IE52269B1 - Antibodies to proteins - Google Patents

Abstract

There is described a collection of monoclonal antibodies the members of which being directed against human leucocyte interferon (HLI) which is natural or obtained by recombinant DNA technology. The monoclonal antibodies are preferably secreted by different hybridomas. It has been shown that individual monoclonal antibodies are not mutually inhibiting, i.e. bind together to HLI while others are mutually inhibiting, i.e. do not bind together to HLI. It has been shown furthermore that the collection consists of groups of antibodies, members of which recognize different epitopes (antigenic determinants) for HLI. Each a member of the different groups of antibodies can be used for a solid phase- sandwich test for HLI. In addition an antibody of the collection can be used for the purification of HLI by affinity chromatography.

Description

The present invention is concerned with antibodies against proteins, namely against human leucocyte interferon (HLI) which is natural or obtained by recombinant DNA technology.

HLI is possibly a valuable therapeutic for the treatment of neoplastic and viral illnesses. The evaluation of this substance and its possible development to a widely-used medicament is restricted by the difficulty of purifying and of characterizing the active substance in the requisite manner. A test which could be carried out in less than 24 hours would be a valuable aid for the desired purification and the requisite characterization of HLI.

In the scope of the present invention it has now been found that for the determination of HLI in an excellent manner there is suitable a pair of monoclonal antibodies which are directed against leucocyte interferon which is natural or obtained by recombinant DNA technology.

According to the present invention, there is provided a pair of monoclonal antibodies which are directed against natural or recombinant human leukocyte interferon (HLI), characterised in that: each antibody binds to a different epitope cf HLI; each antibody is of the IG isotype; o one of the antibodies has ajj heavy chain and does not recognise HLIJf^; and the other of the antibodies has a 2b heavy chain.

Preferably, the antibody with the K heavy chain is immobilised.

Conveniently, the antibody with theϊ χ heavy chain is labelled with radioactivity or an enzyme.

In a second aspect, the present invention provides 15 use of the immobilised and labelled antibodies in a solid phase sandwich test for HLI, and a solid phase sandwich test using these antibodies.

The carrying out of the solid phase-sandwich test for the determination of HLI can be effected according to methods known per se, but a substantial simplification can be met with, in contrast to the known solid phase-sandwich procedure, by incubating unlabelled and labelled antibodies from the beginning together and only once.

The preparation of the monoclonal antibodies is effected according to cell fusion technology known per se, in which the myeloma cells are fused with spleen cells of mice, which have been immunized with HLI. The hybridomas obtained secrete monoclonal antibodies against leucocyte interferon which is natural or obtained by recombinant DNA technology. 53269 It has surprisingly been shown that, according to the solid phase sandwich procedure in accordance with the present invention, human leucocyte interferon is detectable up to a concentration of 2 U/ml in serum.

The following Examples illustrate the invention, and in the following description indicates a Trade Mark.

Example 1 Manufacture of the monoclonal antibodies A) Interferon preparation Partially purified human leucocyte interferon (HLI) was available in sufficient amount for the immunization of the mice. 5 HLI preparations are used: a) HLI v(a) main peak of the fraction in accordance with step 8 of Table 3 of DOS No. 29 47 134 with an approximate purity of 10-15% (estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and specific activity in the antiviral test; b) HLI a; c) HLI β; d) HLI γ; and e) HLI δ.

The HLI α, β and τ fractions consist in each case of 15 mixtures of the individual (σ^, α2), (β^, β2, fig) and (τ , 72' ύ3' ύ4' 75^ sPecies· These are shoulder fractions of the corresponding purified species, which are pooled from different preparations (see step 9 of Table 3 of the previously mentioned DOS). The HLI δ fraction is the shoulder of the δ-species. Besides the species α, β and γ on the LichosorS-diol column (see step 8 of Table 3 of the previously mentioned DOS) there is occasionally seen a δ-fraction. This is purified as in step 9 in accordance with Table 3 of the previously mentioned DOS, whereby it is evident that the species δ shows no sub-species and is uniform. The purity of b), c) and d) is between 20 and 40% in accordance with specific biological activity and in accordance with sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

The purity of e) is at most 5%.

Determination of interferon activity The interferon activity is determined by means of the cytopathic-effect-inhibition (CPE) test in accordance with O.S. Patent Specification No. 4 241 174 (Serial No. 963,256) .

B) Immunization of the mice 3 eight weeks old Balb c/J female mice are firstly immunized with hli Y(a) in Freund's complete adjuvant.

