JPH06213889A - Immune serum diagnostic method - Google Patents

Abstract

PURPOSE:To detect P-B antibody specifically without requiring absorption operation of H-D (Hangautziu-Deicher) antibody and F (Forssman) antibody by using P-B (Paul-Bunell) antigen which is isolated and refined from bovine red cell substrate and mouse T-cell leukemia cell strain. CONSTITUTION:P-B antibody is specifically detected without requiring absorption operation of H-D antibody and F antibody using the P-B antigen which is isolated and refined from bovine red cell substrate and mouse T-cell leukemia cell strain. Namely, two or one sedimentation lines 15 based on the double diffusion method within a gel are formed by reaction of IM serum containing sufficient P-B antibody for example at a concentration of 10-100ng/ml on a disk where P-B antigen, non-absorption mononucleosis (IM) serum, the IM serum after absorption treatment by guinea pig kidney organization, and the IM serum after absorption treatment according to bovine red cell are dispensed to wells 4, 5, 6, and 7, respectively, inside the disk.

Description

Detailed Description of the Invention

[0001]

The present invention relates to infectious mononucleosis (INFE).
P-B (P), which is one of the heterophilic antibodies characteristic of a disease, hereinafter referred to as IM
aul-Bunell) antibody specific detection system, particle agglutination method (PA method) and mixed passive agglutination method (M
PHA method) and reagents necessary for each.

[0002]

2. Description of the Related Art IM is mainly used by young white people (15-
(25 years old) is a frequent (50/10 5) yearly lymphocytosis, which is mainly due to fever, swelling of lymph glands and hepatic spleen, and transient appearance of atypical lymphocytes in the bloodstream. It is a disease that causes symptoms. This disease is called benign or pseudoleukemia because it shows symptoms of acute leukemia during the course of the disease, especially within 2 weeks after the onset, but it is cured within 2 months without leaving any serious aftereffect.

1932 by chance Paul and Bunell
Has discovered a P-B antibody specific for IM. This antibody is a Forssman (F) antibody or Hangautz
Like the iu-Deicher (HD) antibody, it aggregates erythrocytes of different species, such as sheep, horses, and cows, and is therefore collectively called heterophile antibody. For the detection of P-B antibody, in vitro agglutination reaction using sheep blood cells (SRBC) or horse blood cells (HRBC) has been performed. However, an operation (absorption test) for removing the F antibody and the HD antibody before the agglutination reaction was necessary in order to distinguish them from the F antibody and the HD antibody which are other heterophilic antibodies.

Table 1 below shows human heterophilic antibodies (P-B, HD).
And F antibody), ie positive (+) indicates the presence of the corresponding antigen. Based on this reaction pattern, a method for identifying a P-B antibody was established by Davidson by a combination of an absorption test and an agglutination reaction.

[0005]

[Table 1]

That is, when SRBC (or HRBC) is used as an indicator cell for the agglutination reaction, the patient serum is divided into two in advance, BRBC is added to one of them, and guinea pig kidney tissue is added to the other, and the cells are spun down to give SRBC in each supernatant. In addition, the presence or absence of agglutination reaction is checked. If the specimen agglutinated SRBC and not the serum absorbed by BRBC after absorption in guinea pig kidney tissue, the heterophilic antibody in the specimen was P-
Identified as B antibody.

For example, the most clinically used method at present is a method devised by Hoff and Banuer in 1965, which combines an absorption test on a slide glass plate 1 and an agglutination reaction as shown in FIG. Apply patient serum to each well 2 and 3 and in the second step, add BRBC as an absorbent to well 2 and guinea pig kidney tissue to well 3 and allow to stand.
BC is added to wells 2 and 3 to determine the presence or absence of aggregation. B
If the RBC absorption shows a negative image and the guinea pig renal tissue absorption shows a positive image, it can be diagnosed for the first time as an IM patient having the P-B antibody. If both are absorbed and become negative (-) or negative (-), it is the HD antibody, and if positive (+) or negative (-), it is the F antibody. However, the above results are only when each heterophilic antibody is present alone in the serum of a patient. It is difficult to determine when the two or three are mixed in different concentrations in a single patient serum.

