JP3383649B2 - Minimal blood type determination composition, method and kit for criminal discrimination - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to the industry] is, your criminal forensics area
TECHNICAL FIELD The present invention relates to a monoclonal antibody composition for blood group determination for a biological sample, a simple blood group determination method using the same, and a blood group determination kit.

[0002]

2. Description of the Related Art A human blood group is determined by a type-specific antigen-antibody reaction that occurs between a blood group antigen such as red blood cells, saliva and semen and an antibody corresponding to the antigen. In the field of criminal science, human-derived polyclonal antibodies have been used to perform blood group tests on forensic biological test samples (blood stains, saliva, semen, vaginal fluid, tissue pieces, teeth, hair, nails, etc.).
However, due to various reasons, production and sales of human-derived polyclonal antibodies were discontinued. To perform a blood group test,
At present, there is no other way than using a commercially available mouse-derived monoclonal antibody. Mouse-derived monoclonal antibodies do not interfere with blood typing of fresh blood tested in medical institutions, but are legal biological samples in the field of criminal science (blood stains, saliva, semen, vaginal fluid, tissue fragments, teeth, hair). , Nails, etc.), if the monoclonal antibody is directly applied to various test methods using already established polyclonal antibodies, non-specific reaction occurs in all the antibodies and correct blood group determination occurs because indicator blood cells aggregate. I can't.

On the other hand, with the development of analytical instruments, various methods for detecting antigenic substances have been studied, but effective analytical instruments have not been developed yet. At present, in domestic and overseas appraisal institutions or research institutes that have performed ABO blood group tests using polyclonal antibodies, human-derived samples (blood stains,
It has become difficult to perform blood group tests on saliva, semen, vaginal fluid, tissue pieces, teeth, hair, nails, etc.).

[0004]

[Problems to be Solved by the Invention] In the mixed agglutination method, an antibody specifically reacting with an antigen is confirmed by observing an aggregate of indicator blood cells specifically reacting with the antibody under a microscope.
It is a blood type test method for determining the type. According to the mixed agglutination method using human-derived polyclonal antibody (hereinafter abbreviated as conventional method), when a commercially available mouse-derived monoclonal antibody is used as it is, non-specific reaction occurs in all antibodies,
We have experienced that all samples are misjudged as type AB due to the aggregation of indicator blood cells. Therefore, it was necessary to develop a new test method that can completely suppress this nonspecific reaction and correctly determine the blood group. For these reasons, it has been desired to develop a simple test kit having high sensitivity to various blood group antigens. It is an object of the present invention to provide a monoclonal antibody composition having high sensitivity to various blood group antigens, a simple blood group determination method using the same, and a blood group determination kit.

[0005]

[Means for Solving the Problems] The present inventor has proposed a new mixed agglutination method capable of completely suppressing the non-specific reaction that occurs when a mouse-derived monoclonal antibody is used and correctly determining the blood group of all forensic samples. Has been developed and the present invention has been achieved. This method, like the conventional mixed agglutination method, is simple in operation and can observe the presence or absence of agglutination of the indicator blood cells more clearly than the conventional method, and thus requires no skill for determination.

The present invention is a blood containing an antibody additive solution, which causes only a small amount of antigen in a test sample and an antigen-antibody reaction which is a specific reaction originally possessed by the antibody, and does not cause a non-specific reaction. It is an invention relating to a typing monoclonal antibody composition, more specifically, a prepared anti-A antibody, a prepared anti-B antibody, and a prepared anti-H antibody.

[0007] A monoclonal antibody against a blood group substance for accurately revealing the presence of a blood group antigen, in which case the present invention comprises an antibody additive solution and accurately reveals the presence of a blood group antigen. It is a monoclonal antibody against a blood group substance. A monoclonal antibody composition for blood typing, which is characterized by causing only an antigen-antibody reaction, which is a specific reaction originally possessed by a small amount of an antigen and an antibody in a test sample, and not causing a nonspecific reaction, more specifically Specifically, to prepared anti-A antibody, prepared anti-B antibody and prepared anti-H antibody .
It is an invention related to.

That is, it contains a protein selected from the group consisting of a milk component, an albumin component and type AB human serum, and a surfactant. More specifically, the above protein and surfactant are each prescribed in physiological saline. To accurately reveal the presence of trace amounts of antigens and blood group antigens in the test sample, which contains a solution that has been adjusted to a concentration and dissolved, and an antibody additive solution that contains a small amount of preservatives as necessary. Monoclonal antibody composition for blood group determination targeting forensic biological samples in the criminal forensic area that causes only a specific reaction originally possessed by the monoclonal antibody against the blood group substance and does not cause a non-specific reaction, more specifically The outline is prepared A antibody, prepared B antibody and prepared anti-H antibody.

Further, the present invention uses the above monoclonal antibody composition, crime forensics to detect traces of ABH antigens in a test sample, to observe the presence or absence of agglutination of instruction blood cells
The gist is a simple method for determining the blood type of a forensic biological sample in the field.

