NO138182B - ANTIBODIES SPECIFICALLY AIMED AT IMMUNGLOBULIN E (IGE) OR AGAINST FRAGMENT OF IGE - Google Patents

Description

The invention relates to antibodies specifically directed

against immunoglobulin E (IgE) or against fragments of IgE containing class-specific determinants, which antibodies are intended to be used in vitro by immunochemical determination methods based on reactions, in which an allergen is bound to a water-insoluble polymer, IgE directed against the allergen and antibodies directed against IgE or fragment thereof is included, characterized in that the antibodies directed against IgE or fragment thereof are given the ability to emit radioactive radiation or fluorescence radiation by labeling with one or more radioactive isotopes or with a group containing a radioactive isotope resp. with a fluorescent group.

It is known that some immunogenic substances, so-called allergens, many of which are of a protein, peptide or carbohydrate nature, can give rise to allergic disease states, e.g. asthma, hay fever. (Such allergens occur in general and can originate from e.g. hair, plant pollen, micro-organisms, some parasites etc.). This results in a special type of antibodies, so-called reagins, which are made up of immunoglobulins that are directed specifically against the allergen or allergens, usually in very low concentrations, in e.g. the patient's blood and can be detected in blood serum. These special immunoglobulins are called reagin-immunoglobulin or reagin-Ig in the following. (See, e.g., Advances in Immunology, Vol. 3, (1963) pp. l8l - 183). Reagin-Ig is understood here and in the following to mean the group of immunoglobulins that can react with allergens and which thereby,

when the reaction takes place in vivo, causes allergic reactions in humans or animals.' (Now these immunoglobulins are often referred to as IgE).

The present invention is of importance in the detection of specific reagin-Ig directed against allergens in aqueous samples. Detecting reagin-Ig is particularly important in connection with the diagnosis of hypersensitivity in humans and animals.

For this purpose, so-called skin tests have previously been used, as the test allergen is added to the test object, e.g. intracutaneously. If hypersensitivity (allergy) exists, a reaction occurs in vivo between the allergen and reagin-Ig directed against the mentioned allergen, which results in certain symptoms such as redness or swelling of the place where the injection took place, which are observed and assessed with regard to the degree of impact.

In vivo examinations are also so-called provocation tests, as the patient inhales an allergen in aerosol form. If hypersensitivity to this exists, a reaction is obtained, such as e.g. can cause asthma symptoms. Both test types can be said to be cumbersome and time-consuming for the patient, and in certain cases associated with discomfort or the risk of this. In some cases it is not possible to carry them out. Furthermore, multiple in vivo tests are also required in order to be able to diagnose a hypersensitivity to a certain or certain allergens.

Previously tried in vitro methods lack practical significance.

It is admittedly known from J. Immunology 97 (1966), 840-853 and 98 (1967), 490-501 to detect Ig-E with a radiation immunological method, where the use of labeled allergen is an essential feature. The labeled allergen is caused by this method to react with Ig-E in solution, after which the formed complex is precipitated from the solution by the addition of antibodies against Ig-E. The allergen used here is allergen from "ragweed", which is one of the few exceptional cases where the allergen can be isolated in high purity. Allergen-containing extract of other origin, e.g. from dog or horse epithelium or birch pollen does not have sufficient purity to be utilized by this method, which therefore has a particularly limited area of application. Furthermore, the method requires a labeling procedure for each test allergen, which is both costly and time-consuming. With this method, it is also worth noting that the allergen only reacts with Ig-E specifically directed against this allergen, while precipitation of the complex with the antibodies against Ig-E simultaneously results in a precipitation of the reaction products between the antibodies and other reagin immunoglobulins (other Ig- E) than that which is specifically directed against the allergen in question. If, in this system, instead of labeling the allergen, you were to label the antibodies, you would of course also get a solid phase that emits radioactive radiation, but in this case it is impossible to determine whether the allergen is included in any part of this phase or not. Consequently, in this case one would not get the desired information about the possible presence of an Ig-E specifically directed against the allergen.

The purpose of the invention is to produce a reagent which can be used for a simple and reliable in vitro method, whereby the above-mentioned disadvantages are removed and which can be carried out without discomfort or risk for the patient.

