JPS60224067A - Anti-human lactoferrin antibody sensitized blood cell and detection of human lactoferrin using said blood cell - Google Patents

Abstract

PURPOSE:To obtain an index useful for diagnosis of a pancrea disease by conjugating an anti-human lactoferrin antibody with fixed blood via tannic acid to manufacture anti-human lactoferrin antibody sensitized blood cells and bringing such sensitized blood cells and the pancreatic juice into contact with each other. CONSTITUTION:The anit-human lactoferrin (Lf) antibody is obtd. with an animal such as guinea pig processing antibody production ability by using human colostrum and is refined. Tannic acid is then added to the fixed blood of a goat, etc. to conjugate the binder to the blood cell surface and thereafter a liquid contg. the anti-human Lf antibody is brought into contact therewith, by which the anti- human Lf antibody sensitized blood cells are obtd. A specified quantity of the diluted pancreatic juice and standard human Lf are dispensed on a microplate to make a dilution system and a specified quantity of the anti-human Lf antibody sensitized blood cells are added thereto to make the system uniform. The concn. of the human Lf is measured by hemagglutination after resting for specified time. The examination of the pancrea disease is thus elucidated.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to anti-human lactoferrin antibody-sensitized blood cells and a method for detecting human lactoferrin using the same.

More specifically, it relates to anti-human lactoferrin antibody-sensitized blood cells obtained by sensitizing animal red blood cells with anti-human lactoferrin antibodies prepared using human-derived lactoferrin, and a method for detecting human lactoferrin using the sensitized blood cells. .

Lactoferrin; hereinafter rL
fJ) is an iron-containing glycoprotein with a molecular weight of 70,000 to 80,000 that is found in various body fluids, including human breast milk, and is believed to play a role in iron absorption in the gastrointestinal tract. However, its other roles are not well understood.

However, in humans, when chronic pancreatitis occurs, the concentration of Lf in pancreatic juice is 870 to 11,400 (average 2,310) ng/an.
It is known that normal admission pancreatic cancer patients show only a low value of 10 to 387 (average 1105) n/+s sentences (S, S, Federal
tal;-Lancet.

! , 181-182.1978). Currently, early diagnosis of pancreatic cancer is not easy, and in particular, differential diagnosis from chronic pancreatitis has been a very difficult problem, but it has become possible to clearly differentiate between the two by measuring Lf in pancreatic juice.

Currently, Lf concentration is determined using radioimmunoassay method (hereinafter referred to as rRI).
(abbreviated as AJ), but this method
It has excellent sensitivity and has the advantage of being able to measure even the smallest amount of Lf, but on the other hand, there is a risk of handling dangerous substances such as radioactive materials, and therefore requires sufficient equipment and management. The problem is that it can only be implemented in a place where sufficient equipment and management systems are in place.

As the number of specimens continues to increase and daily testing operations intensify, the current state of clinical testing is demanding simpler, more sensitive, more accurate, and more reliable measurement methods. We conducted intensive research to eliminate these difficulties and develop a simple, highly sensitive, and clinically meaningful method for measuring human Lf.

As a result, we succeeded in producing anti-human Lf antibody-sensitized blood cells, and also succeeded in establishing a simple and highly sensitive method for measuring human Lf using the same, thereby completing the present invention.

That is, an object of the present invention is to provide anti-human Lf antibody-sensitized blood cells for measuring human Lf, which are useful indicators for the diagnosis of pancreatic diseases, and furthermore, to provide a method for detecting human Lf using the same. Our goal is to provide the following.

That is, the present invention provides anti-Human Lf antibody-sensitized blood cells comprising blood cells bound to anti-human Lf antibodies via a binding agent, and the sensitized blood cells are mixed with a test solution or a diluted solution thereof according to the microtiter method. The present invention relates to a) method for detecting Lf, which is characterized by contacting the blood cells and observing an image of hemagglutination.

Next, the present invention will be explained in more detail.

The raw materials for preparing Lf used in the present invention include:
Human colostrum is the most suitable.

Its preparation method is well known (B, G, Johans
on;Acta Chew, 5cand,, 14,5
10,1lllf! 0), and commercially available products are also available.

After immunizing an animal capable of producing antibodies, such as a guinea pig, rabbit, or goat, with this human Lf according to a conventional method,
Blood can be collected to obtain antibodies.

