JPH05312808A - Immunity examination method using lipidal antigen - Google Patents

Abstract

PURPOSE:To shorten necessary time for examination by adding antibody sensitized magnetic particles in a reaction vessel, and making antibodies combined with lipidal antigens and the antibody sensitized magnetic particles antigen-antibody react mutually under the existence of a magnetic field. CONSTITUTION:Lipidal antigens are arbitrarily selected depending on a specimen aimed for detection. The specimen is added in a reaction vessel having carriers, where the lipidal antigens are formed into a solid phase, so as to antigen-antibody react the fat antigens and the antibodies in the specimen mutually. When the aimed antibodies exist in the specimen, the antibodies combine with the fat antigens. Then, under the existence of a magnetic field applying magnetism to a reaction unit, the antibodies combined with the lipidal antigens and the antibodies sensitized to the magnetic particles are brought into mutual antigen-antibody reaction. Thereby a complex composed of the fat antigens, the aimed antibodies, and the antibodies sensitized to the magnetic particles is formed, and the magnetic particles are fixed to the carriers so as to form an spreading pattern on the carriers. This case is determined to be positive. When no aimed antibody exists, on the contrary, the magnetic particles are not allow to fix on the carriers, and are attracted by magnetic force so as to accumulate at one place. This case is determined to be negative.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method using a lipid antigen, and more particularly to an syphilis test method.

[0002]

2. Description of the Related Art The conventional test method for syphilis is Tr
The method can be broadly classified into a method using eponema pallidum (TP) cells themselves and a method using lipids containing cardiolipin as an antigen. In a test method for syphilis using a lipid antigen, a so-called biological false positive reaction, which is positive even in diseases other than syphilis, may appear, and in this respect, the method using TP cells is more specific. I can say. However, it is said that an antibody against a lipid antigen (this is called a Wassermann antibody) better reflects the infection state.

The method of using lipids as antigens is described in Mary
It originated in a study by C. Pargborn et al. In 1941. In this study, Pargborn and colleagues found that an alcoholic extract of bovine heart reacted with the serum of patients with syphilis. After this study, lipids such as cardiolipin and lecithin have been used to test for syphilis.

Test methods using lipid antigens include a complement fixation method, a precipitation reaction method, a microscopic cotton-like reaction method, and an indirect passive agglutination method, depending on the method. For these,
"Clinical laboratory 17 Serology", Yoshio Fukuoka et al., Medical and Dental Publishing Co., Ltd. Among these, the glass plate method utilizing the microscopic cotton-like reaction and the Rapid Plasma reagin (RPR) method are mainly performed today.

The glass plate method is based on the above-mentioned "Clinical examination course 17".
As described in "Serology", it is determined by adding a sample to a test solution containing cardiolipin, lecithin and cholesterin on a glass plate and measuring the presence or absence of aggregation of cholesterin crystals. ..

In the RPR method, a reagent in which a lipid containing cardiolipin and lecithin is adsorbed on kaolin or carbon powder is added to a sample, and the presence or absence of aggregation of kaolin or carbon powder is measured to make the determination. .. The reagent used here is also commercially available as a RPR test reagent from KEIKAKEN.

[0007] In both of the above two methods, the determination is made by measuring the presence or absence of aggregation, but attempts have been made to detect the lipid antigen used by the EIA method instead of aggregation, for example, " ELIS of glycolipid antigen
A ", Biochemistry Laboratory Course 4, Carbohydrate Chemistry," Specific Detection Method of Sugar Chain Using Monoclonal Antibody and Its Clinical Application, Clinical Examination, 31, 6, 1987. Also, cardiolipin and lecithin. Anti-phospholipid antibodies and biol is a method of immobilizing lipids on a microplate using EIA and detecting antibodies against the lipids by EIA method.
ogical false positive serological test for Syphili
s in patients with SLE, Clin. exp. Immunol, 56, 19
3-199, 1984.

