Method for detecting blood component using conidiobolus hemagglutinin

A method for detecting blood component in a sample comprising reacting a human erythrocyte membrane band 3 glycoprotein (band 3) in the sample and a hemagglutinin produced by a microorganism belonging to the genus Conidiobolus (CA) and measuring said band 3 glycoprotein contained in a complex produced by the reaction. Because band 3 can be detected specifically, at high sensitivity, and stably by the use of CA, the method ensures qualitative or quantitative, and accurate detection of human blood component in feces or contents of digestive organs, of which the determination of the presence or quantity of human blood component by hemoglobin is difficult.

TECHNOLOGICAL FIELD 
The present invention relates to a method for specifically detecting blood 
component derived from human and, more particularly, to a method for 
detecting a blood component capable of detecting minute bleeding in 
digestive tract for the purposes of screening, for example, the screening 
of colorectal cancer and the like, and a kit using this detection method. 
BACKGROUND ART 
Detection of blood component of human is required in a variety of fields. 
The necessity is especially important in the clinical field. Bleeding, for 
example bleeding in digestive tract, occurs in a variety of diseases. 
Particularly, it is well known that bleeding from digestive tracts is an 
important early stage symptom in malignant tumors of digestive tracts. 
Detection of blood components in feces, especially hemoglobin, is a major 
method currently applied to the detection of digestive tract bleeding for 
diagnosis of diseases such as malignant tumors of digestive tracts. 
Reversed passive hemagglutination, latex agglutination (fixation), gold 
colloid agglutination, enzyme immunoassay, radio immunoassay, and the like 
are included in the methods for detecting the hemoglobin in stools. Their 
principle is preparing an anti-human hemoglobin antibody, causing an 
antigen-antibody reaction with hemoglobin, and measuring or detecting the 
resultant by various methods. 
In the conventional immunological method for detecting hemoglobin using the 
anti-human hemoglobin antibody, however, the hemoglobin to be detected is 
usually supplied to the test together with feces which act as an 
inactivating factor. The antigenic determinant of hemoglobin may be 
inactivated depending on the temperature over time, resulting in a 
drawback of conspicuous deterioration of the detection sensitivity. 
Hemoglobin may be denatured or decomposed, particularly under high 
temperature conditions in summertime, therefore, hemoglobin positive feces 
are often reduced to negative in summer {Takefumi FUJIYOSHI and Mariko 
KOYAMA, Kiso-to-Rinsho CLINICAL REPORT (Basic and Clinical Report)! 
23(15), 6097-6101 (1989), "Basic studies on immune stool occult blood 
reagent"}. The inactivation of hemoglobin is significant particularly when 
its concentration is low. It is difficult to perform hemoglobin detection 
which is meaningful for diagnosis in such low concentration levels. 
Development of a method for properly measuring blood component in samples 
of feces or the like without being affected by temperatures, storage time 
and the like has been required. The object of the present invention is to 
provide such a method. 
DISCLOSURE OF THE INVENTION 
The present inventors have conducted extensive studies in order to detect 
blood or blood component in fecal samples or the like using a blood 
component other than hemoglobin. 
As a result, the inventors have found that human erythrocyte membrane band 
3 glycoprotein (hereinafter referred to as band 3) can be properly 
detected, because it is much mores table than hemoglobin even in samples 
of feces or the like, and forms a complex by binding with a certain 
hemagglutinin specific for human erythrocytes. This finding has led to the 
completion of the present invention. 
Accordingly, the present invention provides a method for detecting a blood 
component in a sample comprising reacting band 3 in the sample and a 
hemagglutinin produced by a microorganism belonging to the genus 
Conidiobolus and measuring the complex produced by the reaction, and a kit 
used for the detection. 
BEST MODE FOR CARRYING OUT THE INVENTION 
Band 3 which is detected as a blood component in the method of the present 
invention is a component which has already been reported as an erythrocyte 
marker (Beppu, M. et al., J. Biol. Chem., 256(6), 3226-3233 (1990), 
"Binding of anti-band 3 autoantibody to oxidatively damaged erythrocytes", 
etc.) and been studied as useful for monitoring erythrocytes. 