This is a method that can be applied as it is to a test method by a mixed agglutination method using a polyclonal antibody. In that case, the present invention uses the above-mentioned monoclonal antibody composition to detect a minute amount of ABH antigen in a test sample. A crime that can be directly applied to the test method based on the mixed agglutination method that uses a polyclonal antibody to detect and observe the presence or absence of the agglutination reaction of the indicated blood cells.
The gist is a simple method for determining the blood type of a forensic biological sample in the crime detection area.

All forensic biological test samples such as blood stains, saliva, semen, vaginal fluid, urine, dirt, nails, hair, and tissue pieces are used as test samples. In that case, the present invention provides blood stains, saliva, semen, vagina. liquid,
The above-mentioned monoclonal antibody composition is used as a test sample for all forensic biological test samples such as urine, dirt, nails, hair, and tissue pieces, and a minute amount of ABH antigen is detected in the test sample to detect the indicated blood cells. A simple method for determining the blood type of a forensic biological sample in the criminal forensic area that can be directly applied to the test by the mixed agglutination method using a polyclonal antibody for observing the presence or absence of an agglutination reaction is summarized. There is.

Further, the present invention is, criminal, wherein the monoclonal antibody additive solution, a combination of a sample adhesive fixing plates which can exert more antibodies simultaneously
The gist is a blood typing kit for forensic biological samples in the field of criminal investigation .

The monoclonal antibody additive solution is characterized in that it contains a protein selected from the group consisting of a milk component, an albumin component and type AB human serum, and a surfactant. More specifically, it is a solution prepared by dissolving each of the above-mentioned protein and surfactant in physiological saline at a predetermined concentration, and adding a small amount of a preservative, if necessary. It is characterized by containing a protein selected from the group consisting of a component, an albumin component, and human serum AB, and a surfactant. More specifically, it is a solution prepared by dissolving each of the above proteins and surfactants in physiological saline at a predetermined concentration and dissolving them, and optionally adding a small amount of a preservative as a monoclonal antibody additive. A blood type determination kit for a forensic biological sample in a crime detection area, which is characterized by a combination of a solution and a plate for adhering and fixing a sample capable of simultaneously acting with a plurality of antibodies. .

The present invention also provides the above-described blood grouping monoclonal antibody composition, more specifically, a prepared anti-A antibody,
Criminal forensics characterized by a combination with a prepared anti-B antibody and a prepared anti-H antibody, or a sample adhesive immobilization plate capable of simultaneously acting with a plurality of antibodies such as anti-H agglutinin
The gist is a blood typing kit for forensic biological samples in the field.

The sample adhesive immobilization plate has three areas for adhering and immobilizing the test sample and dropping anti-A antibody, anti-B antibody, anti-H antibody or anti-H agglutinin respectively to react. In these three areas, the test sample is stuck and fixed,
It is for dropping anti-A antibody, anti-B antibody, anti-H antibody or anti-H agglutinin respectively to react. Crime detection area characterized by a combination of a sample adhesive immobilization plate capable of simultaneously actuating a plurality of antibodies on a transparent plastic plate having three areas and a prepared anti-A antibody, prepared anti-B antibody, prepared anti-H antibody Forensic biological samples in
The subject is a blood typing kit.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention applies immunological detection methods and uses commercially available monoclonal antibodies to forensic biopsy samples of human origin in the field of crime science (blood stains, saliva,
It is a kit that develops a simple method that enables blood group tests for all semen, vaginal fluid, tissue pieces, teeth, hair, nails, etc.).

The present invention is based on the MCAR method (APPLICATION O
The feature is that the plate used for F MIXED CELL AGGLUTINATION REAC-TION) is improved and the adhesive is directly attached to the plastic plate. Due to this improvement, three kinds of antibodies can be simultaneously acted on one plate, there is no concern of die-type contamination from the inspector or the instrument on the adhesive surface, and the cost of the sample can be reduced. .

<< Adhesive Plate (Plate for Adhesive Fixing of Sample) >> A slide glass, a Scotte tape or a Sellotape (registered trademark) can be used, but instead of them, a plastic plate, for example, 0.1 mm thick and slightly elastic. Apply the adhesive directly to three places on a piece cut into a slide glass. There is no particular limitation on the adhesive or pressure-sensitive adhesive to be used, but a cellophane adhesive or a silicone-based medical adhesive was suitable. These adhesives do not become cloudy even when immersed in water for a long time. There is no need to cut clothes, and it is sufficient to use a sample of only 3 mm. Sweat etc. can be easily transferred by direct transfer to determine the type. No test tube or hole glass is used for the equipment, so the equipment is washed. It is a test method that has many advantages, such as the fact that it takes almost no time to spend, and it should be greatly utilized as a method of blood group determination in the future.