[0008]

Both the glass plate method and the RPR method are characterized in that the operation is simple and the inspection can be performed in a short time. However, as described in the above-mentioned “Clinical examination course 17 serology”, these methods require delicate attention because reaction conditions are delicate. The glass plate method also has the drawback that the antigen is unstable. Furthermore, in these methods, since fine agglomeration of cholesterin or carbon powder is performed visually or under a microscope to make the determination, a skill is required for the determination. Further, the judgment result may vary depending on the judge.

In the agglutination method, which is one of the inspection methods, the determination is made by the degree of resuspension of particles after weak centrifugation of kaolin or carbon powder.
Similar to the R method, the subjectivity of the judge is likely to enter. Also, it is an ad hoc measurement and the results cannot be saved.

Furthermore, as can be said for all of the above-mentioned glass plate method, RPR method and agglutination method, in these methods, the prozone phenomenon (zone phenomenon) may occur because the sample is added directly. This is because, when a high-titer sample is used, the antibody in the sample and the antigen on the carrier (in this case, kaolin or carbon powder) cannot be quantitatively balanced and appears to be negative. Is. This prozone phenomenon is said to be a problem generally occurring in the agglomeration method.

Clin. Exp. Immunol, 56, 193-19, cited above.
The measuring method using EIA disclosed in 9, 1984 was developed for the purpose of solving this prozone phenomenon. In this method, as described above, first, a lipid containing cardiolipin or the like is immobilized on the bottom surface of the plate, and blocking is followed by reaction with the sample. Next, after washing, it is reacted with an enzyme-labeled antibody, and a substrate for the enzyme is further added to develop color. In this method, the prozone phenomenon is less likely to occur because the washing is performed after the reaction with the sample.

However, in this method, it is necessary to carry out a blocking treatment in order to prevent a nonspecific reaction. In addition, a washing operation for removing the unbound analyte and the unbound enzyme-labeled antibody is required twice in the procedure. Therefore, the time required for the test is at least 3 hours after the sample is dispensed. This is the glass plate method or RPR
This is extremely long compared to the inspection in the law being completed in about 8 to 10 minutes.

Further, in this method, a biological non-specific reaction may occur due to the above blocking treatment. The blocking process is performed when EIA is performed.
In order to prevent non-specific binding of substances other than the target substance without relying on the antigen-antibody reaction, they are reacted with proteins unrelated to the reaction, such as animal serum, albumin, gelatin, casein protein, etc. It is solid-phased. In fact, even in this method, blocking treatment is performed for a long time using a high concentration of 10% animal serum. However, it is well known that some humans have antibodies that react with certain animal serum components, and when such human serum is used as a sample, it reacts with the animal serum used for the blocking treatment. As a result, the S / N ratio is greatly impaired. Therefore, it is an object of the present invention to provide an immunoassay method that is quick, convenient, and highly objective, and particularly an syphilis detection method.

[0014]

In the immunoassay method of the present invention, a specimen is added to a reaction container having a carrier on which a lipid antigen is immobilized, and the antigen antibody is the antigen contained in the specimen and the lipid antigen. A step of performing a reaction, a step of adding antibody-sensitized magnetic particles to the reaction vessel, and performing an antigen-antibody reaction between the antibody bound to the lipid antigen and the antibody-sensitized magnetic particles in the presence of a magnetic field; And a step of measuring the pattern formed above.

The lipid antigen used in the immunoassay method of the present invention can be arbitrarily selected depending on the antibody to be detected. For example, when syphilis is tested, cardiolipin can be preferably used as a lipid antigen. Those obtained by adding lecithin (phosphatidylcholine), cholesterol and the like to cardiolipin are also preferably used as the lipid antigen. In this case, the content of cardiolipin is important, and the content is preferably 1 to 50% by weight.

