JPH02218959A - Forecasting of sensitivity to injecting x ray contrast medium using histamine discharge test - Google Patents

Abstract

PURPOSE: To predict sensitivity in a test tube test by using an antibody for histamine produced in a cell system having American type collection accession number HB8831. CONSTITUTION: Inspection for measuring discharge histamine in this sensitivity prediction is based on an antibody for histamine produced in a cell system having American type collection accession number HB8831. And a production antibody from the cell system which is hybrid of the spleen cell and myeloma cell of a mouse immunized with a histamine immunogen binder selected from an expression group (R is an aliphatic chain having 1 to 10 carbon atoms, X' is a terminal functional group selected from amino, carboxyl, thiol and hydroxyl, p is 1 to 120, and a carrier is immunogen carrier substance selected from serum albumin or keyhole limpet haemocyavin) is used to perform a histamine discharge test of the whole blood.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to injectable radiographic contrast agents.
A method for predicting sensitivity to trust medium (
Whole blood histamine release (re
lease), including the use of immunoassays.

To summarize the invention, sensitivity to radiographic contrast agents (
The use of histamine release immunoassays as a predictive method for allergic reactions (allergic responses).

Previous studies have shown that radiographic contrast agent testing is associated with a 5-10% incidence of reactions (US Pat. No. 4,717,657). Some studies have noted response rates as high as 14%. These reactions usually consist of mild urticaria, peripheral swelling, rhinitis, conjunctivitis, etc. However, approximately 1-2% of these reactions can be classified as "acute" and consist of more severe symptoms such as organ spasm, laryngeal edema, and hypotension. It is believed that 1/10 to 2/10% of these reactions are extremely serious based on the treatment required.

Studies have shown a mortality rate of 1:12,000 to 1:5,000 (E., C. Lasser).
Lasser) etc., [Pretreatment w
ith Cortpsteroids"1.'+], N
ew Eng, J. Med, 317.845-8
49.1987).

Once a patient suffers an anaphylactoid reaction following administration of a radiographic contrast agent, the risk of suffering a subsequent reaction is significantly increased. In fact, the risk increases approximately three times. Prophylaxis with Benadryl ■ and Verednisone ■ results in an overall rate of less than 5% of repeat reactions, and the majority of these reactions are also very mild. However, it is currently difficult to determine whether an individual patient is at high risk prior to the first contrast agent response.

Several papers have been published regarding the release of histamine associated with the administration of radiographic contrast agents, including but not limited to: Ring (R
ing) et al., Cl1n, exp, Immunol.

(1978), 34.302-309; Ri
ce) et al., J. Allergy C11n, Imm.
unol, (1983) 72:2.180-186
; Salem et al., Am, J, of M
ed, (1986), 80.382-384; and Younger et al., J. AIlerg.
yClin, Immunol, (1986),
94 Methods for predicting adverse reactions to 100° radiographic contrast agents are actively being sought; for example, US Pat.
No. 7.657 suggests a method of complement activation and the measurement of at least one product resulting from complement activation as a method of predicting adverse reactions to the injection of radiographic contrast agents. There is.

However, a convenient whole blood test that can predict whether a patient is likely to respond has not yet been developed. Investigators have tried skin tests and subconjunctival and intravenous provocation tests to no avail. Subjects may have a severe or even fatal reaction to subsequent injections of radiographic contrast agents even if the provocation test is negative. Additionally, subjects may have severe reactions from the provocative test itself. Therefore, a convenient in-tube test that could easily predict sensitivity to intravenous injection of radiographic contrast agents would provide useful and often life-saving information not currently available. .

SUMMARY OF THE INVENTION The present invention provides an immunoassay for use in measuring histamine released in whole blood as a method of predicting sensitivity to radiographic contrast agents, the assay being preferably performed in American type culture.・Collection Accession Number (American Type Cutture Co.
11ection Accession Number
) is based on antibodies against histamine produced by cell lines with HB 8831.

This immunoassay can be used as a predictive test to determine whether a patient will exhibit an adverse reaction when injected with an intravenous radiographic contrast agent.

In the group below R is an aliphatic chain having 1 to 10 carbon atoms, and X' is a terminal functional group selected from amino, carboxyl, thiol and hydroxyl, and p is 1 to 1
20, and the carrier is serum albumin or hemocyanin in keyhole limpet.
t hem.

an immunogenic carrier material selected from cyanin; a histamine immunogenic conjugate selected from
Whole blood was tested for histamine release using a histamine release immunoassay, using antibodies produced from a cell line that is a hybrid of mouse lung cells and mouse myeloma cells immunized with A. ate). Ru. A preferred histamine immunogen conjugate is: where p is 1 to 120 and the carrier is serum albumin or keyhole limpet hemocyanin.