Norwegian patent no. 127-685 describes a method according to which the sample calculated for the test, e.g. a body fluid such as blood serum or blood plasma, in vitro, is brought into contact with a water-insoluble polymer, to which a test allergen is bound, as a reaction between the test allergen and the polymer and reagin-Ig directed against this takes place, so that reagin-Ig binds to the test allergen on the insoluble polymer, and that polymers with adherent test allergen and reagin-Ig are brought into contact with either a) antibodies against reagin-Ig, which antibodies are labeled with a radiation-emitting atom or group,

or

b) antibodies against reagin-Ig and with reagin-Ig, the reagin-Ig being labeled with a radiation-emitting

atom or group

after which the insoluble polymer with adherent substances is separated from the liquid and then the radiation from the insoluble polymer with adherent substances or from the separated liquid is measured.

Thus, if reagin-Ig directed against the allergen is present in the sample, the labeled reagent binds to the insoluble phase, which then emits radiation. The latter increases with increasing concentration of reagin-Ig in the sample. The radiation of the liquid phase instead decreases with increasing concentration of reagin-Ig, as larger amounts of labeled reagent are bound to the insoluble phase. The measured radiation values for the sample can be compared with values on the control sample.

Water-insoluble polymers are used as carriers of the test allergen. They can, for example, be in the form of particles of varying shape and size. Polymers in the form of e.g. flat objects such as disks or bands or also e.g. in the form of the wall of a pipe, e.g. the inside of a test tube. The bond between the polymer and the test allergen must be such that the test allergen cannot be detached from the polymer during normal washing operations.

For this purpose, it can e.g. be of a chemical or possibly also of a physical nature. A suitable form of chemical bonding is to provide bridges of a covalent nature between the polymer and the test allergen.

For this purpose, the polymer is chosen so that

it has suitable reactive groups, e.g. amino groups, hydroxyl groups and carboxyl groups to easily enable a binding of the test allergen to the polymer. Hereby, a chemical bond with bridges with bonds of a covalent nature is preferably chosen.

It is particularly suitable to choose polymers consisting of

of a three-dimensional network held together by bonds of a covalent nature. Such polymers, even if they are swellable in water or water-soluble media, are completely insoluble and thereby cannot release any of the polymer material or substances bound to it, e.g. by washing procedure. Examples of such polymers are polymers formed by crosslinking substances containing a majority of hydroxyl groups such as carbohydrates and sugar alcohols, e.g. dextran, starch, dextrins and other polysaccharides as well as polyvinyl alcohol with a bifunctional substance, e.g. a bifunctional substance of the type X - R - Z, where, for example, X and Z are halogen or epoxy groups and R.. the rest of the bi-functional substance, e.g. an aliphatic radical containing 3-10 carbon atoms. Other examples of insoluble polymer material are cellulose, agarose gel, polyaminostyrene.

For this purpose, grains of the commercially available product "Sephadex" can be used, which consists of dextran cross-linked with glycerol ether bridges, formed by treating dextran with epichlorohydrin. The product can be used e.g. in the form of small grains. "Sephadex" and similarly obtained products are water-swellable but insoluble gel products, e.g. in grain form. They contain hydroxyl groups and can therefore easily be substituted also with other groups, e.g. such containing amino groups or carboxyl groups are therefore well suited for bridging with bonds of a covalent nature to the test allergen.

Appropriately, such a form of the polymer is chosen

that a large contact surface is achieved, e.g. small particles.

The test allergen is bound to this carrier polymer under mild conditions, so that the allergen's immunochemical reactivity does not significantly decrease. In the case of chemical bonding between the allergen and the polymer, reactive groups are utilized in these such as amino groups, hydroxyl groups, mercapto groups, amide groups and carboxyl groups, as a bridge is built with a chemical bond, preferably of a covalent nature, from the test allergen to the polymeric one, e.g. of type:

The bridge between the allergen and the polymer does not need to be structurally determined, and can therefore be chosen of a very variable type as it only has the purpose of preventing the allergen from being washed away from the insoluble polymer.

