at the end of a 2-fold serial dilution, the determination may be inaccurate.

D Method 2 It takes a lot of time to make a determination and is insufficient in terms of sensitivity.

The present invention is based on the results of fFA research conducted on gunshot sounds in an attempt to improve these drawbacks, and the discovery of a simple and rapid method for quantifying antigens with sensitivity <L. That is, the method of the present invention makes use of protein A binding to immunoglobulin G,
After adding a quantitative amount of an antibody against the antigen to a red blood cell suspension containing red J(r+ cells and protein A),
Furthermore, the red blood cells are hemolyzed by adding antigen and complement to make them sizable, thereby indirectly depleting the antigen from the total hemolytic phenomenon.

In the method of the present invention, protein A is usually obtained from Staphylococcus aureus.

In addition, in method 1 of the present invention, 47.9 is used to bind 90 dein A, and this protein Ai
Since there is a large number of people who are immune to clopurin G, many 117i moth antigens are versatile, and the types of antigens include minerals, such as α-fetozolotin, ferritin,
C-JI! It can absorb J protein, HB, immunoglobin G, immunoglobin M, myoglobin, etc. However, rather than being fixed to these antigens, it is possible to use all types of antigens that complement the antigen-antibody reaction.

When protein A is bound to red blood cells in the method of the present invention, it is preferable to bind the red blood cells chemically. Many people have been praying for the secretion of proteins such as antibodies into the red blood cells. For example, 42.

In the method of the present invention, the presence of protein A that sensitizes should not affect the sensitivity of antigen determination, and the presence of a large amount of sensitizing protein A makes it possible to identify antigens with high sensitivity. Ru.

For example, red blood cells], approximately equal to 1010 cells, protein 02
~5rtryjrc addition 1) It is desirable to use the combination function.

Furthermore, red blood cells are limited, so what would happen without them?
Red blood cells of the same type may also be used. In order to increase the strength and short-tubular K, red blood cells that are prone to hemolysis due to complement fixation reaction,
A good example would be sheep red blood cells.

Furthermore, the type of complement is not particularly limited, and a sleeve with a high complement activity is desirable, such as a guinea pig sleeve.

Now, how the method of the present invention differs from the conventional sleeve binding reaction test will be explained.

The sleeve binding reaction test is a method conventionally known as an antigen determination method or an antibody determination method. In other words, when an antibody binds to an antigen, the antibody molecule undergoes a -F induction, and the sleeves bind to the antibody and are activated and consumed. This is called the complement fixation reaction. Utilizing this reaction, it is now possible to indirectly determine the strength of the antigen-antibody reaction from the consumed complement i. For example, if the amount of antigen is kept constant, the amount of antigen can be measured by using the antibody fang as the antibody side, and using the antibody side as f6:-foot. Consumption of the sleeve due to antigen-antibody reaction is an indicator of hemolysis of sheep red blood cells bound to antibodies that are effective against sheep red blood cells. In the conventional sleeve binding reaction test, it is difficult to conduct such an antigen-antibody reaction, and the antibody against the sheep's red n'+ cells is detected by the sleeve. This is for quantifying antibodies. However, in the method of the present invention, the antigen is indirectly determined from the hemolysis of red blood cells caused by the complement fixation reaction, by reacting the original antigen with the antibody on the surface of the massaged blood cells.

In the conventional sleeve binding reaction test, the hemolysis of sheep erythrocytes to which antibodies against sheep erythrocytes have bound due to the external complement extinguished by the antigen-antibody reaction is used as an indicator, so the amount of complement remains constant. The shadow of the existing body in the test serum p#
In order to avoid waste, immobilization was performed at 56°C for 30 minutes.

Therefore, it could not be used for antigens that are unstable to heat. However, in the method of the present invention, if rod complement is above a certain level, the existing complement in the test serum will not be affected and will be immobilized. Antigens that are unstable to fever may also be used. Furthermore, conventional complement fixation reaction tests require complicated techniques such as 2-fold serial dilution of the blood sample to be tested and require a long period of time before making a determination; It is possible to easily and quickly quantify the antigen without having to deal with operations such as excitation.

Next, regarding the embodiment of the present invention, i(, 1 bright).