<< Collection of Sample >> The sample is transferred by lightly pressing the adhesive plate onto the sample. The opposite may be the case depending on the case. Since the plastic plate is thin and deforms, it can be transferred even from an uneven object.

<Immobilization of sample> Generally, it is necessary to perform heat fixation so that the test sample does not elute, but in this method, it is not necessary to fix by heat or a chemical such as methanol.

<< Antibody Sensitization Time >> Antibodies sensitize commercially available mouse-derived monoclonal anti-A antibody, anti-B antibody, and anti-H antibody to a sample, but even if the sensitization time is too long, it is nonspecific. No reaction appears. Generally, the sensitization time is 2 to 4 hours. Sensitization is carried out in a moist box so that the antibody does not dry out. For anti-H, commercially available anti-H (Ulex) agglutinin may be used, but since this reagent is not a monoclonal antibody, it need not be a prepared antibody.

<< Removal of Unreacted Antibody >> Of the antibodies used for sensitizing the test sample, in order to remove unreacted excess antibody, physiological saline stored in a washing bottle and stored at room temperature was poured into the antibody. , Rinse off.

<< Indicating Blood Cell for Judgment >> Normal blood cells are used in all the test samples. It is not necessary to treat blood cells with papain or the like.

<< Sensitization time of indicator blood cells >> In this method, 3 to 5 minutes is sufficient for the sensitization time of indicator blood cells. If it takes more than 10 minutes, it will take time to wash away the blood cells. Sensitization is carried out in a moist box so that the blood cells do not dry out.

<< Removal of unreacted indicator blood cells >> The sample adhesive fixation plate is floated on physiological saline so that the side on which the sample is immobilized is on the lower side, and the unreacted excess indicator blood cells are naturally lowered to be removed. The time required for this is about 10 minutes, but it is better to make a judgment by observing the state of blood cell detachment.

<Judgment> A large number of specimens can be easily judged by a method of taking out a plastic plate (plate for fixing sample adhesion) from physiological saline and observing it under a microscope of 100 times as it is without covering it with a cover glass. However, in the case of hair, the aggregates of indicator blood cells tend to fall off, so it is necessary to gently perform the removal from the physiological saline.

<< Test Material >> 1) Antibodies for Blood Group Determination (1) Mouse-Derived Monoclonal Antibodies Commercially available mouse-derived monoclonal antibodies exemplified below can be used. Imcore Anti-A (Lot 101010, Aggregation Value 256
Anti-B (Lot 201020, agglutination titer 256 times) (Sanko Junyaku) Monoclone BCA anti-A (Lot A8123, agglutination titer 256 times) Anti-B antibody (Lot B8125, agglutination titer 256 times)
(Cosmobio) Bioclone Anti-A (Lot BAA199A21, Aggregation titer 256 times) Anti-B (Lot BBB592A21, Aggregation titer 256)
Fold) (Ortho Clinical Diagnostics) Ceraclon Anti-A (Lot 900091, Aggregation Value 2
56 times) Anti-B (Lot 900101, Aggregation titer 256 times) (Dainippon Pharmaceutical) Wako Anti-A (Lot RL389, Aggregation titer 128 times) Anti-B (Lot RK489, Aggregation titer 256 times) (Wako Pure Chemical) Anti-H serum (Lot 70514, aggregation titer 512 times)
(Diagast) (2) Human-derived polyclonal antibody As a control experiment, the human-derived polyclonal antibody exemplified below was used. Anti-A serum (Lot 004, agglutinin titer 256 times) Anti-B serum (Lot 004, agglutinin titer 256 times) (international reagent) (3) Anti-H agglutinin Eurex seed (Sanko Junyaku, Lot 109001)
Anti-H agglutinin extracted from (home-made, agglutinin titer 64 times) 2) Blocking agent The following commercial products can be used as a reagent for suppressing non-specific reaction. -Protein Block Ace (Lot STK929207, Dainippon Pharmaceutical) Ovalbumin (Lot PAH7069, Wako Pure Chemicals) Bovine albumin (Lot SEG3965, Wako Pure Chemicals) 10% rabbit serum (Lot H711, Nichirei) Horse serum (Lot 8237C, Dainippon Pharma) Pharmaceutical) Human serum (type AB) (Lot 90024, Cosmo Bio) 〇 Surfactant Tween 20 (Lot ACK5505, Wako Pure Chemicals) Tween 40 (Lot ELQ2020, Wako Pure Chemicals) Tween 60 (Lot ELM5800, Wako Pure Chemicals) 3) Indicator blood cells As the indicator blood cells, commercially available products exemplified below can be used. Apharmagen (Ortho Clinical Diagnostic: A1 blood cell and B blood cell, each 3% suspension) Surgescreen (Ortho Clinical Diagnostics: O blood cell 3% suspension) 4) Test sample So far Japan Any legal biological sample (bloodstain, blood stain,
Saliva, semen, vaginal fluid, tissue pieces, teeth, hair, nails, etc.) can be targeted. The following samples were used in the experiment. (1) Sample created in-house <Blood stain> Blood stain with gauze attachment (59 cases of A type, 31 cases of B type, 35 cases of O type)
Examples, AB type 8 cases, A2B3 type 1 case) Diluted blood stain with gauze adhesion (10 times, 20 times, 40 times, 80 times)
Double-diluted A, B, and O blood stains in 5 cases each, 1.5 x 1.5
100 cm of diluted blood stains attached to two cm-sized gauze pieces and naturally dried) <Body fluid spots> Salivary spots with gauze attachment (secretory type of A type, B type, O type: 2 each)
0 cases, non-secretory type of A type, B type, and O type: 4 cases each Semen with gauze adhesion (secretory type and non-secretory type of A type and B type: 1 case each) Vaginal fluid with gauze adhesion (B type) Secretion type: 2 cases) <Nail, hair> Nail (A type, B type, O type: 5 cases each) Hair (root part) (A type, B type, O type: 5 cases each) (2) Site Derived sample <Blood stains> Blood stains collected on gauze (collected from floors, walls, streets, desks, etc. at the murder scene. Collected from glass and floor at the scene of the theft, etc.) <Body fluid spots> Attached to cigarette butts Saliva spots (6 brands made in Japan: Mild Seven, Peace, Seven Star, Hope, Castor, Cabin) (collected from inside cigarette butts in rape and robbery cases) Semen spots on tissue paper (in rape cases (Collected from inside the trash, left for about 1 week outdoors) <Organization ＞ Abraded skin piece attached to a contact vehicle in a false case of road traffic law <Tooth> An unidentified tooth that had been washed up on the shore (using a second molar, plaque and pulp) 5) Other equipment mixture The following materials (commercially available products) were used as sample fixing materials in the agglutination method. (1) Slide glass (Matsunami: White edge frost N)
o. 1) (2) Double-sided tape (Nichiban: CW-18) (3) Plastic patch (A-one: opaque type)

