JPS59136657A - Reagent for measuring eighth factor of blood clotting - Google Patents

Abstract

PURPOSE:To inspect quickly and exactly the presence or absence of missing in the eighth factor and to diagnose hemophilia by using a specific antihuman eighth factor antibody for blood clotting which is sensitized with animal red blood cells, a buffer, and a standard eighth factor positive control and negative control for blood clotting in combination. CONSTITUTION:The measuring reagent ( I ) prepd. by combining a reagent formed by freeze drying the immobilized red blood cells which are the goat red blood cells sensitized with the specific antihuman eighth factor obtd. by immunizing the eight factor for clotting human blood (antifactor for hemopilia disease) with a rabbit, etc. ( I ), a specific phosphoric acid buffer (II), a standard positive control including the human eighth factor subjected to sterilized filtration (III), and a negative control (IV) in which the human eighth factor antibody subjected to the sterilized filtration and the eighth factor include both negative human blood serum is used. The freeze dried reagent of the refined human eighth factor antibody subjected to the sterilized filtration is prepd. for the purpose of specific decision. The above-described sensitized red blood cells are added to the double double dilution column and the simular dillution column of the positive and negative controls of the plasma to be inspected and the pressence or absence of clotting is observed. The max. dilution multiple at which the clotting is observed when compared with the positive control with respect to the test to be examined is determined as the human eighth factor value. The eighth factor value is thus measured exactly.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for detecting the factor-related antigen region (hereinafter referred to as Ag portion) of a component of human blood factor II (hereinafter simply referred to as factor V) in a test solution. 0 to quickly and accurately quantify
This invention relates to a reagent for measuring factor (1).

Factor V is called the antihemophilic factor of plasma globulin, and people with this factor, who think they have hemophilia, are unable to fully protect themselves from severe cases of hemophilia, so they do not receive factor V supplementation. Therefore, it is necessary to detect patients with factor II deficiency through open weighing, and to take prompt preventive measures on the road, which is convenient for physical examination. The development of reagents for this purpose is eagerly awaited.

"Unexploded" means that such a reagent is not available, and the gist is that anti-human blood clotting factor antibody obtained by sensitizing whole morning red blood cells with a specific anti-human blood clotting factor antibody. The present invention is a human plate for reverse receptor antibody hemagglutination reaction characterized by containing sensitized red blood cells, and a reagent for measuring solidification factor.

By the way, factor v1 has an Ag part and a coagulation active part (hereinafter referred to as A
It is composed of three parts: a part C) and an adhesive part. Therefore, since it is the AC part that determines the coagulation activity, it is the AC part that determines whether or not hemophilia occurs.
part must be measured. However, since only a trace amount is present in the constituent components of the Ac moiety il';1' first weak factor, it is extremely difficult to determine hemophilia by measuring this. Furthermore, the AC part and the adhesive part are in extremely small amounts and cannot serve as antigenic parts. On the other hand, the Ag part accounts for most of the factor
It becomes the antigenic part of the factor. Therefore, when Bacillus factor is administered to an animal to prevent antibody production, only antibodies directed against the Ag portion are produced. For example, a patient with hemophilia B
A.
Measuring part g can aid in the diagnosis of hemophilia.

In view of the above-mentioned characteristics of the valor factor, the present inventors have determined that A.
The present invention was completed as a result of repeated attempts at various aF experiments to develop a method or reagent for measuring the g-part.

Since it is convenient for the measurement reagents according to the present invention to be prepared in the form of kits, the present invention will be explained below using the kits as examples.

When the reagents of the present invention are made into a kit, the following reagents are used: Ill, ! will be accomplished. That is, (a) anti-human apical factor sensitized red blood cells obtained by sensitizing animal red blood cells with a specific anti-human factor 1m antibody (hereinafter referred to as sensitized red blood cells), (Lola 0# solution, (c) The kit is comprised of a standard human factor positive control (hereinafter referred to as positive control) and a) standard human factor negative control (hereinafter referred to as negative control).

The sensitized red blood cells consist of sensitized red blood cells that have been sensitized to a specific anti-human factor '4 antibody antibody. Specific anti-human factor (I) is produced by immunizing animals with highly purified human factor Va for immunization and purifying the resulting antiserum. The antiserum may be produced by a known method; for example, a mixed emulsion of heavily purified human factor I and Freund's complete azyubadone is prepared, injected into the animal 2 to 3 times at night, and #, days later, the antiserum is produced. After blood was collected and allowed to coagulate at room temperature, 4°
After standing overnight at C, the antiserum is obtained by centrifugation at 3,000 rpm for 20 minutes. Since the ability to produce antibodies against human factor (I) differs depending on the animal used for immunization, it is economically advantageous to select sensitive animal groups, such as guinea pigs, rabbits, sheep, and chickens.

Purification of the antiserum is carried out, for example, by the method described in Reference 1.2 below. The red blood cells used to sensitize the anti-human factor II antibody do not need to be selected from any particular animal, but in order to produce a stable and highly sensitive reagent, sheep, chicken, and human red blood cells are preferable. After thoroughly washing the original blood cells with physiological saline, they are stabilized by treatment with geltal aldehyde, formalin, etc.