The hemagglutinin produced by a microorganism belonging to the genus 
Conidiobolus (hereinafter abbreviated as CA) is a human 
erythrocyte-specific hemagglutinin produced by a microorganism belonging 
to the genus Conidiobolus such as Conidiobolus lamprauges, Conidiobolus 
nanodes, or the like. This hemagglutinin has been reported to exhibit 
interactions with band 3 (Ishikawa, F. et al., Agric. Biol. Chem., 45(9), 
2105-2110 (1981), "Action of proteases on human erythrocyte glycoproteins 
in relation to hemagglutination by Conidiobolus chitin-binding 
agglution"), but no blood detection method utilizing it has been known 
heretofore. 
The method of the present invention comprises detecting human blood 
component in samples by detecting band 3 contained in a complex of band 3, 
which is a blood component in that sample, and CA. Any detection methods 
can be applicable inasmuch as the band 3 can be detected in that manner. 
One of the embodiments for carrying out the present invention is a 
so-called sandwich method which comprises insolubilizing CA on the surface 
of a solid phase, adding the sample and a labeled anti-band 3 antibody 
which specifically binds with the same band 3 to sandwich the band 3 in 
the sample between the immobilized CA and the labeled anti-band 3 
antibody, and identifying or measuring human blood via band 3 which has 
been present in the sample. 
In this embodiment, it is possible to add the labeled anti-band 3 
antibodies to the sample in advance as mentioned below. 
In the above system, a certain peptide portion of the band 3 in the sample 
is recognized by an added anti-band 3 antibody, and sugar chain portion of 
the band 3 is separately recognized and captured by the immobilized CA. 
Further, another peptide portion of the band 3, which portion is different 
from the peptide portion initially recognized by the added anti-band 3 
antibody, is recognized by a labeled anti-band 3 antibody. In this manner, 
the band 3 in the sample is determined by the anti-band 3 antibody, the 
immobilized CA, and the labeled anti-band 3 antibody, so that the band 3 
can be highly restricted. This prevents cross-reaction with a variety of 
contaminating glycoproteins in samples resulting in an increase of 
sensitivity and the specificity. 
The specific detection system in the present invention is not limited to 
the above embodiments. The immobilized component and the labeled component 
in the above embodiments may be replaced by using a combination of an 
immobilized anti-band 3 antibody and a labeled CA, or CA may be the 
immobilized component and the labeled component as well if a combination 
of an immobilized CA and a labeled CA is used. The methods similar to the 
above described embodiment can be applied to these cases for improving the 
sensitivity and the specificity. 
Another embodiment of the method of the present invention is a detection 
method using a so-called competitive reaction. The method comprises 
capture of a labeled band 3 prepared in advance by the immobilized phase 
competitively along with capture of the band 3 in the sample by the 
immobilized phase, whereby the presence or absence or the quantity of 
blood in the sample can be detected by the label in the former band 3. 
Any labeling method, Such as radioisotope labeling, enzyme labeling, 
fluorescence labeling, or light emitting chemical labeling, can be used 
for labeling the CA or anti-band 3 antibody. Specific methods include a 
method of labeling with biotin and detect it by a labeled avidin, a method 
of using a labeled second (other) antibody to CA or anti-band 3 antibody 
instead of labeling the CA or anti-band 3 antibody. There are no 
limitations to the specific detection system inasmuch as the labeling 
which enables the detection is given. 
In the method of the present invention, it is possible to have the 
anti-band 3 antibody or CA present in the sample in advance, for example, 
by coating the surface of stool collecting sticks beforehand with a 
required quantity of anti-band 3 antibody or CA in a manner where they are 
capable of dissolving out. 
Various buffer solutions, such as acetate buffer, phosphate buffer, 
Tris-HCl buffer, glycine buffer, ammonium buffer, borate buffer, and 
carbonate buffer, are given as examples of the base solution which 
solubilizes and dissolves band 3 from samples of feces or the like 
containing the same. The pH of these base Solution is in the range of 
4-11.5, and preferably 4.5-8.5. 
It is desirable to add sodium chloride to the base solution to make the 
content of it approximately the same as that of physiological saline. 
