JPH0313905B2 - - Google Patents

Description

Detailed Description of the Invention The present invention provides immunoglobulin E (IgE) from blood.
The present invention relates to a substance used for adsorbing and removing Immunoglobulin (E) and a device equipped with the substance.

IgE is considered to be a cause of allergic rhinitis, atopic bronchial asthma, and various types of reagin type (type I) hypersensitivity. IgE to specific allergens
Continuous production of allergen is a characteristic finding of so-called type I allergy, but it occurs when the receptors on the cell surface of mast cells and basophils bind to the Fc portion of the IgE molecule, which leads to re-entry of the allergen. It is thought that the aggregation reaction of the receptor that binds to the Fab part of the IgE molecule progresses, and the cells release various chemical mediators, causing a variety of tissue damage. Therefore, by specifically removing IgE from the blood of allergic patients, it is possible to inhibit or prevent the above reactions, which could be an effective means for treating allergies [The Lancet].
Lancet, Vol. 2, p. 40 (1978)].

In view of these circumstances, the inventors of the present invention
After searching various methods for advantageously removing IgE, we found that anti-human
The inventors have discovered that when blood, particularly plasma, is brought into contact with a combination of IgE and an insoluble carrier, IgE is efficiently adsorbed and removed, and as a result of further research based on this finding, the present invention has been completed.

The present invention is a combination of anti-human IgE and porous glass beads having an aminoalkyl group, which is used as an adsorbent in a device for separating and removing IgE from blood.

The anti-human IgE used in the present invention may be any one produced using known conventional methods.
For example, animals such as rabbits, goats, and horses are
Polyclonal human IgE obtained by immunization with IgE as an antigen [“Biochemistry of Immunity”, edited by the Protein Nucleic Acid Enzyme Editorial Department, pp. 26-33, published by Kyoritsu Publishing Co., Ltd.;
“Bacteriology Practice Summary”, edited by the Institute of Medical Science Alumni Association, Vol.
It can be produced according to the method described in pages 199-222, published by Maruzen Co., Ltd. ” or monoclonal anti-human IgE obtained using cell fusion technology [“Cell Technology” Vol. 1, No. 1, No. 23-
It can be produced according to the method described in "Illustrated Clinical Immunology Course", edited by Junichi Yada, pp. 241-247, published by Medical View Co., Ltd., p. 29, 1982, published by Shujunsha Co., Ltd. ] etc.

Porous glass beads having an aminoalkyl group used in the present invention have an average pore diameter of 100 Å or more, or an average exclusion limit molecular weight for proteins of 1
A porous material having porosity of ×10 ⁴ or more is preferable.
Moreover, the particle size is preferably about 30 microns to 2 mm.

In the following, porous glass beads having an aminoalkyl group may be referred to as an insoluble carrier.

As a method for binding anti-human IgE to an insoluble carrier, a conventional method known per se may be used. In this method, for example, a water-soluble carbodiimide (e.g., 1-ethyl-3
-(3-dimethylaminopropyl)carbodiimide/hydrochloric acid, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidometh-p-toluene-sulfonate, etc.) to attach the carboxyl of anti-human IgE to the aminoalkyl group. Examples include a method of reacting and bonding groups.

The reaction temperature is preferably about 0° to 25°C.

The reaction time is not particularly limited.

The binding strength of the conjugate obtained in this way is strong, but in order to further strengthen this, that is, to more reliably prevent the anti-human IgE from detaching from the carrier, the conjugate obtained in this way has a strong binding strength. can also be crosslinked with dialdehyde compounds such as glutaraldehyde. Glutaraldehyde treatment also performs sterilization.

The thus obtained conjugate of anti-human IgE and insoluble carrier can be stored by suspending it in a buffer solution. Furthermore, it is advantageous to add a protective substance to the suspension and freeze-dry it to preserve it in an anhydrous state.

Examples of the buffer solution include Clark-Lubs buffer, So¨rensen's buffer, Kolthoff's buffer, and Michaelis' buffer.
buffer solutions and the like.

Examples of the protective substance include salts such as potassium phosphate and sodium phosphate, organic acids such as citric acid and tartaric acid, and saccharides such as mannitol, dextrose and dextran.

The blood component separation device used in the present invention includes, for example, (1) a device equipped with an adsorbent through which blood extracted from the human body passes; 2) An example is a device in which blood taken from a human body is separated into plasma using a plasma separator, and the plasma is passed through an adsorption device. The plasma separator in the device (2) above may be any commonly used plasma separator. Examples include a plasma separator based on centrifugation, a membrane-type plasma separator using a permeable membrane, and the like.

The adsorption device itself in the device (1) or 2 above may be any device as long as it can be equipped with an adsorbent and allow a liquid containing the adsorbed substance to pass therethrough. The device of the present invention comprises such an adsorption device equipped with a combination of anti-human IgE and an insoluble carrier as an adsorbent.

In order to remove IgE from blood using the blood component separation and removal device of the present invention, when using the device (1) above, blood taken from a human body is mixed with a combination of anti-human IgE and an insoluble carrier. The IgE present in the blood is adsorbed to the bound substance and removed by passing it through a device equipped with the substance as an adsorbent. In addition, when using the device (2) above, the blood taken out from the human body is passed through a plasma separator to separate the plasma from the blood, and the liquid containing the plasma is converted into a conjugate of anti-human IgE and an insoluble carrier. The IgE present in the plasma-containing solution is adsorbed to the conjugate anti-human IgE and removed. The plasma-containing liquid thus treated by the apparatus (2) can be combined with the plasma-containing liquid described above to form blood with reduced IgE content.

