JPS5994068A - Detection of l.'s cell membrane antibody in blood by rosette forming method - Google Patents

Abstract

PURPOSE:To simply and accurately measure the antibody value in serum, by a method wherein the isolated pancreas L.'s cells of a mouse are adhered to the bottom surface of a well of a test plate and, after serum to be examined is added to said well, labeled particles such as animal red corpuscles are added to count the number of rossettes formed around L.'s cells. CONSTITUTION:The isolated pancreas L.'s cells 7 of a mouse are adhered to the poly-L-lysine layer 5 provided to the well bottom surface of a test micro-plate as antigen cells and a bovine serum albumin layer is subsequently formed to cover the excessive part of the layer 5 and to adjust the surface charge thereof. In the next step, serum to be examined of which the compliance is deactivated by heat treatment is added to react the each cell 7 and the L.'s cell membrane antibody 3 in the serum and a anti-human immune globuline 9 or protein A and animal red corpuscle 10 or a plastic bead are subsequently added. The number of rosettes each formed by adhering the red corpuscle 10 to the cell 7 are counted and, if the number of the rosette to the number of all cells are present 15% or more, it is judged that the presence of the antibody is positive and this result is used in the diagnosis and the treatment of diabetes.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for detecting pancreatic sac cell surrogate cell membrane antibodies, which are often detected in the blood of insulin-dependent diabetic patients, by forming rosettes.

Since pancreatic sac cell membrane antibodies have low species specificity and high organ specificity, isolated pancreatic sac cells from rats and mice have been used as antigens for detection of the antibodies.

Conventional methods for detecting pancreatic sac cell membrane antibodies using isolated pancreatic sac cells include the indirect fluorescent antibody method [authored by Ake Lernynark et al., N.
Eng,,J,fraud,” 299.375.
(1978) :] +25 ■-Protein A method [Ake LernwIark et al., 'Di
abetq-Logia'19.445. (1980
) ) has been used. As shown in the conceptual diagram of Fig. 1, these are surface antigens 2 and 2 of isolated adult bird cells 1 of the pancreatic sac.
The pancreatic sac substitute cell membrane antibody 3 is bound to the antibody 3, and the anti-human immunoglobulin antibody 4 labeled with a fluorescent dye or the like is bound to the antibody 3, and the cells are observed under a fluorescence microscope to determine the number of total antigen cells. However, according to these conventional methods, it is necessary to use a large number of isolated pancreatic cyst substitute cells (approximately 1000 cells/sample).
Searching for antibodies in the serum of diabetic patients is expensive and requires a fluorescence microscope and radioisotopes.
It had the disadvantage of requiring special equipment and facilities and lacking in versatility. However, the fluorescence produced by the fluorescent substance disappears over time, so there are limitations on the inspection time.

Furthermore, due to the nature of the work of observing extremely weak fluorescent light under a microscope, the judgment results are influenced by the examiner's ability and subjectivity, making it difficult to obtain accurate data (i.e., lack of quantitative ability). there were.

In view of the above-mentioned drawbacks of the conventional methods, it is an object of the present invention to provide a method that makes it possible to accurately and easily test for pancreatic sac adult avian cell membrane antibodies using a small amount of pancreatic sac substitute cells.

To achieve this objective, the method for detecting pancreatic sac bird cell membrane antibodies according to the present invention uses isolated pancreatic sac substitute cells dispersed and adhered to the bottom surface of the wells of a microtest plate as antigen cells, and is coated with the antigen cells. A test serum is added to the surface of the antigen cells to bind a pancreatic sac poultry cell membrane antibody, which is an antibody against the surface antigen of the antigen cells present in the test serum, and then an anti-human immunoglobulin antibody or protein A is bound to the surface of the antigen cells. Labeled microparticles made of animal red blood cells or plastic beads are added to form a rosette of labeled cells centered on antigen cells, and the number of rosettes is counted under a microscope to determine the number of rosettes relative to the total number of antigen cells. It is characterized by determining the ratio of the number of cells formed and using this as an index of antibody titer.