Further, it is desirable to add sodium azide or the like in an amount of 
0.05-0.5% by weight as antibacterial agent. It is also desirable to add 
0.05-2.0% by weight of bovine serum albumin or the like as a stabilizer 
for proteins such as antibodies. 
Because the present invention is a method for detecting band 3 contained in 
human erythrocyte membrane, it is possible to use a surfactant as a 
solubilizer for solubilizing the erythrocyte membrane for detecting the 
band 3 more efficiently. 
Examples of the surfactant which can be preferably used as the solubilizer 
in the present invention include, but not limited to, the following 
surfactants. 
Sodium dodecylbenzene sulfonate 
Polyoxyethylene-iso-octylphenyl ether (Triton X-100 etc.) 
Polyoxyethylenenonylphenyl ether (Noident P-40 etc.) 
Polyoxyethylenesorbitol ether (Tween 20 etc.) 
3-(3-Cholamidepropyl)dimethylammonio!-1-propane sulfonate 
3-(3-Cholamidepropyl)dimethylammonio!-2-hydroxy-1-propane sulfonate 
n-Octyl-.beta.-D-glucopyranoside 
Octanoyl-N-methylglucamide 
Nonanoyl-N-methylglucamide 
Decanoyl-N-methylglucamide 
n-Heptyl-.beta.-D-thioglucoside 
n-Octyl-.beta.-D-thioglucoside 
N,N-bis(3-D-gluconamidepropyl)deoxycholamide 
There are no specific limitations to the method for adding these 
surfactants. In the case where the above-mentioned buffers are used as the 
base solution, it is preferable to add a surfactant which can exhibit the 
effect at a concentration of less than 1% by weight. 
The present invention is a method discovered for the first time for 
detecting human blood component in samples such as feces or the like via 
band 3 using CA and an anti-band 3 antibody, and particularly advantageous 
as a method for detecting fecal occult blood. As can be seen in the 
examples hereinafter, the fecal occult blood can be detected using this 
method at a higher sensitivity and more stably than the conventional stool 
occult blood detection method or the like using anti-hemoglobin 
antibodies. 
The present invention will be illustrated more specifically referring to 
examples, which are not intended to be limiting of the present invention.

EXAMPLE 1 
&lt;Method for detecting band 3 in feces Using an enzyme labeled anti-band 3 
antibody and CA&gt; 
(1) Purification and isolation of band 3 
200 ml of O-type human blood to which an anticoagulant was added was 
centrifuged at 320.times.g for 10 minutes to separate plasma. The 
precipitate was suspended in 200 ml of a 0.05M phosphate buffer (pH 7.3) 
containing 1 mM EDTA, 5 mM 2-mercaptoethanol, and 0.03 mM 
phenylmethylsulfonyl fluoride, adjusted to pH 7.5 with 1M NaOH, stirred 
gently for 18 hours at 4.degree. C., and centrifuged at 10,000.times.g for 
1 hour. The precipitate was suspended in ice cooled water which had been 
adjusted to pH 12 with 1M NaOH. The suspension was stirred gently for 30 
minutes at 4.degree. C., and again centrifuged at 10,000.times.g for 30 
minutes. The precipitate thus obtained was suspended in a 40 mM Tris-HCl 
buffer (pH 7.4) containing 1% sodium dodecyl sulfate, 2 mM EDTA and 0.04 
mM 2-mercaptoethanol and allowed to stand at 4.degree. C. for one hour, 
following which the temperature was allowed to raise to room temperature. 
This suspension was passed through a Sepharose 6B column (made by 
Pharmacia, 2 cm.phi..times.98 cm) equilibrated with the above buffer, and 
fractions having absorbance at 280 nm were collected. The band 3 was 
eluted just before the elution of glycofolin A. 
(2) Preparation of polyclonal antibody for band 3 
Band 3 was dissolved in physiological saline to prepare a solution with a 
concentration of 4 mg/ml. The solution was subcutaneously injected into 
back of rabbits 8 times at an interval of 2 weeks, each time at a dose of 
1 ml. Blood was collected 3 weeks after the final subcutaneous injection 
to obtain anti-band 3 antibody serum. 
