JPS60237366A - Blood inspection agent - Google Patents

Abstract

PURPOSE:To distinguish blood between a human being and animals other than human beings by using a red blood corpuscle agglutinin which is produced by microorganism belonging to conidiobolus group. CONSTITUTION:The microorganism used for producing a red blood corpuscle agglutinin can be whatever only belongs to conidiobolus group and has such an ability. For example, the microorganism is inoculated and cultured in a medium which contains those demanded by bacteria used such as glucose, sugar and milk sugar using conidiobolus lamprauges CBS151 and 56 as carbon source, ammonium salt, urea, peptone, yeast extraction and melt extraction as nitrogen source and other inorganic salts to be added. Then, CLA is separated and refined from the culture.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to an animal blood test agent that utilizes the agglutination reaction that is extremely specific to human blood cells caused by hemagglutinin produced by microorganisms belonging to the genus Conidioporus. be.

(Prior art) In recent years, blood test drugs have been widely used in fields such as forensic medicine and genetics, but there are currently no examples of microbial hemagglutinin being used as a blood test drug. do not have. Conventionally, hemagglutinins include plant lectins such as jack bean lectin, red kidney bean lectin, pokeweed lectin, and wheat hygal lectin, and microorganisms belonging to the genus Aspergillus, Streptomyces curvature, Vibrio genus, Neurospora genus, etc. Microbial agglutinins produced by In addition, those that show specificity to human blood types include lima beans (specific for human type A), Fuji peas (specific for human type A), pandera beans (specific for human type B), gorse, and Chinese peas. Dasuma Alfalfa (Hi) O (H) type specific), Iperis amana (') M”K%AI?J)・17”09
9$7 (% different from type N), and some attempts have been made to apply it to blood type testing limited to humans; The present invention is the first test agent that uses blood to distinguish between human and non-human animal blood.

(Structure of the Invention) As a result of our diligent search for novel microbial hemagglutinin, the present inventors discovered a strain isolated from soil and a known strain Conidioporus lamplauges.
s lamprauges) is a novel hemagglutinin (
Other known microorganisms belonging to the same genus Conidioporus, such as C0 nanodes, also produce CLA of the same species (hereinafter simply referred to as CLA)? Please make sure to produce Appl,
Environ, Microbiol, 37,
(A6).

1110 (1979)), and further CL from microbial culture fluid.
C Agric, Biol, C which separated and purified A and revealed that it is a protein with a molecular weight of 86,000
hem. .

47, (AI), 149 (1985)), as a result of subsequent research, they found that a purified preparation of CLA exhibits an extremely specific agglutination reaction for human red blood cells, and using this reaction, We have now completed the present invention by discovering that this method is extremely effective in testing to distinguish between human and non-human animal blood.

To explain the present invention in more detail, first, as a microorganism used for CLA production, CLA belongs to the genus Conidioporus.
Any microorganism that has A-producing ability may be used, such as Conidioporus lamplauges CB5153,
56 are mentioned. Such microorganisms are used as carbon sources, including those required by the bacteria used, such as glucose, sucrose, and lactose.
Inoculate a medium containing ammonium salts, urea, peptone, yeast extract, malt extract, etc. as a nitrogen source, and add other inorganic salts, and inoculate in a conventional manner, for example, for 3 to 7 days, 27 days.
Culture aerobically at C. Various methods are applied to separate and purify CLA from the culture solution depending on the characteristics described below.
For example, after removing bacterial cells from the culture solution, salting out with ammonium sulfate, applying the resulting precipitate to CMSephadex C-50,
mine -coupled 5epharose 4
N-acetyl-D-glu
A purified sample is obtained by elution with cosamine. The physicochemical properties of this specimen include (1) molecular weight: 86,
000 (by SDS polyacrylamide gel electrophoresis), (2) Sugar content = 3.0%, (3
) Isoelectric point: PI≧8.45, (4) Inhibition: Inhibited by N-, (5) Optimum pH: 5-9 (activity is constant during this period), (6) Optimal temperature = 4- It has properties such as 43C (activity is constant over this period), (pH stability: 5 to 9.6, and (8) temperature stability: 50C. It also has substrate specificity as shown in Table 1. are doing.