The carrier for immobilizing the lipid antigen may be any carrier as long as it is usually used as a carrier for immobilization, and the wall surface of the reaction vessel may be used as the carrier. In particular, it is preferable to immobilize on the inner wall surface of the U-bottom or V-bottom microplate well. Immobilization of the lipid antigen can be performed by a conventional method.

As the antibody-sensitized magnetic particles used in the immunoassay method of the present invention, the magnetic particles described in JP-A-63-181119 can be preferably used. The antibody sensitized to the magnetic particles can be arbitrarily selected according to the intended test. When testing for syphilis,
By selecting either an anti-human IgG antibody, an anti-human IgM antibody, or both, IgM, which is a Wasselman antibody that appears in the early stage of infection, and I that appears subsequently.
The fractional measurement with gG can be easily performed.

The antigen-antibody reaction between the antibody bound to the lipid antigen and the antibody sensitized to the magnetic particles is carried out in the presence of a magnetic field. To apply a magnetic field to the reaction system, for example, a magnet may be placed under the reaction container.

After the antigen-antibody reaction between the antibody bound to the lipid antigen and the antibody sensitized to the magnetic particles is completed, the pattern formed by the magnetic particles on the carrier is observed to determine positive or negative.

[0020]

When the desired antibody is present in the sample (positive), this antibody first binds to the lipid antigen immobilized on the carrier. The antibody bound to the lipid antigen is then
By reacting with the antibody sensitized to the magnetic particles, a complex consisting of the lipid antigen, the target antibody and the antibody sensitized to the magnetic particles is formed. Therefore, in the case of being positive, the result is that the magnetic particles are fixed to the carrier, and a spread pattern is formed on the carrier.

On the other hand, when the target antibody does not exist in the sample (negative), the antibody does not bind to the lipid antigen, so that the magnetic particles are not fixed on the carrier. Therefore, when the result is negative, the magnetic particles are attracted by the magnetic force and accumulate at one place.

[0022]

【Example】

Example 1 (1) Immobilization of cardiolipin, lecithin and cholesterol on a microplate

Commercially available lecithin (Sigma, 106F-8380
), The solvent chloroform was distilled off, and ethanol was added. Then, cardiolipin (Sigma, 12
8-8356) and cholesterol (manufactured by Wako, 034-03002)
), 0.27 mg / ml cardiolipin, 2.53
A solution of lipids in ethanol containing mg / ml lecithin and 8 mg / ml cholesterol was prepared. This ethanol solution was gradually added to 10 volumes of PBS (0.01 M, pH = 7.2) under sonication to obtain a cloudy, micellar solution. This micelle solution was dispensed into U-bottom plates (Nunc, Maxisorp type) at 50 μl / well and left overnight under refrigerated conditions. After this was drained, it was blocked with 0.2% BSA and dried to obtain a cardiolipin solid phase plate. (2) Assay As a positive sample, RPR positive or Syphilis (TPH)
A) Positive sera were used, and RPR- or TPHA-negative sera of healthy individuals were used as negative samples.

75 μl of PBS containing 1.0% gelatin was added to the cardiolipin solid phase plate prepared in (1) above.
/ Well was dispensed, and then 25 μl / well of each of the above-mentioned positive and negative sera was added to each well and incubated at 37 ° C for 10 minutes. After incubation, each well was diluted with saline (Bioteck, Washer AMW-2).
Washed twice. Thereafter, 25 μl / well of a suspension of anti-human IgG-sensitized magnetic substance-encapsulated particles (manufactured by Olympus Optical Co., Ltd.) was dispensed, and a pattern was formed on the magnet for 3 minutes.
The obtained pattern is schematically shown in Table 1.

[0025]

[Table 1]

As shown in Table 1, RPR or TPHA
A positive image in which particles were spread over the entire well was obtained for the sample determined to be positive in 1., and a negative image in which buttons were accumulated on the bottom surface of the well was obtained for the negative sample from a healthy person. Example 2 Comparison of titer with RPR card test Based on the same sample, sensitivity comparison between the test method of the present invention using a cardiolipin solid phase plate and the RPR card test was performed.