This anti-band 3 antibody serum was added to a Protein A Sepharose CL-4B 
column (made by Pharmacia, 1 cm.phi..times.14 cm) equilibrated with a 20 
mM Tris-HCl buffer (pH 8.3.) containing 0.2M NaCl and the column was 
eluted with the same buffer solution to collect fractions. The elution was 
continued until the absorbance at 280 nm returned to the initial standard 
level. 
Next, this column was eluted with a 0.1M glycine-HCl buffer (pH 3.0) to 
collect fractions until the absorbance at 280 nm returned to the initial 
standard level. Fractions which was determined to contain the protein 
based on the absorbance was pooled and dialyzed against a 10 mM phosphate 
buffer (pH 7.5). 
The antibody thus obtained was confirmed by the Ouchterlony method to 
possess specificity to band 3 derived from human erythrocyte (this 
antibody is hereinafter referred to as "anti-band 3 antibody"). 
(3) Labeling the anti-band 3 antibody with an enzyme 
Horse radish peroxidase (a product of Sigma Co., hereinafter abbreviated to 
HRPO) was bonded to the anti-band 3 antibody according to the following 
method. 
The HRPO was dissolved into a 10 mM acetate buffer (pH 4.5) to a 
concentration of 15 mg/ml, and sodium metaperiodate was added to this 
solution to a final concentration of 33 mM. The mixture was incubated at 
25.degree. C. for 15 minutes and passed through a Sephadex G-25 column 
(made by Pharmacia, 1 cm.phi..times.14 cm) equilibrated with the same 
acetate buffer as above to collect eluted fractions with a brown color. 
The fraction was confirmed to be the activated HRPO. 
The activated HRPO fractions were adjusted with said acetate buffer as 
above to obtain a final concentration of 1 mg/ml. This was added to an 
equivalent amount of a solution of the anti-band 3 antibody which had been 
adjusted to a concentration of 4 mg/ml with a 50 mM carbonate buffer (pH 
9.5), and the mixture was incubated at room temperature for 4 hours. 
The reaction was retarded by the addition of sodium borohydride to a final 
concentration of 2.6 mM and the reaction was continued for 30 minutes at 
4.degree. C. under the retarded conditions. The reaction of sodium 
borohydride was terminated by the addition of acetone to a final 
concentration of 0.2% (v/v) thus obtaining a HRP0 labeled anti-band 3 
antibody. 
(4) Preparation of CA 
Conidiobolus lamprauges CBS 153,56 stocks were inoculated into test tubes 
with an internal diameter of 16 mm, each containing 5 ml of 0.05M 
phosphate buffer (pH 7.0) containing 1% lactose, 0.5% peptone, 0.3% yeast 
extract, and 0.3% malt extract, and cultured for 3 days at 27.degree. C. 
on a reciprocal shaker at 120 rpm. 5 ml of the culture broth was 
inoculated into each of 500 ml Sakaguchi shaker flasks to which 100 ml of 
the same phosphate buffer as above was added, and further cultured for 5 
days at 27.degree. C. on a rotating shaker at 120 rpm. 
The culture broth was filtered through a filter paper to separate cells. 
Solid ammonium sulfate was added to 3000 ml of the filtrate at 0.75 
saturation with ice cooling and the mixture was allowed to stand overnight 
at a low temperature. The precipitate produced was collected by centrifuge 
for 20 minutes at 10,000.times.g, dissolved in a 0.05M phosphate buffer 
(pH 6.0), and dialyzed against the same buffer overnight. Insoluble 
matters produced were removed by centrifuge for 20 minutes at 
10,000.times.g, thus obtaining 142 ml of supernatant. 
This solution was passed through CM-Sephadex C-50 column (made by Sigma 
Co., 2 cm.phi..times.98 cm) equilibrated with the same buffer as above to 
collect a fraction eluted gradiently with 0.05-0.5M of the above buffer 
solution. Hemagglutination activity of CA of the fraction was determined. 
67 ml of this fraction was applied to a Sepharose 4B column (made by 
Pharmacia., 1 cm.phi..times.14 cm) with .beta.-N-acetyl-D-glucosamine 
bonded and equilibrated with a 1M NaCl -0.05M phosphate buffer (pH 6.0). 
After washing the column with the same buffer, the column was eluted 
gradiently with 0-0.36M N-acetylglucosamine solution. Among the fractions 
eluted, a portion obtained initially in the gradient elution was used as a 
purified CA sample. 