Test Method 1) Preparation of Blocking Agent (1) 6% Block Ace Solution Adjust to 6% concentration using physiological saline, and add sodium azide to a final concentration of 0.01%. , Stored at room temperature. (2) 0.3% Tween 20 solution: Adjust to 0.3% concentration using physiological saline, add sodium azide to a final concentration of 0.01%,
Stored at room temperature. 2) Preparation of antibody for preparing blood group with addition of blocking agent A commercially available monoclonal anti-A antibody, anti-B antibody and each blocking agent were added in equal amounts and mixed, and used as an antibody for preparing blood group. 3) Inspection procedure (1) Stick double-sided tape on a slide glass, and stick three plastic patches side by side on the tape. (2) A sample is attached to an adhesive surface in a circle surrounded by a plastic patch and having a diameter of about 6 mm in the form of a single fiber, and is pressed and fixed. (3) Prepared anti-A, anti-B antibody and anti-H antibody or anti-H
Eurex agglutinin (homemade, 64 times agglutinating titer) is dropped onto the sample and left to stand in a wet box for about 4 hours for sensitization. (4) Preparation of unreacted solution by pouring physiological saline into the sample (5) Drop 3% indicator blood cells corresponding to each agglutinin on the sample and let stand for 3 to 5 minutes in the wet box to react (6) Slide glass with sample surface facing down The sample is allowed to stand in physiological saline for 10 to 15 minutes, and the unreacted indicator blood cells are spontaneously dropped and removed.
(0 times) and make a pattern determination. (8) All inspection operations are performed at room temperature.

<Results> 1) Characteristics of this method In the conventional method using a polyclonal antibody, some non-specific reaction may remain, and it may require skill in the determination. However, in this method, since non-specific reaction does not occur at all, it is easy to observe the presence or absence of the aggregation of indicator blood cells.
Therefore, no skill is required for the determination. 2) Results of examination of commercially available mouse-derived monoclonal antibodies Anti-A and anti-B antibodies were manufactured by Sanko Junyaku and Cosmo Bio.
Consistent with the results using human-derived polyclonal antibodies in all forensic samples. Other companies' products are non-secretory type B
B antigen in type saliva (1 of 4 cases) and A2B3 type blood stain (1 case) could not be detected. Diagast anti-H antibody could be used without problems. 3) Results of examination of detection sensitivity The detection sensitivity was equivalent to that of the conventional method using a human-derived polyclonal antibody. (1) Sample amount required for inspection It was possible to determine the type from an extremely small amount of blood stains and body fluid spot samples (sample attached to 3 mm cotton thread, 0.1 x 1 mm piece of tissue). (2) Detection limit of diluted bloodstains Up to 20 times diluted bloodstains could be detected. Distilled water and CM
It was possible to test up to a 40-fold dilution by extracting with a (chloroform / methanol) extract and concentrating (Table 2). (3) Detection limit of diluted saliva In the secretory type, each type was detectable up to 1600-fold dilution. The non-secretory type could be tested up to a 40-fold dilution although there were individual differences.