The determination method for reagin-Ig is based on the fact that reagin-Ig can bind both to the allergen that it is specifically directed against and to antibodies generally directed against reagin-Ig. Such antibodies are at least bivalent, i.e. they can bind at least two molecules of reagin-Ig. Antibodies directed against reagin-Ig and also reagin-Ig itself can be labeled with a radiation-emitting atom or group, e.g. with a radioactive isotope, or a fluorescent group.

Compared to the known methods with execution of. skin tests and provocation tests, the above method has the advantage that with a blood sample from the patient in a completely harmless way in vitro with a very sensitive and simple method, the presence of and also the amount of reagin-Ig directed against a specific type of allergen or a certain group of allergens, e.g. animal allergen and plant allergen. Essential to the method is that the test allergen against which the reagin-Ig to be determined is directed is very firmly bound to a water-insoluble carrier. Reagin-Ig that reacts with the allergen can thus be easily separated from non-allergen-bound reagin immunoglobulins. During the further working steps of the method, the antibodies or the reagin-Ig that is not bound to the polymer is very easily separated from those that are bound to the polymer.

Separation of the polymer with adherent substances from the liquid can be easily carried out. If the polymer is present in particulate form, the separation can take place through e.g. simple centrifugation or filtration. The separation is insensitive to variations in salt and protein concentration in the liquid within physiological limits. The entire determination procedure including the separation of free labeled antibodies resp. reagin-Ig and polymer-bound labeled antibodies resp. reagin-Ig can e.g. carried out in the same test tube with the addition of a precipitating agent or similar.

The above method requires access to isolated

reagin-Ig and antibodies against these.

Reagin-Ig can be extracted from serum from allergic patients and in special cases from serum from patients with tumors in the plasma cells that form reagin-Ig. Reagin-Ig can be purified by different methods for separating proteins such as gel filtration, ion exchange chromatography and electrophoresis. Like other immunoglobulins, reagin-Ig can be cleaved chemically or enzymatically into an antigen-binding part and a part that carries the class-specific determinants.

The invention relates to antibodies against reagin-Ig or fragments thereof, which antibodies have been labeled with the ability to emit radiation and are intended to be used in radiation-immunological methods, for example as indicated for the determination of reagin-Ig..

Antibodies against reagin-Ig can be produced according to methods known per se for immunization of laboratory animals by e.g. repeated subcutaneous injections of small amounts of reagin-Ig or fragment of reagin-Ig in mixture with a suitable adjuvant, e.g. Freund's mineral oil suspension. Those in the animals, e.g. rabbit, antibodies formed can be recovered from the animals' blood serum.

Specific antibodies, i.e. antibodies directed against the class-specific determinants in reagin-Ig, can be obtained by immunization with fragments of the reagin-Ig molecules containing these determinants or also by "isolation of these specific antibodies from a mixture of antibodies formed by immunization with whole reagin-Ig.

Specific antibodies directed against reagin-Ig can, if desired, be isolated from the antiserum by conventional immunosorbent technique, reagin-Ig being coupled to e.g. bromoacetyl cellulose and the antibodies that are bound to coupled reagin-Ig are released by lowering the pH.

It is an advantage to use specific antibodies against the class-specific determinants in reagin-Ig in order to achieve high safety and accuracy in the determinations.

Labeling with a radioactive isotope of reagin-Ig resp. antibodies against reagin-Ig can be made in the usual way, choosing an isotope suitable for the purpose, e.g. <125>I (see e.g.

the method of Hunter and Greenwood, Nature, vol. 194 (1962)

page 495). Likewise, the labeling with a fluorescence-emitting group can be done in the usual way, e.g. with a fluorescein derivative such as fluorescein isothiocyanate.•

Allergen-containing extracts, e.g. from dog or horse epithelium, timothy or birch pollen, are commercially available.

The radioactivity determinations can be carried out using usual methods, e.g. using scintillation detectors. Likewise, fluorescence measurements can be carried out using common methods.