Any animal can be used in this case as long as it has the ability to produce antibodies, but it is preferable to use large animals in order to obtain a large amount of antibodies. Rabbits and goats are usually used, but are not limited to these.

To purify anti-human Lf antibodies from antisera containing anti-human Lf antibodies obtained from these animals, any commonly used method can be used. For example, the antiserum is salted out with ammonium sulfate and the insoluble antigen There are methods of purification by repeating adsorption and dissociation using a method of purification, methods of purification by salting out ammonium sulfate, ion exchange chromatography, and gel filtration.

The blood cells used to produce the sensitized blood cells of the present invention are, for example, red blood cells from birds such as chickens and turkeys, and mammals such as sheep, goats, and rabbits, and among them, red blood cells from sheep, chickens, and turkeys are preferred. In addition, blood cells obtained from these animals may be sensitized with anti-human Lf antibodies on the surface of the blood cells as they are, that is, live blood cells, or may be sensitized after being made into fixed blood cells. In consideration of the shelf life of hematopoietic cells, it is preferable to use fixed blood cells. Fixed blood cells can be prepared according to a conventional method using fresh blood and aldehydes such as formalin and glutaraldehyde.

In order to carry anti-human Lf antibodies on blood cells, live blood cells or fixed blood cells obtained as described above are brought into contact with a solution containing a binding agent to obtain blood cells with a binding agent bound to the surface of the blood cells,
A solution containing anti-human Lf antibody was brought into contact with this, and anti-human Lf antibody was added.
f Obtain antibody-sensitized blood cells. As the binder, for example, tannic acid, glutaraldehyde, chromium chloride, carbodiimide, etc. can be used, but tannic acid is particularly preferred.

The anti-human Lf antibody-sensitized blood cells thus obtained showed specific activity against human Lf even after repeated washing with physiological saline and a dilute salt solution near neutrality. If blood cells are not used, even if blood cells are treated with a solution containing anti-human Lf antibodies, they will not show any activity against human Lf. H)
It is bound to Lf antibody. Furthermore, the sensitized blood cells of the present invention are completely new and unprecedented.

Using the thus obtained anti-Human Lf antibody-sensitized blood cells, Lf in pancreatic juice can be easily measured by a microtiter method. That is, a certain amount of diluent is dispensed into a microplate, then a certain amount of pancreatic juice is added to the first plate, and the diluent is sequentially diluted with a diluter. On the other hand, standard H)L
Similarly, make a dilution series using f. These include anti-human Lf
After adding a fixed amount of antibody-sensitized blood cells to make them homogeneous and leaving the tube open for a fixed period of time, the end point of agglutination can be observed and the absolute amount can be calculated from the standard antigen series.

The method for measuring human Lf using anti-human Lf antibody-sensitized blood cells of the present invention can measure human Lf if 1 ng or more of human Lf is present per ml, and the detection method of the present invention has extremely high sensitivity. It can be said that the sensitivity is equivalent to or higher than that of radioimmunoassay, which is said to be the most sensitive method of conventional analysis.

Next, the present invention will be explained in more detail with reference to Preparation Examples and Examples, but the present invention is not limited by the following Examples unless it exceeds the gist thereof.

Human Lf (Hoechst) was dissolved in physiological saline at a concentration of 4 mg per l + Jj, and after mixing with an equal amount of Complete φ Freund 0 adjuvant, the body weight was 2.3 to 2.
．． Using 4 healthy rabbits weighing 5 kg, 0.1+su was injected subcutaneously into the foot of each limb, and 3 doses were administered every week.
After the two injections, 1 ml of 0.1% human Lf physiological saline solution was injected one week later. Three weeks after the last injection, whole blood was collected from the carotid artery of the rabbit, centrifuged according to a conventional method, and the obtained serum was kept at 56°C for 30 minutes to obtain about 200 mM of antiserum.

The obtained antiserum was confirmed to be a single antibody because it formed a single sedimentation line with the antigen by the Ophthalonyx method and the immunoelectrophoresis method.

1. 5mg of human Lf was added to 0.2M acetate buffer (pH 5,0).
After adding about 0.5 m of 5% BSA, 5% glutaraldehyde was added dropwise until a precipitate formed. The precipitate was collected, homogenized, washed with a mixture of 1 volume of 1/15 M phosphate buffer (pH 7, 2) and 3 volumes of physiological saline (hereinafter abbreviated as rPBsJ), and washed with a glycine-medium acid buffer. After washing with solution (p)l 2.8), further wash with PBS.
and stored at -20°C.