Table 1 Aggregation inhibitory effect of each compound (values represent the minimum concentration showing aggregation inhibition) (Note 1) Hemmagglutination-Inhib using ammonium sulfate saturated precipitate as CLA
ition Te5t: Add an equal amount of solution B to solution A below, leave it at room temperature for 10 to 30 minutes, then add an equal amount of solution A, and add 50
Observation with the naked eye for ~90 minutes Solution a: 2n times diluted solution of physiological saline containing each compound (pH
6,0) Solution b: Physiological saline containing CLA (CLA titer: 2-4F: indicated, pH
6,0) Solution a: 2.5 ml of human red blood cells suspended in physiological saline (pH 6.0) (Note 2) Measurement method for titer: 1 p of 2.5% human red blood cells
Physiological saline suspension of H6,0 and p containing CLA) (equal volumes of 6,0 in 0.9% saline (2n fold dilution) were mixed on a m1crotiterplate and left at room temperature; Observe with the naked eye for 10 to 90 minutes.1-2.
Add an equal volume of CLA solution to 5% bioid cell suspension, and determine the titer as the maximum dilution factor at which aggregation is observed after 60 minutes.

Next, Table 2 shows the agglutination activity for human red blood cells of the purified sample of CLA obtained as described above.

CLA has been found to agglutinate human red blood cells, regardless of type A%B or 01AB, at extremely low concentrations.

Table 2 (Note 1) Numbers indicate the number of samples showing coagulated gold at that concentration of acLA.

(Note 2) Aggregation activity measurement method a, Red blood cell suspension: Red blood cell i Washed twice with 0.05M phosphate buffer (p) (6,0), and added 2% to the same buffer.
Suspend at 1C.

b, Hemagglutinin solution 20, 05M'J acid buffer (
pH 6.0).

Mix equal amounts of a and bi on a m1crotiter plate and observe at room temperature for 1 hour.

Furthermore, Table 3 shows the results of measuring the agglutination activity for various animal red blood cells using the same method as above.

In addition, an example of a plant lectin is also shown as a comparison control.

Ku Table 5 *WGA is a plant lectin (wheat germ lectin).

(Effects of the Invention) As described above, it is clear that Ic and CLA specifically exhibit agglutination activity against human red blood cells, and can be used as test agents for distinguishing between human and non-human animal blood.

(Example) Conidioporus lamplauges CB8153.56 strain was mixed with 1% lactose and 0 polypeptone (Dabutsu Nutrition).
．． 0.05M phosphate buffer (pa7.o) containing 5 ml, yeast extract (Difco) 0.5 ml, malt extract (Difco)
6111 test tube and cultured for 5 days at 27T on a reciprocating test tube shaker at 120 rpm. Next, the culture solution 5- of the kernels was dispensed into 100- portions of the above-mentioned composition solution.
A 00-capacity shoulder shake flask was inoculated and cultured for an additional 27C55 days on a rotary shaker at 120 rpm. The culture solution was filtered through Toyo Togami paper A2, and the bacterial cells were separated from each other.
'CF liquid 3000m/j - While cooling with ice, solid ammonium sulfate was added to saturation of 75% and left at low temperature overnight. The precipitate i10,0002 was collected by centrifugation for 20 minutes and added to acid buffer (pH 6,
0) and dialyzed against the same buffer overnight. The resulting insoluble material was removed by centrifugation for 10.0002.20 minutes to obtain 142 ml of supernatant.

The solution was divided into several times and CM-5epha equilibrated with 0.05 MIJ acid buffer (pH 6,0).
Pour into dex C-50 color A (2X 98 pieces),
67 ml of eluate containing the active portion eluted in the void volume was collected.

This eluate was diluted with 1M sodium chloride. β-N-acet equilibrated with 05 M IJ acid buffer (pH 6,0)
yl-D-glucosamine-coupl
ed 5epharose 4 B column (I
14 cm) to adsorb the active ingredient, and after washing with the same buffer, it was developed using a 1inear gradient method using a 0 to 0.56 M N-acetylglucosamine solution, and the active ingredient was absorbed in the area where the eluate exceeds about 190 me. Since this was observed, 9.4 mi of this fraction was collected and used as a purified sample of CLA. This preparation contained 53 mg of a as protein and showed a titer of 2,400 using CLA titer (measured using the method described in Table 1, note (2) above).

Next, using such specimens, blood samples from humans (type 0), dogs (mongrels), pigs (landraces), cows (Holsteins), horses (thoroughbreds), and chickens (white leghorns) are selected from each sample. Five types of red blood cells were collected,
The minimum concentration for aggregation was determined according to the method for measuring aggregation activity in Note (2) of Table 2 above, and the results shown in Table 4 were obtained.

Thus, using CLA, it was possible to clearly distinguish between human blood in which an agglutination reaction was observed and non-human animal blood in which an agglutination reaction was not observed.

Table 4

Claims (1)

[Claims] A test agent that distinguishes between human and non-human animal blood using hemagglutinin produced by microorganisms belonging to the genus Conidioporus.