<Discussion> 1) When commercially available mouse-derived monoclonal anti-A and anti-B antibodies of one company and anti-H antibody of one company are used in accordance with the conventional method, nonspecific reaction occurs in any of the antibodies. As a result, all the samples were erroneously determined to be AB type because the indicator blood cells aggregated in all the samples. 2) In the mixed agglutination method, when a mouse-derived monoclonal antibody is used, a nonspecific reaction occurs, and the indicator blood cells agglutinate in all the samples, so that the blood type cannot be determined at all.
Such a phenomenon was not seen in human-derived polyclonal antibodies or human-derived monoclonal antibodies, and was considered to be unique to mouse-derived monoclonal antibodies. Since this phenomenon may be caused by substances other than the antibodies contained in the monoclonal antibody, the antibodies were treated with an ultrafiltration membrane having a molecular weight of 10,000, 30,000, 50,000, 100,000 in order to remove the substance. But it didn't work. That is, it was considered that the non-specific reaction caused by the monoclonal antibody was not caused by a substance other than the antibody but by the reactivity of the antibody itself. 3) On the other hand, for the sample, methanol, chloroform,
A treatment with an organic solvent such as formaldehyde and a heat treatment with a drier were performed, but there was no effect. 4) Block Ace and Tween, which are blocking agents
Although the effect was recognized when 20 was added to the antibody alone, some nonspecific reactions remained and it was sometimes difficult to judge.
In particular, the saliva sample may be difficult to determine depending on the sample. Therefore, when these two types of blocking agents were added to the antibody, the non-specific reaction of indicator blood cells could be completely suppressed in all the samples. This phenomenon is 2
It was considered that the mixing of the types of blocking agents resulted in a synergistic blocking effect that could not be achieved by itself.

<< Summary >> The new mixed agglutination method of the present invention was examined. 1) By adding Block Ace and Tween 20 to a mouse-derived monoclonal antibody, a non-specific reaction peculiar to the mouse-derived monoclonal antibody was completely eliminated. I was able to suppress it. 2) This method can perform ABO blood group tests on all forensic samples. As compared with the conventional method, since non-specific reaction of indicator blood cells does not occur at all, it is easy to observe the presence or absence of indicator blood cell aggregation, and no skill is required for the determination. The inspection time is
Since the sensitization time of blood cells and the removal time of unreacted blood cells can be shortened, it is about 1 hour shorter than the conventional method.

[0032]

[Operation] By performing a blood group test using this kit, the following operations and effects can be obtained. (1) Using a commercially available monoclonal antibody, the blood sample of all forensic biological test samples can be easily tested without requiring special pretreatment of the test sample. (2) The determination was easy because the sensitivity was high and the presence or absence of aggregation of indicator blood cells was clear. (3) The inspection time is as short as 4 hours and a half. (4) All operations can be performed at room temperature. Also,
It is not necessary to cool physiological saline for washing excess serum and excess blood cells, and it is possible to use it even if it is stored at room temperature. (5) The work process is simple and no special skill is required. By using the present invention, a commercially available monoclonal antibody can be used to perform A
The BO type blood group can be clearly determined with high sensitivity.

By using the present invention, forensic biological samples (blood stains, saliva, semen, vaginal fluid, tissue pieces, teeth, hair, etc., which have been used in appraisal institutions or research institutions in Japan and overseas.
Blood typing of the nails, etc.) can be continued.

By improving the sample adhesive immobilizing plate so that the adhesive is directly adhered to the plastic plate, three kinds of antibodies can be simultaneously acted on one plate, and the inspector on the adhesive surface can There was no fear of mold contamination from the instrument, and the cost of the sample could be reduced. A transparent and slightly elastic plastic plate of a slide glass size was used as the base plate of the sample adhesive fixation plate. For this reason, if the sample is sweat stains or blood sperm semen that has adhered to clothing, it can be easily transferred by pressing it directly onto the adhesive surface of the plate, and there is no need to cut clothes. Have advantages. Since all the inspection work is performed using this plate, there is no need to spend time cleaning the equipment, since no test tubes or hole glasses are used for the equipment.

[0035]

The details of the present invention will be described with reference to Examples. The present invention is not limited to these examples.

Reference Example 1 (Experiment 1) Sample: Unused gauze piece inspection method: Gauze fiber was attached to double-sided tape, and anti-A, anti-B,
Anti-H monoclonal antibody is sensitized for 4 hours, and indicator blood cells not corresponding to each are added to observe the reaction. Results: Monoclonal antibodies do not aggregate except in the corresponding indicator blood cells. That is, the agglutination reaction of indicator blood cells that occurs when a mouse-derived monoclonal antibody is used in the mixed agglutination method is caused by a specific antigen-antibody reaction, and the non-specific reactive group and the indicator contained in the antibody It was confirmed that the blood cells did not cause non-specific reaction.
As described above, since the monoclonal antibody causes a normal antigen-antibody reaction with blood cells, it can be used for inspection in the medical field without any problem.