Such red blood cells preferably have a diameter of about 5 to 15 μm. It is known to sensitize these red blood cells with purified antibodies (Reference 36).
It is possible to come and give alms using the following method.

This is preferably done by sensitizing red blood cells and antibodies in a buffer solution, and is generally carried out by mixing red blood cell suspensions and an anti-inflammatory solution.

This operation may be carried out at a pH of 6.8-8.5 and a temperature of 20-60°C.The sensitized red blood cells may be lyophilized, for example in a vial, with the addition of a preservative, preferably sodium azide. , it is preferable to enclose it.

The positive control related to the present invention is used to create a standard calibration curve, and usually consists of a lyophilized product containing human factor (I). Such a person
As a factor, for example, AH sold by Midori Juji Co., Ltd.
F-Midori etc. are used.

Human factor Ⅰ, which is a positive control, is used after being dissolved in a buffer solution, and divided into portions for use in creating a standard calibration curve.

The positive control is sealed, for example, in a vial, preferably in a preservative such as sodium azide.

Negative control is a liquid that is negative for both human factor II antibody and human factor I (e.g. human serum, immediate saline solution, etc.)
For example, it is preferably sealed in a vial, preferably with the addition of a preservative such as sodium azide.

Buffers are used to dilute sensitized red blood cells, positive controls, and negative controls, and include, for example, phosphate buffers. etc. may be added. It is also preferable to add a preservative such as sodium azide.

In the reagent kit of the present invention, a confirmation reagent for determining the specificity of hemagglutination (hereinafter referred to as confirmation reagent) may be further combined. The confirmation reagent consists of anti-human factor 1.

Example 1 Using a commercially available anti-human factor II antibody manufactured by DaK (Sweden) as a material, it was diluted to 0.01 at pH 6.3.
The cellulose was purified by dissolving it in 75 MIJ acid and passing it through DEAE-cellulose equilibrated with the same buffer to elute the remaining grams in the cellulose with the same buffer. This eluate was dialyzed and concentrated using a vacuum dialysis method, and then a physiological saline concentration was added to the same buffer solution.
Gel filtration was performed using gel swollen with ceftudex, and the first fractionated peak portion was used for sensitization as a purified human chorofactor antibody.

Animal red blood cells for sensitization are prepared by washing sheep red blood cells thoroughly with phosphate buffer and adding geltaraldehyde to a final concentration of 0.
After adding 5% w/v K, the mixture was treated at room temperature for about 1 hour, and the glucuranothehyde was removed with the above buffer to obtain fixed red blood cells.

5% w/v Fixed red blood cell suspension and equivalent amount of the above purified human factor antibody (approximately 0.01 for E28onIT1)
and tannin 25-201Nj/at pH 7.2.
ml was added and stirred, and the mixture was left at 4°C overnight for sensitization to obtain anti-human chorion factor antibody-sensitized red blood cells. Then, it was thoroughly washed with the phosphate buffer solution described above. The sensitized blood cells were dispensed and freeze-dried to obtain a reagent.

The lyophilized red blood cells sensitized with enemy human factor
H7,2) was fermented and adjusted to 0.5% with a phosphate buffer containing 2% or more of sheep red blood cell stroma, and the agglutination reaction against human factor Ⅰ was measured using a microplate method. It was possible to measure up to 0.005L of human factor (I) contained in the specimen.

Example 2 Kit composition The following five types of reagents make up the kit.

■ Anti-human factor ■ sensitized sheep red blood cells.

The contents of one vial are dried sensitized sheep red blood cells and when suspended by adding 5 rnl of phosphate buffered 1 (PBS) with preservative, a 0.5% (w/v) red blood cell suspension is obtained. . Sodium azide l rq is added as a preservative.

■ Human Factor ■ Positive Conotrol 1 vial contains 1 ml of ultrafiltered human Factor ■. The amount of human factor factor by RPHA method of this solution is l:
It is 64 or more. Sodium azide 1 as a preservative! (
0.1%) is added.

■ Negative control: 1 rn human serum or physiological saline that is negative for both sterile-filtered human factor ■ antibody and human factor 1 in one vial.
Contains l. Sodium azide lrng as preservative
(0.1%) is added.

(2) Phosphate buffer YL = 1 vial containing 50 ml of sterile-filtered isotonic phosphate buffer with added sodium chloride, pH 7.2 (PBS).

PBSK- contains solubilized stroma and WJ animal serum to prevent non-specific reactions from occurring due to failure of the sample.

Composition (in 50mf) Disodium phosphate (anhydrous) 395mr phosphoric acid -
Potassium 155 m? Sodium chloride 225 ■Stroma
3% animal serum 1
% sodium azide 50μ pH 7.2 101nl of phosphate buffer is used for suspending the anti-human factor 1 sensitized sheep red blood cells, and the rest is used for diluting the test solution. It is preferable to add sodium azide lη(0,i)k as a preservative.

■ Confirmation reagent for specificity determination.