The labeled antibodies according to the invention can be used in an aid for the determination of specific reagin-Ig directed against allergens in aqueous samples, described in Norwegian patent no. 127,685, which comprise a first reagent, which includes a water-insoluble polymer to which, by means of covalent bonds, ionic bonds or adsorption bonds a test allergen is bound another reagent containing antibodies against reagin-Ig, since the antibodies against reagin-Ig are given the ability to emit radiation by labeling, since the first reagent is intended to come into contact in vitro with an aqueous sample containing reagin-Ig to bind•reagin-Ig to the test allergen on the insoluble polymer, and the second reagent being intended to contact the insoluble polymer with the adjacent test allergen and reagin-Ig to form a mixture containing insoluble material that radiates and which consists of insoluble polymer and by this stuck substances and radiant liquid phase, the radiation of the soluble material and the liquid phase each being a function of the concentration of reagin-Ig directed against the test allergen in the sample.

The insoluble polymer is conveniently present

in the form of particles.

The reagent can form part of a reagent combination which

is intended to be used for testing samples from allergists.

The reagent combination may include an ampoule

or similar with labeled antibodies against reagin-Ig, preferably in dried form, e.g. lyophilized form.

The determination method for reagin-Ig shall in the following be described with an example, which shows in more detail the production of labeled antibodies against reagin-Ig and how they can be used for the determination of reagin-Ig.

Example 1.

Determination of reagin-Ig directed against a plant allergen (Bjerkepollen) A. Production of particles with chemically bound allergen.

As starting material, fine-grained particles consisting of dextran cross-linked with glycerol ether bridges ("Sephadex G 25", superfine) and substituted with isothiocyanatophenoxyhydroxypropyl groups are used (R. Axén and J. Porath, Acta Chem. Sean., 18 (1964), page 2193). To a suspension of 100 mg of the particles in a water solution of sodium hydrogen carbonate was added 1 ml of allergen solution (commercially available allergen extract) with an approximate concentration of 0.2 mg of allergen per ml. The mixture was incubated for 3 days at room temperature under constant slow vertical rotation. The particles were then centrifuged and washed twice each time with 0.5 M NaHCO 3 , 0.1 M acetate buffer, 0.1 M Tris buffer with 1% bovine serum albumin. The particles were homogenized and suspended in 0.1 M Tris buffer with 1% bovine serum albumin.

B. Preparation of reagin-Ig

Sample of thawed or recently collected plasma from

a patient with myelomatosis was diluted with 0.15 M sodium chloride to a content of about 15 mg/ml with respect to the M component (reagin-Ig). Precipitation with anhydrous sodium sulfate (18 g/100 ml) was done at 25°C. After one hour, the precipitate is collected by centrifugation (10,000 x g, 20 minutes, 25°C). The sediment was preserved, washed with sodium sulfate solution

(18 g/100 ml) and was dissolved in 0.8 volume of 0.1 M sodium phosphate buffer, pH 7.5 - The material was reprecipitated once, as mentioned. After the last precipitation, the sediment was dissolved in 0.1 M tris-HCl buffer, pH 8.0 (20°C) and placed on a column (3.2 x 30 cm)

with DEAE-"Sephadex" A-50 equilibrated.with the same buffer. Elution of the material was performed with a continuous gradient from 0.1 M to 1 M tris-HCl at a constant pH of 8.0 at 20°C. Fractions containing reagin-Ig were saved and concentrated and applied to a column (3.2 x 95 cm) of "Sephadex" G 150 equilibrated with 0.1 M tris-HCl - 0.2 M NaCl - 0.02 M EDTANa2 pH 7.7 and containing 0.02% sodium azide. The material was passed through the column three times (recirculation chromatography method): Fractions containing reagin-Ig were saved and concentrated by ultrafiltration (Visking tubing 8/32") at 4°C.