According to the study by the inventors, the P-B antibody is not single but two BRBC and SRBC (H with different specificities).
RBC) that reacts with a common antigen (BS antibody) and B
It is divided into those that react with RBC but not SRBC (HRBC) (B antibody). In about 5% of patients, only B antibody is detected but BS antibody is not detected (Cl
in. Immun. & Imm. Pathol. Two
7, 296-299, 1983).

Therefore, in a test using SRBC (HRBC) as an indicator cell, some IM patient sera may show false negatives.

[0010]

DISCLOSURE OF THE INVENTION The present inventors have tried to solve the following problems inherent in the above conventional inspection methods.

The conventional method is a complicated method consisting of an absorption test with two types of antigenic substances and an agglutination reaction after that, which is not a rapid and reliable method required in clinical practice.

[0012] The determination depends on the pattern of negative (-) and positive (+), and there is a possibility that the sample is difficult to determine.

Since SRBC (HRBC) is the indicator cell, a small number of BS antibody negative IM patients (false negatives) may be missed.

[0014]

[Means and Actions for Solving Problems] A method (J. ofS) previously established by the inventors for solving the above-mentioned conventional problems.
upramol. Str. 6, 275-290), P-B antigen extracted from BRBC is used as the target antigen. This P-B antigen has the following serological performance. Mouse T cell leukemia strain; L5178Y, EL4 and L # 2
Modified from cells (J. of Immunol. 12
7, 5-8, 1981) may be used.

62% of the glycoprotein having a molecular weight of about 26K is a protein, and the main amino acids are glutamic acid, proline, glycine, isoleucine, leucine and threonine.
The sugar content is 9.2% and the main ones are sialic acid, galactosamine and galactose.

This P-B antigen is contained in the wells 4, 5, 6, 7 in the disk as shown in FIG.
The non-absorbed IM serum, the IM serum after the absorption treatment with guinea pig kidney tissue, and the IM serum after the absorption treatment with SRBC (for the removal of BS antibody) were dispensed on a disc.
IM containing sufficient P-B antibody at a concentration of 100 ng / ml
By reacting the serum, two or one settling line 15 based on the in-gel double diffusion method is formed. This sedimentation reaction is not affected by pre-absorption of IM serum with guinea pig kidney tissue homogenate. Further, no reaction with the HD antibody was observed at this antigen concentration.

This P-B antigen is 4 units of SRBC aggregate P
-The antibody activity of IM serum diluted with B antibody is
It was completely suppressed at a concentration of 10 ng / ml, and H
No inhibitory effect on -D antibody is observed.

The above-mentioned specificity of the P-B antigen is similarly proved in the test by the enzyme antibody method. That is, this P-B antigen naturally does not react with any other heterophilic antibody, F antibody, nor does it react with the H-D antibody at the above-mentioned concentration, but only with the P-B antibody. Is an antigenic substance.

<Particle agglutination inspection method (PA method)> P-
Sensitized particles having B antigen adsorbed on the surface of various carrier particles have pH
Prepare a 1 to 5% (v / v) suspension (PA reagent) with 7.2 to 7.4 phosphate buffer. The carrier particles used have a particle size of 0.0
Various materials of 5 μm to 20 μm can be used. For example, latex synthesized from polystyrene or the like, gelatin particles formed from gelatin and water-soluble polysaccharides, acrylamide particles, silica, activated carbon, kaolin, etc., organic or inorganic materials. Examples of the artificial particles include human red blood cells, and animal red blood cells in which H-D antigen and F antigen have been reduced by enzyme treatment. Among them, latex, gelatin particles and the like are preferable.

The method of sensitizing particles to an antigen includes physical adsorption,
Chemical crosslinking agents such as tannic acid, aldehydes and carbodiimides can be used. Sensitized particles may be mixed with IM serum on a slide and the degree of particle aggregation may be observed, or the particles may be added to a reaction vessel at the U or V bottom such as a microplate to aggregate / non-aggregate like a mixed passive agglutination reaction. You may judge from a pattern.

When the above PA reagent and IM serum are mixed on a glass slide, an agglutination reaction is visually observed in 5 to 30 minutes and does not react with human serum containing F or HD antibody and normal human serum. . The agglutination reaction with this PA reagent is a method suitable for testing a small number of samples in clinical sites such as clinics and hospitals, and a technician who does not have the knowledge of the complicated absorption operation of the conventional method can easily and definitely make a judgment. Can be done. The antibody titer of the IM serum by the PA slide method is 160 to 5120, and that of the serum of various patients other than the normal control and IM is 10 or less.