A blood component separation and removal device equipped with a combination of anti-human IgE and an insoluble carrier of the present invention as an adsorbent is a device for separating and removing IgE present in blood taken from a human body.
can be effectively and specifically separated and removed.

The present invention will be explained in more detail by referring to Reference Examples and Examples below.

Reference Example 1 Human IgE is used as an antigen, and 1 mg of it is administered subcutaneously to a goat together with Freund's complete adjuvant at a time. This immunization procedure is performed a total of 5 times every 2 weeks to obtain antiserum. γ-globulin is isolated from this antiserum by ammonium sulfate precipitation. Further, the γ-globulin is passed through a column containing diethylaminoethyl cellulose ion exchanger, and the effluent from the column is collected to obtain an anti-human IgE solution (8.33 mg/ml protein concentration).

Example 1 Porous glass beads having an aminopropyl group (aminopropyl CPG-1400, manufactured by Electronucleonics, USA, pore size 1400 Å, particle size 74-147
micron) 2.5% glutaraldehyde solution in 1g
Let 10 ml react at room temperature for 1 hour, then remove the reaction solution.

Add 6 ml of the anti-human IgE concentration obtained in Reference Example 1 to 1 g of the glass beads treated above and react at 0° C. for 4 hours. The Schiff base product thus obtained is reduced with 10 ml of 1% sodium borohydride solution. Then, the same ethanolamine treatment and washing operations as in Example 1 are performed to obtain a combination of anti-human IgE and porous glass beads.

An adsorption/removal test is conducted using 0.5 g (as the dry weight of the carrier) of the thus prepared bond as an adsorbent. That is, 10 ml of plasma containing 860 U/ml of 1 gE is added to this, and stirred for 1 hour at room temperature using a magnetic stirrer. After stirring, measure the IgE concentration (Ct) in the plasma using an IgE measurement kit (IgE "MITSUI", IR-1200, manufactured by Mitsui Pharmaceutical Industries, Ltd.), and calculate the removal rate (%) from the following formula. do.

Removal rate (%) = Co-Ct/Co x 100 Co is the initial IgE concentration of the adsorbed liquid, which in this example is 860 U/ml.

The adsorption removal rate of IgE is 92%, and most IgE
can be removed by adsorption.

Example 2 Specific examples of the adsorption device of the present invention include those shown in FIGS. 1 to 3 and 5 to 6, for example.

In FIGS. 1 and 2, 1a and 1b are
2 is the inlet of the blood or plasma-containing liquid, 3 is the inlet of the anti-human IgE and insoluble carrier used as an adsorbent, and
4 represents an outlet, 4 represents a column, and 5 and 6 represent a filter, respectively.

In Fig. 3, 1 is the conjugate of anti-human IgE and insoluble carrier used as an adsorbent, 2 is the inlet of blood or plasma-containing liquid, 3 is the outlet, 8 is the cylinder, and 7 and 7' are the filters. , 9 and 9' represent packing, and 10 and 10' represent caps, respectively.

Example 3 Figure 4 shows a state in which IgE present in the blood is removed by passing blood through the blood component separation and removal device of the present invention to separate plasma, and passing it through an adsorption device to process the plasma. Diagrammatically shown.

In FIG. 4, 11 is a plasma separator, 12 is a plasma separator, and 12 is a plasma separator.
13 and 14 are pumps, 15 is a tube, 16 is the flow direction of blood, and 17 is the flow direction of plasma-containing liquid. , 18 indicates the flow direction of the plasma-treated solution, and 19 indicates the flow direction of the concentrated blood cell-containing solution.

Blood containing high IgE is introduced into 11 by pump 13 and is separated into plasma. The separated plasma-containing liquid is introduced into 12 by a pump 14, and after selectively adsorbing and removing IgE in the plasma-containing liquid, the concentrated blood cell-containing liquid and the plasma-treated liquid are combined in a confluence section 20.

Example 4 Specific examples of the blood component separation device of the present invention include those shown in FIGS. 5 and 6, for example.

FIG. 6 shows a cross section taken along the line AA in FIG. 5. Fifth
In the figure and FIG. 6, 1 is a combination of anti-human IgE and an insoluble carrier used as an adsorbent, 2' is a blood inlet, 3' is an outlet, 25 and 25' are filters, 22 and 22 ′ indicates porous membrane material,
21 and 21' represent collection passages, 23 represents a differential pressure control throttle valve, and 24 represents a housing, respectively.

[Brief explanation of drawings]

Figures 1 to 3 are cross-sectional views of the adsorption device in the blood component separation and removal apparatus of the present invention, Figure 4 is a schematic diagram of the state in which blood is passed through the apparatus of the present invention, and Figures 5 and 6 are of the present invention. 3A and 3B respectively represent cross-sectional views of the blood component separation and removal device of the invention.

Claims (1)

[Claims] 1. A combination of anti-human IgE and porous glass beads having an aminoalkyl group, used as an adsorbent in a device that separates and removes IgE from blood.