Use of the detailed histamine release test of the present invention is generally described in the Examples below. A patient's whole blood sample is used, and separation of cells from the sample is unnecessary. Basophils (ba) from patient whole blood samples
The release of histamine from sophils is used to predict when adverse reactions may occur to any or a particular radiographic contrast agent. X-ray contrast agent (
RCM) is widely used in diagnostic procedures such as IVP and Cat scans. There are many contrast agents. Contrast agents that are widely used are Hypaque, Conray (C.

co-pending application USSN No. 7 filed July 3, 1985.
No. 52,320 (European Patent Specification No. 208.95)
The histamine release test described in the US Pat. No. 3, January 21, 1987) is based on the use of antibodies against immunogenic conjugates of histamine prepared in accordance with that disclosure.

The antibody is an aliphatic chain having from 1 to 10 carbon atoms, and X' is a terminal functional group selected from amino, carboxyl, thiol and hydroxyl, and the carrier is serum albumin (BSA). ) or keyhole limpet hemocyanin, from a cell line that is a hybrid of mouse lung cells and mouse myeloma cells immunized with a histamine immunogen conjugate selected from produced. As used in the above formula,
It must be pointed out that p'' represents the number of histamine moieties bound to the carrier. The number p is referred to as the epitope density of the immunogen, which is usually on average from 1 to about 120, More typically from 1 to about 50. However, these densities can vary widely depending on the particular carrier material used.

A preferred antibody is an immunoconjugate in which R is -CH,CH,-, X' is -NH-, p is 1 to 120, and the carrier is serum albumin or keyhole limpet hemocyanin. This is what was raised against. Budapest Treaty (th
e Butapest Treaty Convent
ion) on May 31, 1985,
Most preferred are antibodies produced by cell lines having the American Type Culture ψ' Collection Accession Number HB 883.1.

Histamine release test tests free histamine and histamine-
It is a competitive system in which protein conjugates compete for monoclonal antibodies that are highly specific for histamine. Suitable monoclonal antibodies have been shown to have little cross-reactivity with histamine. High free histamine concentrations in a blood sample prevent monoclonal antibodies from binding to histamine-protein conjugates, and conversely, low histamine concentrations in a patient's blood sample prevent high concentrations of monoclonal antibodies from binding to histamine-protein conjugates. It would be possible to combine with The relative amount of monoclonal antibody that binds to the histamine-protein conjugate can be easily quantified by labeling the monoclonal antibody with an enzyme. A particularly useful format for this exam
t) is the use of an enzyme in which the addition of a chromogenic substrate to the enzyme results in the development of a color in the presence of the enzyme and its covalently bound monoclonal antibody. A useful combination is the use of a peroxidase and one of its chromogenic substrates. However, other enzymes well known to those skilled in the art may also be used. This is the basis for the use of histamine release immunoassays as a method of predicting adverse reactions to intravenous administration of RCM.

In the first step, the histamine-protein conjugate is simultaneously adsorbed onto a 94 polystyrene peg. It contains 96 holes (we) containing a solution of histamine-BSA conjugate.
ll) by immersion into the holes of a microtiter plate (referred to as an adsorption plate). Two microtiter wells contain only BSA and are used as negative controls. Incubate for 1 hour at room temperature (1 ncu
bate), then the stopper is removed and washed to remove non-adsorbed conjugates.

A second 96 containing various dilutions of RCM or histamine.
A hole microtiter plate (referred to as a release grate) is also provided. Fresh whole blood (patient sample) is treated with peroxidase, preferably horseradish (horseradish).
sh) Dilute in a buffer containing a histamine monoclonal antibody covalently bound to peroxidase. Diluted blood (54/ml) per microtiter well
46: Blood/diluent (v/v)) in equal volume (50 μQ)
Add to each well, mix, then incubate at 37°C for 30 minutes. During this incubation period, histamine is released from basophils in the blood sample if the patient is susceptible. Polystyrene stoppers with adsorbed histamine-BSA are immersed into the microtiter wells of the release plate for 30 minutes at room temperature. During this period, free histamine and histamine-BSA conjugates compete for the monoclonal antibody. The polystyrene stopper is then removed and washed.