(Experiment 2) Sample: Blood stain, saliva (secretory type and non-secretory type) in each case 1) A sample is attached, the antibody is used as it is, and an indicator blood cell is added to make a determination. 2) Attach the sample, blocking agent (Block Ace 1
After 4 hours of blocking with 5%), the antibody is used as it is, and sensitization is performed for 4 hours. 3) Attach sample and blocking agent (Tween20 1.5%)
After blocking for 4 hours, the antibody is used as it is, and the cells are sensitized for 4 hours. 4) Attach a sample, block with a blocking agent (0.3% of Tween20 and 6% of Block Ace in an equal amount) for 4 hours, then use the antibody as it is, sensitize for 4 hours, and add an indicator blood cell for determination. 5) Attach a sample, sensitize with the prepared antibody (antibody mixed with Tween20 0.3% and Block Ace 6% in an equal amount) for 4 hours, and then judge by adding indicator blood cells (method of the present invention). Results and discussions 1) -4) cannot prevent non-specific reactions,
In 5), only a normal antigen-antibody reaction occurred and a non-specific reaction could be prevented. That is, even if any pretreatment is performed, the non-specific reaction cannot be prevented by using the antibody as it is, and the specific antibody originally possessed by the antibody prepared by adding the additive to the antibody is used. It was found that only the antigen-antibody reaction, which is a reaction, occurred, and non-specific reaction did not occur.

(Experiment 3) (Results of Examining Additives) One of the reasons why the test method used for the polyclonal antibody cannot be used for the monoclonal antibody is that the protein concentration of the monoclonal antibody is low. This point will be examined by adding various proteins to the antibody. The results are shown in Table 1.

[0039]

[Table 1]

<Discussion> The non-specific reaction in the mouse-derived monoclonal antibody was considered to be due to the lack of protein concentration due to the method for producing the mouse-derived monoclonal antibody. Then, the content of the protein was measured with a visible ultraviolet absorptiometer, and it was found that the content of the protein was smaller than that of the conventionally used human-derived polyclonal antibody. Then, although various proteins were added to the antibody, it was found that this non-specific reaction can be prevented by adding not only the proteins but also various proteins and a surfactant (Tween 20, 40, 60; trade name). Tween 80 had no effect. Among them, Block Ace (brand name, 1% milk protein and organic acid buffer as the main components) and Tween20,40,60 had the best results, but good results by changing the concentration of each additive. I think there is a possibility of getting In addition, the above proteins and
After blocking with Tween 20, 40 and 60, a non-specific reaction appears when the mixed agglutination method is performed using only the monoclonal antibody. This means that the blocking agent does not exert its original blocking effect, but the environment of the reaction system is changed by adding an additive to the antibody,
It is reasonable to assume that the non-specific reactive groups possessed by the antibody itself will not be able to react and only the specific antigen-antibody reaction that should exist should occur. From the above, FIG.
A reaction system such as Although the experiment was carried out in a physiological saline system, the optimum pH of the monoclonal antibody was 7.2.
Is. When handling general samples, it is not necessary to use PBS, but for septic samples and the like, a more stable reaction system can be secured by using PBS (phosphate buffered saline).

Example 1 (Contents of Kit) (1) Sample Adhesive Fixing Plate (FIG. 1) The sample adhesive fixing plate is approximately 7 cm × 2 cm ×
A double-sided tape was attached on a transparent plastic having a size of 0.3 mm (thickness), and three thin water-repellent plastic rings were attached on it. Fig. 1 shows what was pasted at three locations. One end of the plastic is preferably ground glass so that the sample name can be entered. The inspection is carried out by fixing the sample on the adhesive surface in three plastic rings attached to the sample adhesive fixing plate and then in the ring. To prevent anything other than the sample from adhering to the adhesive surface for adhering the sample adhesive, attach protective paper of double-sided tape to the entire adhesive surface. (2) Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) in an antibody additive solution physiological saline solution at 6%
Above, Tween20 (manufactured by Wako Pure Chemical Industries) is prepared to a concentration of 0.3% or more and dissolved, and sodium azide as a preservative is added to
Added to be 1%. (3) Hemagglutinin (commercially available) Mouse-derived monoclonal anti-A antibody and mouse-derived monoclonal anti-B antibody (manufactured by Sanko Junyaku), mouse-derived monoclonal anti-H antibody (Diagast) Anti-H agglutinin (Eurex seed extract) , (Homemade) (4) Directed blood cells A-typed blood cells, B-typed blood cells, O-type blood cells