<Mixed passive agglutination test method> IM causes hundreds of patients at the peak of epidemic in university dormitories and the like. Therefore, an inspection system having a processing capacity for a large number of samples is required. To meet this need, the inventors have previously developed a mixed passive agglutination method (Mixed Pass), which was developed for the purpose of screening a large number of samples of platelet and erythrocyte alloantibodies.
We have succeeded in applying the principle of iv Agglutination (MPHA method) to the detection of P-B antibody. As shown in FIG. 3, a buffer solution (100 ng) containing the P-B antigen 9 described above in each well 8 made of plastic such as polystyrene, polyvinyl chloride, and polymethylmethacrylate.
˜1 μg / ml) (200 μl) and adsorbed at 4 ° C. for 16 hours, and washed 3 times with phosphate buffer (PBS-Tween 20) pH 7.2 containing 0.05% Tween 20 to form an antigen plate. 200 μl of IM serum serially diluted with PBS-Tween 20 solution was added to each well of the antigen plate, and allowed to stand at room temperature for 2 hours, and then PBS-T.
Wash 3 times with ween 20. Whether or not the P-B antibody 10 is bound is determined by the positive pattern image 12 and the negative pattern image 13 appearing on the bottom surface of the well of the anti-human IgG sensitized magnetic particles 11.

As the antibody-sensitized magnetic particles, latex particles, gelatin particles or the like in which magnetic particles are encapsulated can be used, but gelatin particles are particularly preferable. Anti-human IgG and anti-human IgM may be mixed-sensitized or may be separately sensitized,
Human IgG and human IgM may each have characteristics.
This enables screening of specific antibodies and classification of antibodies. In addition, anti-human IgG for sensitized particles
Alternatively, P-B antigen may be sensitized instead of IgM, and IgM antibody may be preferentially detected to eliminate the need for washing after the reaction with IM serum.

As shown in FIG. 4, in the positive reaction, the indicator cells show a pattern spreading over the entire bottom surface of the well, and in the case of a negative reaction, the indicator cells show a pattern in which the indicator cells gather in a center on a dot. The P-B antibody titer of IM serum of this MPHA method is 320.
-20720, that of the normal control is less than 10.
The antibody titers of patient sera other than IM (including F and HD antibodies) were 10 or less, and rarely 20.

In the present invention, the construction of a test system targeting antigenic substances isolated and purified from BRC derived from F-negative species and mouse T cell leukemia strains involves the involvement of F antibodies contained in almost all human sera. It could be excluded from the reaction system in advance. Next, in the purification process, the mixing of HD antigens was suppressed to below the detection sensitivity (1 to 10 ng / ml), the possibility of false positives due to HD antibodies was eliminated, and the positive of the test system was immediately detected.
-Results in the identification of the B antibody. This is 5 of IM serum
0% or more contains high titer HD antibody, and H- is present in acute leukemia, various liver diseases, etc., which present with IM-like symptoms.
Considering that D antibody is detected, it is extremely important for immunoserodiagnosis of IM.

Next, in the inspection system of the present invention, by removing the absorption operation which is an essential step in the conventional method, the IM
The immunoassay of was simplified. Furthermore, the heterologous hemagglutination reaction of IM sera after absorption required a good knowledge of heterophile antibodies in interpreting the results, which, in combination with the complexity of the operation, was sometimes a source of error. In the test system of the present invention, the positive reaction immediately proves the existence of the P-B antibody as described above, so that there is a great merit that a non-specialist can easily perform an immunoserodiagnosis of IM.

Both the PA method and the MPHA method of the present invention,
The time required for the inspection by the conventional method could be greatly shortened. In both cases, the time required for the absorption operation is shortened, and in the PA method, the reaction is completed in 5 minutes to 5 to 30 minutes. In the MPHA method for a large number of samples, the time required for testing 96 samples is only 30 minutes due to the use of magnetic particles.