The relative concentration of monoclonal antibody-horseradish peroxidase conjugate bound to histamine-BSA conjugate on a polystyrene stopper was determined by the chromogenic substrate of horseradish peroxidase, 2,2'-azinodi-(3-ethyl-benzothiazoline). -6-sulfonic acid (A B
It is measured by dipping the stopper into a third microtiter plate (referred to as the chromogen plate) containing T S■). High color expression indicates a given blood sample/RCM
This means that there is almost no free histamine present in the microtiter wells. The opposite is true if color expression is low. A low color development indicates that the patient whose blood is being tested is sensitive to the test ROM. Color development takes 10 to 30 minutes and a marked inhibition of color development, ie high histamine release, is often visible to the naked eye. Quantitative assessment of histamine release can be performed using a microtiter ELISA reader.

This test method requires approximately 2 hours to obtain results, compared to conventional histamine release tests that require one day to obtain results. Therefore R
Patients exhibiting adverse reactions to administration of OM can be conveniently tested in in vivo studies with provoking agents without endangering the patient.

Example Preparation of histamine-bovine serum albumin immunogen conjugate Near 5 mg bovine serum albumin (BSA), 38°4I
1 g of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 184+ ng of histamine were mixed in a 0.1 molar boric acid solution to a total volume of 4.0
mQ (pH was kept at 4-5). - Night stirring (
After (room temperature) the mixture was extensively dialyzed against phosphate buffered saline. Absorption at 280 nanometers (nm) was measured and protein concentration was calculated. Histamine epitope density was measured by a scintillation counter using a tritiated histamine tracer. According to two representative experiments, the epitope density was 17 and 53
Met. The conjugates from the two experiments were combined and used as described in the Test Methods below.

Histamine Release Test The histamine-BSA crystal produced as described above was added to a 0.05M NaHCO3 solution at pH 9.6 to give a concentration of 40 μg/mQ. 75μQ of this solution
Place an aliquot of the sample into the well of a round-bottomed microtiter tray (referred to as a "coated pyrate"). 0.05M NaHC in two wells to serve as a control.
O, bovine serum albumin (BSA, 4
Fill with 0μg/m(2). Lid with 96 stoppers (NU
NCTSP■ or Falcon [Falcon] F A
S T @lid-like) is placed on the coated plate and allowed to stand at room temperature for approximately 1 hour. Next, remove the stoppered lid and add 0.05% Tween 2 to distilled water.
Total 5050- per l plate using a mixture of 0.
Wash 8 times with 100. A second microtiter tray (referred to as the "release plate") is then prepared for the histamine release process as described below. A histamine standard curve was created by sequentially placing 10-20 μa of histamine standard solution into two wells. These standard solutions were prepared such that the final histamine concentration in the wells was in distilled water or in Tris-ACM dilution (i.e., 25 millimolar (mM) Tris (pH 7,6); 0.12 molar NaCl; 5 mM), respectively. KC1 of M; 0.3
mg/i+Q human serum albumin; 1mM CaC
I! ; and 0.5mM g Ctx).
11, 37, 12.3 and 4.1 nanomoles (nM).

Radiographic contrast media (RCMs) tested as allergens
dilute into 8 to 9 serial dilutions using Tris ACM or distilled water as diluent.

An aliquot (lO-
20 μQ) were placed into two consecutive wells of the release plate. Finally, in all wells of the release plate desired, add 10-20 μa of histamine standard solution, RCM#
This method can be used to test each desired R
Repeated for commercials. The release plate is now ready for use.

Heparinized blood (3,0+*n) was removed from the test patient and mixed with 2.56 mM Tris ACM buffer containing a monoclonal antihistamine antibody-horseradish peroxidase conjugate. This is sufficient for one plate. Monoclonal antihistamine antibody
Horseradish peroxidase conjugate was prepared by Paul Nakane R, Dit-[Dit
o] and E. S. [Tucker] ed.) Ala
n R, Li5s Inc., New York (1978).
ng) method. Monoclonal anti-histamine antibodies were prepared as described in co-pending [USSN No. 752,320].

A preferred monoclonal antibody is American Type Culture Collection Accession Number H88.
Produced by a cell line with 831. Adding a small amount of this antibody-enzyme conjugate to the Tris ACM/blood mixture,
A suitable predetermined conjugate concentration (approximately 0.5-1.5μ
g/key Q). An aliquot of this mixture (50 μQ
/hole) into each well of the release grate and incubated at 37°C for 30
Incubate for a minute to release histamine.

The lid with the 96 stoppers described above is then placed on the release plate so that each stopper is introduced into a well. The arrangement was then incubated at room temperature for 30 minutes, after which the lid with stopper was removed and Tween was incubated as above.
) Washed 8 times with 3030-50/water.