(5) Test procedure Mouse-derived monoclonal anti-A antibody, anti-B antibody, anti-H
An equal amount of antibody additive solution is mixed with each of the antibodies, and they are used as prepared anti-A antibody, prepared anti-B antibody, and prepared anti-H antibody. Anti-H
Since agglutinin is not a monoclonal antibody, it is not necessary to add additives. The sample is divided into three parts, and the sample is adhered to the adhesive surface in the plastic ring at three points (A, B, and C) on the sample adhesive fixing plate, and pressed to fix. Of the samples fixed at 3 points,
Prepared B antibody is dropped on B and prepared H antibody or anti-H agglutinin is dropped on C, and they are reacted in a humid box for about 4 hours. Remove from the moist box and inject physiological saline to remove unreacted antibody. After returning to the wet box, add 1 drop of A type blood cells to A, B type blood cells to A, and 0 type blood cells to C, and then cover the wet box for 3 to 5 minutes. Place it and let it react. The sample adhesive fixation plate is left still in physiological saline for 10 to 15 minutes with the sample surface facing downward, and unreacted indicator blood cells are spontaneously dropped and removed. Check the presence or absence of agglutination of the indicated blood cells on the sample under a microscope (× 10
Observe in 0) and make a pattern decision. All operations are performed at room temperature.

Example 2 Regarding commercially available monoclonal anti-A and anti-B antibodies from 5 companies,
Blood stains 134 cases (including A2B3 type 1 case), saliva (secretory type 80)
Examples, non-secretory type 16 cases) Semen (secretory type 2 cases, non-secretory type 2 cases)
Example), vaginal fluid (secretory type 2 cases), hair (15 cases), nails (15 cases)
Example), tissue pieces attached to an accident vehicle (1 case), teeth of a water carcass (1 example), cigarette butts (6 brands) left behind at the crime scene, etc. were examined and compared with the results obtained with the polyclonal antibody. did. The results are shown in Table 2. Monoclonal anti-A,
Among the products of 5 companies, the anti-B antibody manufactured by Sanko Junyaku Co., Ltd. and Cosmo Bio Inc. was correctly typed in all the test samples. Anti-H antibody was typed correctly in all test samples.

[0044]

[Table 2]

Example 3 The detection limit obtained by examining diluted blood stains (0 to 80 times) by this method and the detection limit obtained with a polyclonal antibody were compared and examined. The results are shown in Table 3. This method has the same detection sensitivity as the test method using a polyclonal antibody, and was detectable up to 20-fold dilution.

[0046]

[Table 3]

Example 4 The detection limit obtained by examining diluted saliva (0 to 6400 times) by this method was compared with the detection limit obtained with a polyclonal antibody. The results are shown in Table 4. This method had a detection sensitivity equivalent to that of a test method using a polyclonal antibody, and was 1600 times as high as the secretory type and 40 to 320 times as high as the non-secretory type although there were individual differences.

[0048]

[Table 4]

Example 5 Using the monoclonal antibody composition for blood typing of the present invention, all samples (type, age, decay,
Results of tests for contamination, subtype, fetus, dilution, etc.,
Everything that could be tested with the polyclonal antibody could be detected. Then, although some non-specific reaction may remain in the polyclonal antibody, in the method using the blood group determination monoclonal antibody composition of the present invention, since no non-specific reaction occurs at all, the determination can be made for all samples. It was easy. 1) Subtype blood stain inspection Monoclonal preparation antibody: Mouse-derived monoclonal antibody (manufactured by Imcore), 6% Block Ace, and 0.3% Tw
Equal amount of een20 added Polyclonal antibody: Kokusai Reagent α: Anti-A antibody β: Anti-B antibody sample: Blood attached to gauze and naturally dried. 2) Examination of old blood stains Monoclonal antibody, polyclonal antibody, α, β
Is the same as 1) above. The same applies below. Sample: 23 years ago, blood was attached to gauze and air dried,
It was stored in a tea envelope in a tea drawer. 3) Test sample for blood stain of septic blood: Blood was put in a tester, attached to gauze at room temperature for 2 weeks, naturally dried, put in a tea envelope and stored in a drawer of a desk. 4) Fetal heart test sample: A sample of a baby infant's body that had been left on the beach, dissected, and stored at -80 ° C for 3 years. 5) Semen sample soaked in soil: Table 5 shows the test results for semen subtype blood stains soaked in soil, Table 6 shows the test results for old blood stains, Table 7 shows the test results for septic blood stains, and fetal heart Table 8 shows the test results, and Table 9 shows the test results of the semen soaked in the soil.