[0028]

Example 1 <Identification of P-B antibody by PA method> Using a P-B antigen-sensitized PA reagent separated and purified from a BRBC substrate, sera of IM patients and other patient sera and normal adolescents (15-25)
A) tested serum. The IM serum was collected during the period of 1 to 4 weeks after the onset of illness. These sera are 10 times and 10 times
The 0-fold dilution was examined. 1 as seen in the results in Table 2
Of the 47 IM sera, 145 were positive and the positive rate was 98.4.
%Met. This was higher than 96% in the conventional method in which IM serum containing only B antibody was missed. The two negative samples were both collected one week after the onset of illness, and the two samples of the same patient after two weeks were both positive. In Table 2, * indicates a sample collected one week after the onset of illness, and ** indicates a sample containing leukemia, lymphoma, solid cancer, hepatitis, lepra, and rheumatism patient serum.

[0029]

[Table 2]

[0030]

Example 2 <Detection of BS Antibody and B Antibody by PA Method> As shown in FIG. 2, the P-B antibody is usually a BS antibody against an antigen common to BRBC and SRBC or a B antibody against an antigen present only in BRBC. Two types are mixed in IM serum. The inventors examined the titers of BS antibody and B antibody in the sera collected over time from two patients, sister A and sister B, who developed IM at the same time. As shown in Fig. 4, sister B produced BS antibody (dotted black circle) and B antibody (dotted white circle) P-B antibody after the onset of illness.
Sister A produced only B antibody (solid white circle), while BS antibody (solid black circle) was not detected during the entire observation period.

Similarly, a random IM patient serum 10
As a result of performing the same search for 0 cases, 5 cases were found to lack the BS antibody and only the B antibody was detected as in the case of sister A in FIG. However, in the conventional Davidson method, SRB is used.
A small number (5-6%) of false negative cases were generated that depended on the C-aggregation reaction. Therefore, according to the present invention, it became clear that such a small number of false negative cases would not be overlooked.

[0032]

[Example 3] <P-B antibody test by MPHA method> P-B antibody titer was measured by MPHA method on 47 IM patient sera and 80 normal human sera collected at random. I
44 cases of M serum showed an antibody titer of 80 times or more, and the average was 1
It was 280 times. On the contrary, the normal serum was 10 times or less in all cases, and from this result, the method of the present invention was used for P
It has been shown to be fully useful for the identification of the -B antibody.
Note that the remaining 3 cases are undecided because samples of blood collected over time are not available. However, in the conventional method using SRBC as an indicator cell, one negative case was found in the 44 IM patient sera. It was found that this one example was an example in which only B antibody was lacking BS antibody.

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, the P-B antibody can be specifically detected without the need for the absorption operation of the H-D antibody and the F antibody, and further, the P-B antigen and the artificial particles extracted from BRBC are obtained. By carrying out the particle agglutination reaction and the mixed passive agglutination reaction using, it is possible to reliably detect the BS antibody negative IM patient.

The aggregation results are negative (-) and positive (+).
It is possible to reduce the human error because it can be judged directly from the presence or absence of aggregation, instead of the judgment from the combination.

Furthermore, by conducting the examination by the particle agglutination reaction and the mixed passive agglutination reaction, it is possible to perform a quick and large number of specimen inspections without requiring a special and expensive device.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a monospot test of Orthodiagnostics,

FIG. 2 is a diagram showing an example of detecting P-B antibody by an in-gel double diffusion method,

FIG. 3 is a diagram for explaining a mixed passive agglutination method using a P-B antigen-immobilized microplate.

FIG. 4 is a diagram showing the time course of BS and B antibody in two IM patients, sister A and sister B,

[Explanation of symbols]

 1 slide glass 2, 3 well 4, 5, 6, 7 well 15 sedimentation line 8 well 9 P-B antigen 10 P-B antibody 11 sensitized particle 12 positive pattern image 13 negative pattern image

Claims (3)

[Claims] 1. Bovine red blood cell (BRBC) substrate and mouse T
A test characterized by using P-B antigen isolated and purified from a cell leukemia cell line as an antigen, and specifically detecting P-B antibody without requiring an operation of absorbing H-D antibody and F antibody Method. 2. A test characterized in that the P-B antibody specific detection can be directly judged by the presence or absence of agglutination, not by the combination of positive (+) and negative (-) agglutination reaction results. Method. 3. An inspection method, wherein the agglutination reaction is performed by a particle agglutination reaction method and a mixed passive agglutination reaction method.