As the sample, an RPR-positive sample diluted 2-fold with negative serum was used, and this was further diluted at the dilution rate shown in Table 2. The test was also conducted on the negative serum.

The test using the cardiolipin solid phase plate was carried out in the same procedure as in Example 1 above. Also, RP
For the R card test, use RPR test blood laboratory (lot No. QN-1) manufactured by Institute of Chemistry and Serum Therapy,
The test was performed according to the instructions of this kit. The results are also shown in Table 2.

[0029]

[Table 2] The judgment in Table 2 is based on the following criteria. In addition, the determination of the RPR card test is based on the instruction of the RPR card test KEIKEN. a) RPR card test ++: Large aggregates are present ++: Medium aggregates are present +: Small aggregates are present −: No aggregates are observed b) Cardiolipin solid phase plate ++: All over the bottom surface of the well Particle spread pattern +: Slightly collapsed spread pattern ±: Button-like pattern but bleeding around the periphery-: Button-like sedimentation pattern From Table 2, the test method of the present invention using a cardiolipin solid phase plate Is clearly more sensitive than the conventional RPR card test. Example 3 Comparison with inspection method using EIA

A cardiolipin solid phase plate was prepared by the same procedure as in (1) of Example 1, and E was prepared using this plate.
A comparison was made between the IA method and the method using magnetic particles. As samples, RPR method positive serum diluted 10 to 160 times with negative serum and eight negative serums were used. (1) Inspection by EIA method

In each well of cardiolipin solid phase plate
PBS with 1% gelatin (0.01M, pH = 7.2) 75μ
1 / well was dispensed, 25 μl / well of the sample shown in Table 3 below was added, and the mixture was incubated at 37 ° C. for 10 minutes. After incubation, the plate was washed 6 times with physiological saline.

Then, a peroxidase (POD) -labeled product of anti-human IgG antibody (manufactured by Jackson, lot No. 101)
38) diluted 2000 times with 0.2% BSA / PBS to 1
00 μl / well was dispensed, incubated at 37 ° C. for 30 minutes, and then washed 6 times with physiological saline. After washing, the substrate buffer (0.014% H ₂ O ₂ , 0.1 M citric acid, 0.2 M phosphate buffer pH = 5.0) was added with ortho-phenylenediamine (OP
D) Dissolve 0.5 mg / ml (manufactured by Sigma) of the coloring solution prepared in 100 μl / well, and develop color for 10 minutes at room temperature. Add 50 μl / well of 2N H ₂ SO ₄ to stop the reaction. Then, OD _{490 nm} was measured. The results are also shown in Table 3. (2) Inspection using magnetic particles

In each well of the cardiolipin solid phase plate
PBS with 1% gelatin (0.01M, pH = 7.2) 75μ
1 / well was dispensed, 25 μl / well of the sample shown in Table 3 below was added, and the mixture was incubated at 37 ° C. for 10 minutes. After incubation, the plate was washed 6 times with physiological saline.

Next, 25 μl / well of a suspension of magnetic particles encapsulating magnetic material sensitized with an anti-human IgG antibody was dispensed, and pattern formation was performed on the magnet for 3 minutes. The results are also shown in Table 3. The criteria for determining the pattern are the same as in the second embodiment.

[0035]

[Table 3]

As shown in Table 3, when the test is performed using magnetic particles, positive can be judged as positive up to 4-fold dilution, and all 7 negative cases and blanks can be judged as negative. did it.

On the other hand, when the test was carried out using the EIA method, the color development was strong throughout and the baseline increased, and the color development degree among individual wells was variable, and the result was positive × 1. However, it could not be clearly determined to be positive. As described above, the inspection using the magnetic particles can make the determination more clearly than the inspection by the EIA method.