(5) Immobilization of CA 
A 2 .mu.g/ml CA solution (.0.1 mol Tris-HCl buffer, pH 8.4) was charged 
into a microplate in an amount of 150 .mu.l per well and allowed to stand 
overnight at 4.degree. C. to adsorb and immobilize (insolubilize) CA on 
the microplate surface. 
(6) Preparation of fecal samples 
Sample 1 was prepared by mixing 2 g of feces of a healthy adult and 8 .mu.l 
of human blood. Sample 2 was prepared by diluting Sample 1 to a 1/4 
concentration, i.e., by mixing about 0.5 g (1/4) of the sample with 1.5 g 
of the feces of the healthy adult. Sample 2 was diluted in the same manner 
to a concentration of 1/4 to prepare Sample 3 (1/16 dilution of Sample 1). 
Feces containing no blood was used as a sample blank (Sample 4). 
Accordingly, the amounts of blood added were 4 .mu.l 1 .mu.l, 0.25 .mu.l, 
and 0 .mu.l per 1 g of feces. 
(7) Preparation of sampling solution 
A 0.1 mol/1 Tris-HCl buffer solution (pH 8.0) containing 1% Triton X-100 
(made by Sigma Chemical), 0.1% sodium azide, 0.1% bovine serum albumin, 
and 0.9% sodium chloride was prepared and charged to test tubes to be used 
as the solution for the samples, in an amount of 2 ml per tube. 
(8) Collection of samples and preparation of sample solutions 
Feces sampling sticks were inserted into sample feces to collect samples, 
each about 10 mg. Each sample was then suspended in 2 ml of the sampling 
solution and incubated at 37.degree. C. for 5 minutes to prepare sample 
solutions. 
(9) Measurement 
The immobilized CA plate obtained in (5) mentioned above was washed with 
0.1 mol Tris-HCl buffer (pH 8.4). The sample solutions prepared in (8) 
were added to the plate in an amount of 100 .mu.l/well, followed by the 
addition of 50 .mu.l/well of 0.5 mol acetate buffer (pH 4.5). The mixtures 
were incubated at 37.degree. C. for one hour to capture band 3 in the 
sample solutions. 
After thoroughly washing each well with a 0.1 mol phosphate buffer (pH 
6.8), a HRPO labeled anti-band 3 antibody solution (a solution of 1 
.mu.g/ml HRPO labeled anti-band 3 antibody in 0.1 mol phosphate buffer (PH 
6.1) containing 2% bovine serum albumin) was added in an amount of 150 
.mu.l per well, and the mixture was incubated at 37.degree. C. for one 
hour. 
Next, after again thoroughly washing with a 0.1 mol phosphate buffer (pH 
6.8), a substrate solution (0.05M acetate buffer (pH 4.5) containing 0.03% 
o-phenylene-diamine and 0.01% hydrogen peroxide) was added in an amount of 
150 .mu.l per well, followed by incubation at 37.degree. C. for 30 
minutes. The reaction was terminated by the addition of 50 .mu.l/well of 
4N hydrochloric acid. Absorbances were measured at two wavelengths, 492 nm 
and 630 nm, using a spectrophotometer (a microplate reader manufactured by 
Corona Co.). Band 3 in Samples 1-3 showed absorbance of 0.5 or larger 
against the sample blank, so that these were apparently positive. 
COMATIVE EXAMPLE 1 
&lt;Detection of fecal occult blood using anti-hemoglobin antibody 
(conventional method)&gt; 
(1) Preparation of a latex reagent sensitized with anti-human hemoglobin 
antibody 
10 ml of 1 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide was added 
to 10 ml of 5% carboxylated polystyrene latex and the mixture was reacted 
for 20 minutes while stirring. After separating out supernatant by 
centrifuge, an equivalent amount (10 ml) of 0.01 mol/l borate buffer was 
added to the precipitate latex. The mixture was stirred to homogenize, 
centrifuged to remove the supernatant. This procedure was repeated again, 
and finally the latex was dispersed in 10 ml of said buffer solution. 