Example (1) Washed with physiological saline and added 1 volume of sheep erythrocyte sediment to 1 volume of chromium botanicals ((14 ml/7 ml)) and 70 lotiine A.
(2.5m?/ηze, Pharmacia Fine Kumikarume + lJ!!) 2 units and #7? 30 J in the whirlpool
li4 & After a while, work 3. ! I': Food" 4. - Washed with liquid to make it 8% floating. To 50 μl of sheep red blood cell rX fluid was added 50 μl of anti-hill α-fetogloline woolly antibody to 50 μl of the sheep red blood cell rX fluid, and 8 ml of fluorine A as shown in Table 1 and Figure 1 was added. Concentration of human alpha-phetone 6
0 tin delivery name KI℃ tree I50 itl ro 8 force 11
shi, page to ra)--le 5 Ji lil liquid pIl 7
．． 2 i+c'T-100C (diluted mol ~ 3. (l ml), 37℃ + 303
After j time & response 7'iC, a-k Eh i rcj
t'j1'r VL X.

The ψ' and luminosity (41 bmm) of the supernatant C hemocropin were determined.

Also:, 4+: The temperature of human α-fetoproline and ll! ! , Luminosity and C Tomari line Sekiiga (′・3 1 human α-fetoprobuin 26
We were able to.

Example (2) Actual M1 - Same as example (]) K 1Lll'A I, Proline A along sheep red JrII FJi floating i'ij? l+
5 (anti-HBg antibody (200μ5L for 1pi)
/III/) 50 μl'f: After addition, 50 μl of a sample containing a moderate concentration of HB antigen as shown in FIG. 2 was added, and the guinea pig cuff 3. Add Q ml and heat at 37°C.

After 30 minutes of reaction, the luminous intensity (4169 m) of hemoglobin in the supernatant was measured by low-speed centrifugation.

The results are shown in Figure 2.The HB antigen was determined by the reverse passivation hemagglutination method (R-PHA method). I was able to do it.

Example (3) After adding 50 μl of anti-human immunoglobulin G (γ chain discriminating) rabbit antibody (200 μy/g) to 50 μl of protein A-sensitized sheep red blood cell suspension prepared in the same manner as in Example (1). 50 μl of samples containing human immunoglobulin G at various concentrations as shown in FIG. 3 were added, and guinea pig complement 3. (l
ml and reacted for 30 minutes at 37°C, the absorbance of hemoglobin (41611
11) 'i was measured.

As a result of step 1, as shown in FIG. 3, it was possible to determine the amount of human immunoglobulin G based on the surface line relationship between the concentration of human immunoglobulin G and the absorbance.

As mentioned above, it is possible to quickly deplete the antigen 'jL through the 11-mediated method, which contains excellent sensitivity and is effective against many types of antigens. Significant effect
lose one's fangs.

[Brief explanation of drawings]

] The surface shows the relationship between the antigen of the present invention and the absorption (4 degrees), the first figure, human α
- Ni1Si, the relationship between the concentration and absorbance of Fetoproline
Figure 1, Figure 2 shows the relationship between the concentration of HB antigen and absorbance, and Figure 3 shows the relationship between the concentration of human immunoglobulin G and absorbance. B'Kiyoshi + 1 person as a littoral engineer Takehiko Suzue Director General of the Patent Office Kazuo Wakasugi Membrane antigen quantification method 5, voluntary amendment 6 Specification subject to amendment 7, content of amendment (1) Specification page 1, No. 19 In the line "Oxygen immunoassay (B), J", correct it as "Enzyme immunoassay (B), J." +21 q % In line 12 of page 4, "Protein"
Correct the statement to read "Buitin A." (3) On page 5, line 12, "antigen-antibody reaction" is corrected to "antigen-antibody reaction." (4) On page 7, line 12, page 8, line 6, and page 9, line 1, the words "hemoclobin" are corrected to "hemoglobin."

Claims (1)

[Claims] To the erythrocyte suspension containing prodin A bound to red blood cells, t
After adding an antibody against the antigen to be defeated, the antigen and complement are further added to cause a reaction, causing hemolysis of the red blood cells, and this dissolution phenomenon indirectly leads to an erection. Antigen amount l method.