In the inspection method of the present invention in which the magnetic force of the magnet is used to promote the sedimentation of magnetic particles, when a U-bottom or V-bottom microplate well or the like is used as the reaction container, the well is distant from the magnet. Since the magnetic force is weaker than that in the central portion, the positive pattern tends to spread, and conversely, the magnetic force becomes stronger and the negative pattern becomes clearer as it approaches the well central portion (bottom portion).

When the EIA method is used, there is no method for more clearly distinguishing such positive and negative, and it is possible to use a means such as broking treatment, an increase in protein concentration, the use of a surfactant, and an increase in the number of washings. S / by reducing non-specific binding
There is no choice but to increase the N ratio. However, these means damage the antigen (here, lipid) immobilized on the carrier, causing problems such as variation and increase in baseline, and as a result, reduce the S / N ratio. Thought to be a thing. Example 4 Optimal Ratio of Cardiolipin in Lipid The difference in reactivity was observed by changing the content of cardiolipin in the solid-phased lipid.

Cardiolipin (Sigma), lecithin (Sigma) and cholesterol (Wako) were each dissolved in ethanol, and all cholesterol concentrations were
A lipid ethanol antigen solution was prepared which was 8 mg / ml, the total amount of cardiolipin and lecithin was 2.8 mg / ml, and the ratio of cardiolipin / lecithin (CL / PC) was changed to 50-0%. With these liquids,
A cardiolipin solid phase plate was prepared by the same procedure as in (1) of Example 1.

Using this solid phase plate, RP as a sample
R-positive serum diluted with negative serum, and negative serum
The assay was performed in the same procedure as in Example 1 using 6 cases. The results are shown in Table 4. The criteria for determination in Table 4 are the same as in Example 2.

[0042]

[Table 4]

As is clear from Table 4, no reactivity can be found when the cardiolipin content is 0%. Also,
Nonspecific reactions tend to increase even when the ratio of cardiolipin to lecithin is high. Therefore, in the test method of the present invention, the ratio of cardiolipin to lecithin is preferably in the range of 1 to 50%, particularly preferably about 10%.

[0044]

As described above, in the immunoassay method of the present invention, the reaction is carried out in the presence of a magnetic field, whereby the precipitation of magnetic particles can be accelerated and the time required for the test can be shortened. As described above, the detection using the EIA method required several hours for the inspection, but according to the inspection method of the present invention, 10
The test can be completed in about a minute. This is about the same time as the glass plate method and the RPR method, which are characterized by their rapidity. In addition, not only was the inspection time reduced,
It is possible to suppress non-specific reactions and obtain stable test results with a high S / N ratio.

Further, since the positive / negative determination is made based on the pattern formed by the magnetic particles, the determination can be made easily and objectively by visual observation, and no skill is required. In addition, the formed pattern can be stored and the determination can be made at any time if desired.

Furthermore, when a U-bottomed or V-bottomed microplate well is used as the reaction vessel, a positive pattern is generated due to the difference in the magnetic field strength between the well peripheral portion and the central portion.
It is possible to form a negative pattern more clearly.

Claims (4)

[Claims] 1. A step of adding a sample to a reaction container having a carrier on which a lipid antigen is immobilized to carry out an antigen-antibody reaction between the lipid antigen and an antibody contained in the sample; A step of adding magnetic particles to carry out an antigen-antibody reaction between the antibody bound to the lipid antigen and the antibody-sensitized magnetic particles in the presence of a magnetic field; and a step of measuring a pattern formed on the carrier. An immunoassay method comprising: 2. The immunoassay method according to claim 1, wherein the lipid antigen contains cardiolipin, lecithin and cholesterol, and the ratio of the cardiolipin to the lecithin is 1 to 50% by weight. 3. The immunoassay method according to claim 1, wherein the reaction vessel is a U-bottomed, flat-bottomed or V-bottomed microplate well. 4. The immunoassay method according to claim 1, wherein the antibody sensitized to the antibody-sensitized magnetic particles is either anti-human IgG or anti-human IgM, or both of them.