Add an equal amount of saturated ammonium sulfate solution to the antiserum, mix well, and leave at room temperature for 30 minutes. The resulting precipitate is separated by centrifugation. After washing with 0.5 saturated ammonium sulfate solution, add it to PBS. Dialyzed. Next, insoluble H)Lf was added to the dialysis fluid, left at room temperature for 30 minutes, centrifuged to separate the supernatant and precipitate, and insoluble H)Lf was added to the supernatant again and treated in the same manner, and both precipitates were separated. After washing with PBS, glycine-hydrochloric acid buffer (pH 2,8) was added, and after shaking for 5 minutes,
After centrifugation, the precipitate was treated again with glycine-hydrochloric acid buffer in the same manner, and the resulting supernatants were combined and dialyzed against PBS to obtain purified antibodies.

The fixative was prepared by adding formalin to 5% sodium citrate in physiological saline to give a concentration of 1%.

Add an equal volume of Arsepar solution to fresh chicken blood and -
After blowing in carbon oxide gas for 30 minutes, an equal amount of fixative was immediately added, kept at 37°C for 24 hours, centrifuged to collect blood cells, and washed with physiological saline to obtain fixed blood cells.

The obtained fixed blood cells are suspended in PBS containing 0.1% sodium azide to a concentration of 10% and stored at 4°C.

Add an equal volume of tannic acid diluted 100,000 times to a solution diluted with PBS so that the fixed blood cells are 2.5%, and incubate at 37°C.
The blood cells were collected by centrifugation and washed with PBS, suspended in an equal volume of PBS, added with an equal amount of purified antibody, kept at 37°C for 30 minutes, centrifuged, and collected. P
After washing with BS and then diluent, add 0.5% to diluent.
It was suspended so that

When human Lf solution was added to these sensitized blood cells, agglutination occurred.
No aggregation occurred even when albumin, IgG, IgM, and IgA were added.

In addition, as a diluent, 0.07% bovine serum albumin,
M/60 PBS (pH 7.2) containing 0.005% gelatin and 0.1% sodium azide was used.

Add 0.025+J of diluted solution to each hole of the V-shaped microplate.
0.025 μm of pancreatic juice diluted 10 times with a diluent was added to the 16th volume, and the mixture was successively diluted with Guiluta. On the other hand, add a standard solution containing 84 ng of Lf per 1 m to the 1st 6th column of another column.
Diluted in the same way. Next, 0.025 μl of anti-Human Lf antibody-sensitized blood cells were dispensed into each well, thoroughly mixed, and left at room temperature for 2 hours or more, and the end point of agglutination was observed. When an antigen-antibody reaction occurs, blood cells are dispersed over the entire tube bottom, and when no reaction occurs, blood cells gather in the center of the hole, so the end point of agglutination can be easily determined.

According to the above method, the Lf concentration was measured in the pancreatic juice diluted 10 times. The results are as shown in the table. As is clear from the table, the Lf amount in the pancreatic juice of patients with chronic pancreatitis is
The value was much higher than that of pancreatic cancer patients. Therefore, it can be said that measuring the amount of Lf in pancreatic juice is extremely useful for differential diagnosis of these diseases.

If the anti-human Lf antibody-sensitized blood cells of the present invention are used, 0.03 m
If a very small amount of sample (human pancreatic juice) of less than a sentence is available, the Lf content can be easily measured within a short period of time. Moreover, the sensitivity is extremely high and it is possible to measure up to 1ng of Lf per 1111 specimens. From this, chronic pancreatitis and pancreatic cancer can be distinguished very clearly.

Claims (4)

[Claims] (1) Anti-human lactoferrin antibody-sensitized blood cells, which are obtained by binding anti-human lactoferrin antibodies to blood cells via a binding agent. (2) The sensitized blood cells according to claim 1, wherein the blood cells are fixed blood cells. (3) The sensitized blood cells according to claim 1 or 2, wherein the binder is tannic acid. (4) Anti-human lactoferrin antibody-sensitized blood cells, which are made by binding anti-human lactoferrin antibodies to blood cells via a binding agent, are brought into contact with the test solution or its diluted solution according to the microtiter method, and a hemagglutination image is obtained. A method for detecting human lactoferrin characterized by observation.