Reagin-Ig prepared as described was contaminated with less than 0.1% of total protein by other immunoglobulins (IgA, IgD, IgG and IgM). Purified reagin-Ig gave a single peak

by electrophoresis pH 8.6 I = 0.05 wander in the B-a area. Ultra-centrifugation studies showed a single boundary line and the sedimentation constant S°£o. U n i W, was calculated at 8.20s. The molecular weight of reagin-Ig was calculated to be 200,000 ± 5,000 using

of a partial specific volume of 0.713 cm^/g determined on the basis of the amino acid and carbohydrate composition of reagin-Ig. The diffusion constant, D°on . was calculated at 3.71 x 10 cm/sec.

you, w

The reduction experiment showed that reagin-Ig consists of two types of polypeptide chains and thus resembles other serum immunoglobulins. Evidence has emerged for the presence of two light polypeptide chains with molecular weights of 22600 in reagin-Ig. The molecular weight of the heavy polypeptide chain including its prosthetic carbohydrate group(s) was calculated to be about 77%, assuming a molar ratio of 1:1 for heavy and light peptide chains in native reagin-Ig. The amino acid and carbohydrate composition is shown in Table 1.

C. Isolation of fragment of reagin-Ig containing class-specific determinants (so-called Fc fragment)

100 mg of reagin-Ig prepared according to example 1 B in 0.1 M sodium phosphate buffer (pH 7.0) with 0.1 M cysteine was incubated at 37°C for 4 hours with 1 mg of papain, after which the reaction was stopped by the addition of iodoacetamide solution to a final concentration of 0.05 M. The reaction mixture was fractionated by gel filtration ("Sephadex G 150"), using a

copolymer of dextran and epichlorohydrin with a water absorption capacity of 15 g per g dry matter and elution of the gel mass took place with 0.1 M tris-HCl, 0.2 M NaCl buffer (pH 7.7). By analysis, the fraction containing the Fc fragment was determined, which was then isolated and used for immunization. D. Production of antibodies against the Fc fragment of reagin-Ig.

Rabbits were injected intramuscularly with each a mixture of 0.5 mg Fc fragment of reagin-Ig in 0.2 ml, 0.15 M sodium chloride solution and 0.8 ml complete Freund's adjuvant. The immunization was repeated after 14 days and then every week for three weeks. After a further 10 days, the rabbits were bled and antiserum was prepared from the blood by allowing it to coagulate and the blood coagulum was then separated.

The antiserum was made specific by absorption for less than 1 hour at +37°C and 16 hours at +4°C by adding one part of normal human serum to one part of the antiserum. After centrifugation, the specificity of this antiserum was tested by immuno-electrophoresis against normal human serum and with Ochterlony gel diffusion analysis against pure immunoglobulins A, G and M, so-called light polypeptide chains from normal polar immunoglobulin G as well as Bence-Jones proteins, whereby it proved to be specific for reagin-Ig.

From this antiserum, antibodies directed against the Fc fragment were isolated in the following way: 2 grams of a complex between bromoacetyl cellulose and reagin-Ig was mixed with 34 ml of solution containing approx. 95 mg immunoglobulin isolated from antiserum directed against Fc fragment by ion exchange chromatography. The resulting suspension was activated at pH 7 and 4°C

for 2 hours, after which it was centrifuged at 20,000 xg for 20 minutes at 10°C. The supernatant was separated, the cellulose was washed with 0.15 M NaCl - 8 M carbamide, pH 6 - 7, centrifuged

and the supernatant separated. This washing procedure was repeated until the supernatant became free of protein. The cellulose protein complex was mixed with 9 ml of 0.1 M glycine-HCl buffer (pH 3.1) and incubated with agitation at 37°C for 40 minutes. The whole was centrifuged and the supernatant dialyzed against 1200 ml of 0.01 M tris-HCl 0.1 M NaCl (pH 7.1) at 4°C for 48 hours. This solution contained antibodies in an amount of approx. 1 mg per ml, which are specific for the Fc fragment of reagin-Ig and thus also against

whole reagin-Ig. This antibody material is sufficient for some 10s of millions of determinations.

E. Labeling of antibodies specific to the Fc portion of reagin-Ig.

Labeling was carried out with I according to a method described by Hunter and Greenwood, Nature, vol. 194 (1962), pp. 495-

The labeling can also be carried out in the same way as described below in example 2 for antibodies against reagin-Ig.

F. Provision:

1. 0.5 ml suspension of particles with chemically bound plant allergen formed according to point A above, which suspension contains 1 mg of particles per

ml was introduced into each of two test tubes.

2. 0.05 ml of the patient serum intended for testing

was added to one of the pipes.