To 10 ml of the latex suspension (concentration: 5%) was added 7 ml of 
anti-human hemoglobin antibody (Rabbit IgG, concentration: 5 mg/ml), 
prepared by immunizing rabbit with purified human hemoglobin A.sub.o, and 
the mixture was reacted for 5 hours while slowly stirring. The supernatant 
was removed by centrifuge. 10 ml of a 0.01 l borate buffer (pH 8.0) 
containing 0.1% bovine serum albumin was added to the precipitate latex. 
The mixture was stirred and then centrifuged to remove the supernatant. 
This procedure of removing supernatant by centrifuge was repeated again. 
Then, after adding 10 ml of said buffer, the mixture was stirred to obtain 
a latex reagent sensitized with anti-human hemoglobin antibody (latex 
concentration: 1%). 
(2) Immunological latex agglutination reaction 
Fecal sample solutions prepared in Example 1(8) were placed on each 
serological reaction slide glass plate in an amount of 100 .mu.l each. 25 
.mu.l of the latex reagent as above was added to each, and, after 5 
minutes mixing with rotation, the agglutination images were observed by 
naked eyes (macroscopically). As a result, the agglutination images were 
seen in Samples 1 and 2, but not in Samples 3 and 4, indicating that the 
sensitivity is inferior to the method of the present invention. 
The results of Example 1 and Comparative Example 1 are summarized in Table 
1. 
TABLE 1 
______________________________________ 
Blood Conventional 
Method of 
concentration 
method this invention 
(.mu.l/g stool) 
(Comp. Ex. 1) 
(Example 1) 
______________________________________ 
Sample 1 4 + + 
Sample 2 1 + + 
Sample 3 0.25 - + 
Sample 4 0 - - 
______________________________________ 
-: Fecal occult blood, negative (samples exhibiting negative agglutinatio 
reaction in the conventional method or samples exhibiting absorbance of 
0.4 or less against the sample blank in the method of the present 
invention. 
+: Fecal occult blood, positive (samples exhibiting positive agglutinatio 
reaction in the conventional method or samples exhibiting absorbance of 
0.5 or more against the sample blank in the method of the present 
invention. 
EXAMPLE 2 
&lt;Comparison of the present invention and conventional method&gt; 
Sample 1, Sample 2, Sample 3, and Sample 4 (sample blank) prepared in 
Example 1(7) were incubated at 37.degree. C. for 6 days, while measuring 
and comparing the presence of blood by the method of the present invention 
in Example 1 and the conventional method of Comparative Example 1 every 
day. The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Incubation at 37.degree. C. (days) 
Measurement method 
0 1 2 3 4 5 6 
______________________________________ 
Sample 1 
Invention method 
+ + + + + + - 
Conventional method 
+ + - - - - - 
Sample 2 
Invention method 
+ + + + + - - 
Conventional method 
+ - - - - - - 
Sample 3 
Invention method 
+ + + + - - - 
Conventional method 
- - - - - - - 
Sample 4 
Invention method 
- - - - - - - 
Conventional method 
- - - - - - - 
______________________________________ 
-: Fecal occult blood, negative (samples exhibiting negative agglutinatio 
reaction in the conventional method or samples exhibiting absorbance of 
0.4 or less against the sample blank in the method of the present 
invention. 
+: Fecal occult blood, positive (samples exhibiting positive agglutinatio 
reaction in the conventional method or samples exhibiting absorbance of 
0.5 or more against the sample blank in the method of the present 
invention. 
The above results shows that, when incubated at 37.degree. C., according to 
the method of the present invention the period of time for which a blood 
component can be detected at higher sensitivity was extended by 
utilization of band 3 while according to the conventional method 
hemoglobin was detected for only one day or so. It was confirmed that 
stable detection was possible according to the method of the present 
invention over a long period of time even in the case where the sample was 
continuously held at 37.degree. C. 
Industrial Applicability 
As illustrated above, according to the detecting method of the present 
invention band 3 can be detected specifically, at high sensitivity, and 
stably by the use of CA. It is thus possible to qualitatively or 
quantitatively detect human blood component in feces, contents of 
digestive organs, and other materials in which the presence of human blood 
is questioned, or, so detect digestive tract bleeding, occult blood in 
feces, and the like, at high accuracy.