Put the lid with this stopper into the following: 1.61119/111
A solution consisting of 2.2'-azinodi-(3-ethyl-benzothiazoline) (ABTS@) and 10 mM urea peroxide in 0.1 M citrate buffer (pH 4,3) was added to each well at 50 μα Place in a third microtiter tray (referred to as "chromogen plate 7").

After an appropriate incubation period (approximately 15 minutes at room temperature), the stopper was removed and the absorbance (415 nM) of the contents of each well of the chromogen plate was measured using an ELISA reader. Alternatively, evaluation of histamine release can be performed, especially when the stopper is first coated with a histamine-protein conjugate in a plate.
Preferably, this can be done by visual inspection when coated with the histamine-bovine serum albumin conjugate produced as described above. The % reduction in color or the actual concentration of histamine released obtained from the standard curve (prepared as described above) can be obtained and/or plotted against dilution of ROM if desired. be able to. From such a plot, a judgment can be made regarding the patient's susceptibility to RCM allergens. sparse RO
The release of histamine that occurs at M concentrations shows high sensitivity;
The opposite is also true.

An exploratory clinical study was conducted on 20 patients with a past history of reactions to radiographic contrast agents. Patients were tested with four different radiographic contrast agents: Conray, Hyvac, Omnivac, and Isobu.

Each is an injectable radiographic contrast agent that can be used for the same diagnostic procedure or for different procedures, such as IVPs, Cat scans, etc.

Studies have shown that the histamine release test described above can measure histamine released from patient's basophils when their blood is incubated with an X-ray contrast agent. Compared to the control group, a significant amount of histamine was released in the group of those who had previously reacted to the injection of radiographic contrast agent when their cells were incubated with the radiographic contrast agent Haivac. There were several patients who showed.

This suggests that the described histamine release test can be used to diagnose (predict) increased sensitivity to radiographic contrast agents. This also predicts that these are patients who will have a significant response to radiographic contrast agents.

The advantages of this test method in terms of time and convenience make it practical to use histamine release as a predictor of sensitivity (allergy) to radiographic contrast agents.

A clinical study was conducted to test the hypothesis that sensitivity (allergy) to radiographic contrast agents can be predicted by the consequences of histamine release described in the text. Histamine release was measured using a radiographic contrast agent in blood samples taken from patients who were subsequently tested. The results were later compared with the occurrence of allergic reactions to the administration of radiographic contrast agents. Of the 16 patients used in the study, - patients had allergic reactions to injections of the radiographic contrast agent, Haivac. That patient was also the one who showed the highest degree of histamine release as measured in the test.

Many modifications and variations may be made to the practice of the invention without departing from its spirit and scope, and the invention is defined only by the claims.

The main features and aspects of the invention are as follows.

1. American Type Culture Collection φ accession number H8 for measuring histamine release in whole blood as a method for predicting sensitivity to radiographic contrast agents.
Use of an immunoassay based on antibodies against histamine produced by cell lines with 8831.

, 2. In the group below R is an aliphatic chain having 1 to 10 carbon atoms, and X' is a terminal functional group selected from amino, carboxyl, thiol and hydroxyl, and p is 1 to 1
20 and the carrier is an immunogenic carrier material selected from serum albumin or keyhole limpet hemocyanin. Mouse lung cells and mouse myeloma cells immunized with a histamine immunogen conjugate selected from A histamine release immunoassay using antibodies produced by a cell line that is a hybrid of the A predictive test that measures whether or not.

3. The predictive test according to 3 above, wherein the histamine release immunoassay method is produced by a cell line having the following histamine immunogen conjugate: 31.

where p is 1 to 120 and the carrier is serum albumin or keyhole limpet hemocyanin.An antibody produced by a cell line that is a hybrid of mouse lung cells and mouse myeloma cells immunized with The predictive test described in 2 above, using

Claims (1)

[Claims] 1. American Type Culture Collection Accession No. HB for measuring histamine release in whole blood as a method for predicting sensitivity to radiographic contrast agents.
Use of an immunoassay based on antibodies against histamine produced by cell lines with 8831. 2. The following groups ▲ Numerical formulas, chemical formulas, tables, etc. ▼ In the carrier formula, R is an aliphatic chain having 1 to 10 carbon atoms, and X' is a terminal functional group selected from amino, carboxyl, thiol, and hydroxyl. group, p is 1 to 1
20 and the carrier is an immunogenic carrier material selected from serum albumin or keyhole limpet hemocyanin. A histamine release immunoassay using antibodies produced by a cell line that is a hybrid of the A predictive test that measures whether or not.