3- 0.05 ml control serum from a non-allergic person was added

to the other pipe.

4. The tube contents were incubated for 5 hours at room temperature while the tubes were allowed to rotate slowly

the vertical plane.

5- The particles were centrifuged down at 3000 revolutions per minute for 1 minute, after which the above

liquid was removed.

6. The particles were washed three times with 0.1 M tris buffer pH 7.4 with 1% bovine serum albumin. The supernatant was aspirated after each centrifugation. At each aspiration, it was ensured that solid particles did not follow. 7. 0.1 ml of the solution of antibodies against the Fc part of

125

reagm-Ig and labeled with I were added to all

the tubes.

8. The tube contents were incubated for 15 hours at room temperature, the tubes being brought to a slow boil

rotate in the vertical plane.

9. The particles were centrifuged down at 3000 rpm. minute for 1 minute, after which the above

liquid was separated.

10. The particles were washed three times with 0.1 M tris-

HCl buffer (pH 7.4) with 1 wt# bovine serum albumin with centrifugation down and suction of the supernatant after each washing. At each sucking was

it was ensured that solid particles did not accompany it.

11. The tubes were placed in a scintillation detector for measuring gamma radiation.

For evaluation, the value obtained was compared with the radiation from one tube with the value of the radiation from the other tube containing control serum from non-allergic patients.

Alternatively, after centrifugation according to point 9, a certain volume of the supernatant can be transferred into a rain tube, after which the gamma radiation from labeled antibodies against the Fc part free in the solution is measured.

For quantitative determination, in point 2 above, 0.05 ml of sample serum in different dilutions can be added to each of a number of test tubes. A number of tubes are similarly equipped with different dilutions of a standard serum. The activity can then be expressed in relation to that of the standard serum. Example 2.

Labeling of antibodies against reagin-Ig.

Antibodies against reggin- Ig.

These were produced by immunization of rabbits

with reagin-Ig in a similar way as discussed in example 1 D. Radioactive iodine (125 I).

Iodine-125, carrier-free, was dissolved as Nal in 0.1 M NaOH and is used free of reducing agent.

Iodine monochloride.

Iodine monochloride was prepared according to J.L. Izzo,

W. F. Bale, M.J. Izzo and A. Roncone, J. Biol. Chem., 239, no.

11 (1964) 3742 and the iodine content was determined by reduction of all iodine to iodide with arsenic trioxide and subsequent titration with 0.1 M AgNO-j. The stock solution of ICI contained 2.54 mg I/ml in 0.02 M KCl, 2.0 M NaCl and 1.0 M HCl and is stable at room temperature for at least 18 months. Before use, the stock solution of ICI was diluted to the required concentration. As a diluent, an HC1-NaCl solution was prepared, the concentration of which was adjusted individually for each individual experiment to be well above the minimum concentrations of HC1 and NaCl at which ICI is known to be stable (R.W. Helmkamp, M.A. Conteras and W.F. Bale: Int. J. Appl. Radiat. Isotopes, 18 (1967) 737).

Marking procedure.

The iodination is based on Helmkamp's modification of McFairlane's iodine monochloride method (A.S. McFairlane, Nautre, 182

(1958) 53) with further modifications (J.L. Izzo, W.F. Bale, M.J. Izzo and A. Roncone, J.Biol.Chem. 239, No. 11 (1964). 3743, W.F. Bale, R.W. Helmkamp, T.P. Davis, M.J. Izzo , R.L. Gooland, M.A. Contreras, and I.L. Spar, Proc.Soc. Exp.Med. 122