The present invention will be explained below with reference to Examples. The method of the present invention includes:
This method uses a microtest plate, in which isolated pancreatic cyst substitute cells are evenly distributed and adhered as antigen cells to the bottom surface of each well of the microtest plate. In the examples, poly-luridine was dissolved in phosphate buffer (NaCl 8000, Kcl 200"7f, KH
2PO42001Wy, Na2HPO4・2H2014
34,6# buffer solution (Ph7.
4) Add 1πg/me of solution to the well.
μe, left at room temperature for 30 minutes, and washed with pure water to form a poly-L-lysine layer 5 as shown in Figure 3(a).
was formed on the bottom of well 6, and then the mouse pancreatic sac isolated live cells (antigen cells) were suspended in the above phosphate buffer solution.
Add 1μβ of the antigen cells adjusted to 10' cells/ml (that is, approximately 1000 antigen cells per well), and place the microtest plate in a centrifuge at 200f.
Centrifuge for 5 minutes. As a result, as shown in FIG. 3(b), the isolated live pancreatic sac cells 7 are dispersed and adhered to the poly-L-lysine layer 5.

Next, ME with bovine serum albumin (BSA)
Add M culture solution containing 10 μe to 10 μe well 6 and leave it at room temperature for 30 minutes.
, HEPESo, 4.8y, L-glutamine (glu
tamine) 29m! , NaHCO342mW
with water to 100ml1. It is washed repeatedly with a 4% BSA-MEM culture solution, thereby forming a bovine serum albumin layer 8.

This covers the excess portion of the poly-L-lysine layer 5 and adjusts the surface charge so that unreacted sheep red blood cells, which will be described later, can be easily detached. Instead of bovine serum albumin, a simple serum or other processed material can be used.

Next, 5 to 10μ4 of the test serum whose complement had been inactivated (inactivated) by heating at 56°C for 30 minutes was added to the well, and the isolated living cells and the pancreatic sac bird cell membrane antibody in the serum were added to the wells. The cells were allowed to react at 37°C for 30 minutes, and then unreacted immunoglobulin (mainly IgG) was removed by repeated washing with the 4% BSA-MEM culture solution. Through such treatment, as shown in FIG. 4, the pancreatic sac adult bird cell membrane antibody (immunoglobulin) 3 in the test serum bound to the isolated living cells 7 having the surface antigen 2.

Next, sheep red blood cells 10 to which protein A (i.e., Staphylococcus aureus cell protein) or rabbit, goat, etc. anti-human immunoglobulin antibody 9 was bound to the surface with chromium trichloride etc. were cultured in the above 4% BSA-MEM. 5 x 1 in liquid
08 cells/ml was added into 5 μ4 (approximately 2×106 cells) wells and reacted at 37°C for 30 minutes. As a result, as shown in FIG. 3(d), FIG. 4, and FIG. 5, for those to which the pancreatic cyst substitute cell membrane antibody 3 was bound,
Sheep red blood cells 10 bind to the antibody 3 via the protein A and anti-human immunoglobulin antibodies 9. After the reaction, the microtest plate is inverted and left for about 1 hour to remove unreacted sheep red blood cells, which are then observed using an optical microscope. The actual image recognized by this observation is as shown in FIG. In other words, if there is a large amount of antibody 3 in the test serum, 10 sheep red blood cells per 7 isolated live cells.
However, since the sheep red blood cells 10 have a diameter of approximately % of the isolated living cells 7, it is easy to determine whether the sheep red blood cells 10 are combined to form a rosette. be able to. Here, for example, a cell in which several or more sheep red blood cells 10 are bound is regarded as a rosette-forming cell, and it is determined whether the rosette-forming cells are present in, for example, 100 cells 7 or not. do. When the ratio of rosette-forming cells (positive cells) is, for example, 10% or less, it is negative, for example, 10% to 1.
It is determined that 5% is a false positive, and for example, 15% or more is determined to be positive.

Note that the present invention is not limited to the above embodiments, and various modifications are possible. For example, instead of the above-mentioned living cells, the antigen cells are isolated pancreatic cyst substitute cells fixed with 3.0-5.0% valaformaldehyde and 30%-50% water-soluble carbodiimide reagent. You can also use Further, if the fixation treatment is performed by adding glucose and bovine serum albumin to this fixative solution, excellent fixation can be achieved. By this method, isolated pancreatic sac substitute cells can be cryopreserved in the presence of fetal bovine serum and dimethyl sulfoxide.