In one vial, add sterilized purified human factor antibody (P
It is a lyophilized product containing HA value of 1:512 or more. Dissolve this with five drops of physiological saline.

Example 3 A quantitative test was conducted using the reagents shown in Example 2 as follows.

■ Anti-human chorion factor sensitized sheep red blood cells are suspended in 5 ml of 1K phosphate buffer per vial.

■ Put 25 μt of phosphate buffer into each hole of the U-plate using a dropper.

■ Use a diluter to add 25 ml of test solution to each hole in row A.
Add μt each. dilut using mixture from row A on top
Use er to move to row B, then use length to move from row B to row 6, and move one after another until you reach row 1. As a result, the test solution is serially diluted 2 times, and the dilution factor for one row is 1024.
It has doubled.

■ Separately, create a dilution series for the positive control and negative control instead of the test solution.

■ Using a dropper, add 25 µl of a suspension of anti-human protease factor-sensitized sheep red blood cells in phosphate buffer to all the holes.
Add by t.

■ Shake the plate with a micromixer for about 10 seconds and then incubate at room temperature for 2 hours.

■ Observation of controls For positive controls, agglutination was observed at 64-fold dilution or higher. In the negative control, sediments of red blood cells are observed at the bottom of the wells in all AmJs (if the measurement is good, the negative image is small and clear).

■ Test results For the test solution, the highest dilution ratio at which an agglutinated image was observed compared to the negative control was defined as the human choral factor value.

To precisely quantify the content of the choral factor, create a standard calibration curve by plotting the dilution factor and the diameter (m) of the corresponding aggregate image on a logarithmic graph using a solution with a known content of human choral factor. . Based on this calibration curve, the content of the choral factor is determined from the dilution factor of the unknown test liquid and the diameter of the aggregated image.

Example 4 Comparative experiment on sensitivity and specificity Comparative quantitative test of the primary factor using human plasma test fluid. RPHA method using the reagent of the present invention, Mancini method and Laurell method (J, Cl1n.

Invest, 50, 244-254 (197
1) ) As a result of conducting k, the lowest value of % in the detection window was 0.005 μ/m1 for the reagent of the present invention. , others are 0.062
5 - Failed at 0.125μ/-.

In addition, in a test conducted to check the specificity of human plasma test fluid, when the human plasma test fluid was neutralized with antibodies and retested, less than 3% of the results were determined to be false positives. It was found that this measurement reagent has extremely high specificity.

Literature 1. J,Am,Chem,Soc,,62.
3386 (1940) Reference 2. Fed, Proc., 17.1116 (1
958) Literature 3. History of medicine, 78.759 (197
0) Patent Applicant Midori Juji Co., Ltd. Procedural Amendment (Dereliction) Showa Yu and March 2016/Otsuji Patent Office Director General 14 patents 1982 Patent Application No. 011076 2 Title of Invention Blood Reagent for measuring coagulation factor 3 Relationship with the case of the person making the amendment Patent applicant trap” 8 (ZM,) Mitri Juji Co., Ltd. (1) “Ag” on page 2, lines 3 and 20 of the specification Of courserR:Ag,
Correction to J
h and m8 lines (1) [Ac Jhrvnl: CJIC
Correction 0 (3) In the same book, page 3, line 9, ``obtainable'' is corrected to ``difficult''.

(4) Circular page 3, lines 10, 15 and 18
The line r Ag Jhr R:Ag J has been corrected.

(5) ``When, hgJx'' on page 3, line 1z of the same book, corrected to Ag J, page 3, line 14. FB patient is AgJr.
[Among A, especially Von Vuillebrand (von
Patients with a disease called R.
: Corrected by Ag J.

(7) Doho No. 3 Negative, line 16, “Hemophilia” t “Fuo R Konoko Nito, Fur” Filate JIJ 口入ゐゐ.

(9) Dobara page 9, line 5 r O,005J 7 r
Corrected to O, 001J.

uq r dilter on page 11, line 15 of the same book
J meeting arropper J .

(6) Used above p. 11, line 16, 1 of the same book, "r deleted".

(2) “Test liquid” on page 13 of Douyo, line 3 is “test liquid”
Correction 'Tゐ.

345

Claims (3)

[Claims] (1) A reverse receptor material characterized by containing an anti-human blood g-coagulation factor sensitized red plate obtained by sensitizing a red plate with a specific anti-human plate and coagulation factor antibody. Antibodies and human blood for agglutination reactions
Reagent for measuring coagulation factor 1. (2) Anti-human blood coagulation factor sensitized red plate prepared by sensitizing a specific anti-human blood coagulation factor I antibody to the blood clotting factor I antibody;
The reagent for measuring human blood coagulation factor 1 according to claim (I), which is in the form of a kit comprising a buffered sample, a standard human dish fluid-fixed factor 1 positive control, and a negative control. (3) Furthermore, in order to ensure the specificity of the hemagglutination reaction, human dishes as described in paragraph (2) above, including all confirmation reagents consisting of human blood test and pupil factor anti-infection, are provided. #Gate V1
Factor measurement reagent.