(1966) 407 and R.W. Helmkamp, M.A. Contreras and W.F. Bale, Int. J. Appl. Radiate. Isotopes, 18 (1967) 737). Antibodies to reagin-Ig (100 µg) were labeled with a molar ratio of ICI to antibodies to reagin-Ig of 2 : 1 and ICI to 12^;I of 1 : 1. The following solutions were used: Solution.I: Antibodies to reagin-Ig was dissolved in 0.2 M tris-HCl buffer pH 8 to a concentration of 10.0 mg/ml. Solution II: The diluent for the stock solution of ICI was prepared by dissolving 5.84 g of NaCl in 35 ml of 2.0 HCl and 65 ml of distilled water. Solution III: The final iodine monochloride solution was prepared by diluting 100 µl of the stock solution of ICI with 20.2 ml of solution II. Solution IV: 44.29/µl of the radioiodide (4.0 mCi) was added to 25/µl of solution III and placed in an ice bath before use. All solutions are kept cold and the iodination reaction was carried out at ice temperature. The reagents were added in the following order in a small glass tube: To 200 µl borate carbonate buffer (0.4 M, pH 9.1) and 10 µl solution I (100 µg antibodies against reagin-Ig) 50 µl of solution was added IV and mixed immediately vigorously. After 60 seconds, 20 µl 0.1 M Na2S2°3' 5°/µl 2# Kl and 200/µl 5% human serum albumin blue were added

(HSA blue). HSA blue was prepared according to Melani (F. Melani, K.M. Bartelt, R. Conrads, E.F. Pfeiffer, Z.klin.Chem. 4th year 1966, booklet 4). This was added to reduce radiation damage and adsorption of antibodies against reagin-Ig to the glass walls.

125

Preparations with I-iodinated antibodies against reagm-Ig can be purified by "Sephadex" cross-linked dextran or "Sepharose" gel filtration (agarose gel) or with "Dowex" or "Amberlite" ion exchangers. Fractions from the column were stored at about 4°C in 0.1 M tris-HCl buffer pH 7.72 containing 5% HSA. The fractions containing the labeled antibodies against reagin-Ig were pooled and stored frozen.

This method gave 81,355 binding of the total amount of iodine to antibodies against reagin-Ig. The labeled immunoglobulin contained about 1 atom "1"^-I and about 1 atom <12>^-1 per molecule (molecular weight 150,000) and had a specific activity around 16 mCi/mg.

Example 3»

Labeling of antibodies specific to the Fc fragment of reagin-Ig (labeling with a fluorescent group).

For labeling with a fluorescent group, antibodies against the Fc fragment of reagin-Ig prepared according to example 1 D can be used. 2 ml of 0.9% aqueous solution of NaCl and 2 ml of 0.5 M aqueous solution of sodium carbonate-sodium hydrogencarbonate buffer of pH 9 were mixed. The antibodies were dissolved in this mixture in such an amount that a one percent solution of antibodies appeared. 3 mg of fluorescein isothiocyanate was adsorbed on 30 mg of diatomaceous earth ("Ce.lite") is added. The mixture was shaken for 3 minutes, after which it was centrifuged at 2000 rpm. minute for 5 minutes. The supernatant clear solution, which contained the labeled antibodies, was saved. In order to purify the labeled antibodies that had reacted with the fluorescein isothiocyanate from unreacted fluorescein isothiocyanate, gel filtration was performed to separate the high molecular weight labeled antibodies from low molecular weight contaminants. For this purpose, a column (length 15 cm, diameter 1 cm) was used, which was filled with gel particles of an insoluble but water-swellable gel filtration material consisting of dextran cross-linked with epichlorohydrin ("Sephadex G 25"), the gel mass being brought into equilibrium with a 0.9% water solution of NaCl. The elution took place with a 0.1% aqueous solution of NaCl.

They labeled antibodies that were eluted in a fraction before the low-molecular-weight contaminants were stored, in a frozen state. The formed, labeled antibodies had strong fluorescence in ultraviolet light and can be used for the detection and determination of reagin-Ig.

Claims (2)

1. Antibodies specifically directed against immunoglobulin E (IgE) or against fragments of IgE containing class-specific determinants, which antibodies are intended to be used in vitro by immunochemical determination methods based on reactions in which an allergen is bound to a water-insoluble polymer., IgE directed against the allergen and antibodies directed against IgE or fragments thereof are included, characterized in that the antibodies directed against IgE or fragments thereof are given the ability to emit radioactive radiation or fluorescence trawling by labeling with one or more radioactive isotopes or with a group containing a radioactive isotope or with a fluorescent group. 2. Antibodies according to claim 1, characterized in that they are labeled with a radioactive iodine isotope.