In addition, it is also possible to use sheep red blood cells whose surfaces are coated with protein A or anti-human immunoglobulin antibodies that have been fixed with formalin or a water-soluble carbodiimide reagent, or their freeze-dried products. Can be saved.

In addition, instead of the sheep red blood cells whose surfaces were coated with protein A or anti-human immunoglobulin antibodies, beads made of synthetic resin such as polyacrylamide resin or styrene resin were coated with protein A or anti-human immunoglobulin antibodies. can be used.

However, in the present invention, it is of course possible to use a fractionated immunoglobulin (IgG, IgA, IgM, or all of these) instead of the test serum as the test specimen; The present invention also includes the case where a picture is used.

Furthermore, it goes without saying that the composition of each reagent, blood cells, etc., the temperature and time during reaction and standing, etc. may be varied as necessary.

As described above, the method of the present invention requires only a fraction of the amount of isolated pancreatic sac substitute cells compared to conventional methods, and does not require the use of special equipment or facilities such as a fluorescence microscope. Because there is no
The cost required for the test is significantly reduced, making it economical and making it possible to improve the versatility of the test. Also,
In the method of the present invention, it is possible to clearly recognize whether or not rosette formation is occurring in the image obtained with the microscope, eliminating subjective differences among examiners, and since no change occurs over time, the data does not change depending on the elapsed time. , accurate data is obtained and quantitative nature is ensured. Furthermore, since rosette-forming cells can be preserved, observation can be carried out at a separate facility or location, which eases operational constraints.

[Brief explanation of drawings]

Fig. 1 is a conceptual diagram of the conventional method, Fig. 2 is an explanatory diagram of the observed image when using the conventional method, and Fig. 3 (a) to (d) are the images in the microplate when the method of the present invention is implemented. FIG. 4 is a conceptual diagram of the method of the present invention, and FIG. 5 is an explanatory diagram of an observed image obtained when the present invention is implemented. 2... Surface antigen, 3... Immunoglobulin, 5...
Poly L-lysine, 6...well, 7...isolated living cells, 8...bovine serum albumin, 9...protein A
t or anti-human immunoglobulin antibody, 1o...Sheep red blood cell patent applicant Sogo Biomedical Research Institute Co., Ltd. Agent Patent attorney Waka 1) Katsu - Procedural amendment (voluntary) December 22, 1980 Commissioner of the Japan Patent Office 1. Indication of the case 1982 Patent Application No. 203999 2. Title of the invention Detection method of blood membrane caries cell membrane antibody by rosette formation method 3. Relationship with the person making the amendment Patent applicant address Tokyo Sogo Biomedical Research Institute Co., Ltd. 4-25-10 Chuo, Nakano-ku, Tokyo Name Representative Director Kenji Kondo 4, Agent I 6 Column 7 for detailed explanation of the invention in the specification subject to the amendment Contents of the amendment As shown in the attached sheet The following amendments will be made to the specification. (1) r Diabet9-Logia on page 2, line 12
DiabetoI! Correct as ogia J. (2) “Poly L-lysine” on page 4, line 17
-lysine] and rNaclJ to rNac6J. (3) Correct rKclJ on page 4, line 18 to rKcnJ. (4) Correct r Ph J on page 4, line 19 to rPHJ. (5) Delete "excess fetal bovine serum" from page 5, line 16 to line 17. that's all

Claims (1)

[Claims] Isolated pancreatic sac adult bird cells dispersed and adhered to the bottom surface of a well of a microtest plate are used as antigen cells, and test serum is added to the antigen cells, and the antigen present in the test serum is displayed on the surface of the antigen cells. A pancreatic sac substitute cell membrane antibody, which is an antibody against a cell surface antigen, is conjugated thereto, and then labeled fine particles made of animal red blood cells or Plasnac beads conjugated with an anti-human immunoglobulin antibody or protein A are added.
As a result, a rosette of labeled cells centered on antigen cells is formed, and the number of rosettes is estimated to be about 10 or more under a microscope.
A method for detecting blood membrane substitute cell membrane antibodies by a rosette formation method, characterized in that the ratio of the number of rosette-forming cells to the total number of antigen cells is determined and this is used as an index of antibody titer.