[0050]

[Table 5]

[0051]

[Table 6]

[0052]

[Table 7]

[0053]

[Table 8]

[0054]

[Table 9]

<Results> Regarding the examination of subtype blood stains, there are A type, B type, etc. among blood types, and among A types, A1, A2, A3, Am, Ax, Ah, Amh. There are also types such as types. It is possible to detect up to A3, but it cannot be detected any more, and it becomes O type or inspection impossible. For example, Am
Neither type B nor type Bm reacts with anti-A antibody (α) or anti-B antibody (β). Therefore, blood stains are determined to be O type. For such a subtype, blood cells and serum are tested with blood, and judgment is made based on the test results of both, and it is not possible to perform detailed classification with a sample that has blood stains. Regarding the examination of old blood stains (type A, type B each 2 cases and type AB 1 case), the result was correct. About the test of old blood stains, the sample was that blood was attached to gauze, dried naturally 23 years ago, stored in a tea envelope and stored in a drawer of the desk, and the statute of murder is 15 years. Therefore, the sample handled by the present inventor has passed sufficient years. The rotten sample and the heart tissue of the infant in the abandonment of the infant infant corpse were also able to be determined without any problems. In addition, it was possible to determine the blood type from the semen that had soaked in the soil.

[0056]

INDUSTRIAL APPLICABILITY By using the present invention, a commercially available monoclonal antibody can be used to easily and clearly determine the ABO blood group from all human-derived samples by a simple operation. By using the present invention, forensic biological samples (blood stains, saliva, semen, vaginal fluid, tissue pieces, teeth, hair, which have been conducted in domestic and overseas appraisal institutions or research institutions, etc.
Blood typing of the nails, etc.) can be continued.

[Brief description of drawings]

FIG. 1 is a plan view of a sample adhesive fixation plate.

FIG. 2 is a drawing explaining the principle of the mixed agglutination method using the monoclonal antibody composition of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 33/80 G01N 33/577

Claims (13)

(57) [Claims] 1. A milk component, an albumin component and AB.
And a protein selected from the group consisting of type human sera
In the test sample containing the antibody additive solution containing the sexing agent
Accurately reveals the presence of trace amounts of antigens and blood group antigens
Monoclonal antibody against blood group substance for
Initiate only specific reactions that have been present, and non-specific reactions
Strained not a blood typing monoclonal antibody composition to act dropwise to test sample was adhered affixed to the plate to remove excess antibody unreacted by the action of instruction blood cells, to remove unreacted instruction blood cells to detect minute amounts of ABH antigens in a test sample is adhered affixed to the plate, simple method for determining blood type intended for law biological sample in criminal forensics area to observe the presence or absence of agglutination of instructions blood cells. 2. The above-mentioned blood group-determining monoclonal antibody
2. The simple method for determining blood group according to claim 1, wherein the antibody additive solution in the composition is a solution prepared by dissolving the above-mentioned protein and surfactant in physiological saline at a predetermined concentration and dissolving them. 3. The above monoclonal antibody for blood group determination
The method for easily determining the blood type according to claim 2, wherein the antibody additive solution in the composition is a solution to which a small amount of a preservative is added. 4. The monoclonal antibody for blood group determination as described above
The simple method for determining the blood group according to claim 1, 2 or 3, wherein the monoclonal antibody in the composition is a prepared anti-A antibody. 5. A monoclonal antibody for blood group determination as described above
The method for easily determining the blood group according to claim 1, 2 or 3, wherein the monoclonal antibody in the composition is a prepared anti-B antibody. 6. The above-mentioned blood group determination monoclonal antibody
The simple method for determining the blood group according to claim 1, 2 or 3, wherein the monoclonal antibody in the composition is a prepared anti-H antibody. 7. is a method that can be applied directly to the test method by mixing agglomeration method using a polyclonal antibody according to claim 1
A simple method for determining the blood type of any one of 1 to 6 . 8. Blood stains, saliva, semen, vaginal fluid, urine, dirt, nails,
A simple method for determining a blood type according to any one of claims 1 to 7, wherein all biological test samples of hair and tissue pieces are used as test samples. 9. A plate mixed with a monoclonal antibody
In order to prepare a monoclonal antibody composition for blood typing for dropping and acting on a test sample adhered and adhered,
Combination of a monoclonal antibody additive solution containing a protein selected from the group consisting of a milk component, an albumin component and human serum AB and a surfactant, and a sample adhesive immobilization plate capable of simultaneously acting with a plurality of antibodies A blood typing kit for forensic biological samples in the criminal forensics field. 10. The blood group determination kit according to claim 9, wherein the monoclonal antibody additive solution is a solution prepared by dissolving the protein and the surfactant in physiological saline at a predetermined concentration and dissolving them. 11. The blood typing kit according to claim 10, wherein the monoclonal antibody additive solution contains a small amount of a preservative. 12. The criminal record according to any one of claims 1 to 6.
Simple blood group analysis for forensic biological samples in the field of knowledge
Determination method for carrying out, and said blood typing monoclonal antibody composition, blood typing kit characterized by a combination of a sample adhesive fixing plates which can exert more antibodies simultaneously. 13. The sample adhesive fixation plate is a transparent plastic plate on which a test sample is adhered and fixed, and three areas for forming anti-A antibody, anti-B antibody, and anti-H antibody by dropping are reacted. The kit for determining a blood group according to any one of claims 9 to 12.