Each mouse receives approximately 150 pg total protein containing 20-25 pg of interferon in 0.25 ml at 5 different positions (0.05 ml in each position): subcutaneously in the left and right iliac region and in the left and right axilliary region as well as an intraperitoneal injection. days later a second immunization is carried out as follows; interferon HLI v(a)· is separated by preparative sodium dodecyl sulphate-polyacrylamide (15%) gel electrophoresis in three 0.6 x 11 cm cylindrical gels. Approximately 300 yg total protein in 0.3 ml (dialyzed against assay buffer) containing approximately 40-50 yg of interferon are loaded per gel. [This purification is effected essentially according to the method of Laemmli, U.K. (1970) Nature 227, 680-685]. After the electrophoresis, the gels are sliced into 2 mm thick discs. These are immersed for 10 minutes in 0.5 ml of phosphate-buffered sodium chloride solution (0.01 M potassium phosphate buffer, pH 7.3; 0.14 M sodium chloride) containing 0.1% Triton*X-100 in propylene test tubes.

The buffer is investigated for interferon activity in serial dilution according to the CPE inhibition test.

The discs 6 and 7 (numbered from the lower end of each gel) show the highest interferon activity (in each case approximately 25% of the activity of the total gel). In each case one disc with highest activity is finely sliced with a razor blade on a glass plate, transferred into a 1 ml syringe containing 0.2 ml of 0.15 M sodium chloride and injected into each mouse. The gel suspension is injected intraperitoneally, whereupon 0.2 ml of BCG (Bacillus Calmette-Guerin; Serum Institute Berne) are injected. δ After 12 days, the serum of each mouse is investigated for interferon neutralization activity. The serum of mouse No. 3 shows a 50% neutralization of 10 units/ml of HLI r(a) at a dilution of 1:72 000, but does not neutralize the same concentration of crude HLI (< 0.1% purity) even at a dilution of 1:100. The mice No. 1 and No. 2 show neutralization titres of less than 1:1000 against HLI y(a). days after the second immunization mouse No. 3 receives on three successive days an intraperitoneal booster injection with a mixture of HLI α, β and y containing approximately 50 yg of interferon and 15 yl of normal mouse serum as the carrier protein (in 0.2 ml of 0.15 M sodium chloride). 48 hours after the last injection the mouse is killed and the spleen is removed for the preparation of the monoclonal antibodies.

C) Cell cultures and cell fusions The following materials and media are used. Iscove's modification of Dulbecco's modified Eagle medium (IMDMEM) is obtainable from Gibco. It is made up freshly with sodium pyruvate (1 mM), glutamine (1.5 mM), 2-mercaptoethanol (5 x 10 ^M), penicillin (100 units/ml) and streptomycin (100 yg/ml). Complete HAT medium consists of thus-completed IMDMEM as well as hypoxanthine (10 M), aminopterin (4 x 10~7M), thymidine (1.5 x 10_^M) and 15% foetal calf serum. A 50% (w/v) solution of polyethyleneglycol 4000 (PEG 4000, Merck) .in IMDMEM is prepared.

The fusion with a non-producing azaguanine-resistant myeloma cell line is carried out, with small modifications, according to the method of Stahli et al. in J. Immunol. Methods 32, 297-304. In this fusion 48 x 10® nucleated spleen cells are fused with 25 x 10® myeloma cells of the line FO (Fazekas de St. Groth and Scheidegger in J. Immunol. Methods /5, 1-25 (1980). The spleen cells and myeloma cells are washed in serum-free IMDMEM. They are then re-suspended in the same medium, mixed in the above-mentioned ratio for the fusion and sedimented in 40 ml of serum-free IMDMEM in a 50 ml conical polypropylene test tube at 200 x g for 15 minutes, followed by complete sucking off of the supernatant. To the cell sediment there are added dropwise with constant stirring 0.5 ml of 50% PEG 4000 in order to re-suspend and to disperse the cells. After approximately 90 seconds, 10 ml of serum-free IMDMEM are added dropwise at room temperature with constant stirring during 4-5 minutes.

After a further 15 minutes without stirring, large cell clumps are separated by cautious pipetting with a 10 ml pipette. The fusion mixture is diluted to 250 ml with 52268 ίθ complete HAT medium and then placed in 240 Costar cluster wells (1 ml/well), which already contain 1 ml of complete HAT medium and 10^ peritoneal mouse cells as the nutritive layer. The cultures are incubated in a 5% CO2/95% air atmosphere at 85% humidity. The cultures are fed twice a week by replacing half of the medium (1 ml) with fresh HAT medium.

D) Detection of interferon-specific hybridomas by a solid phase-antibody binding test (SABA) This method is adapted from the principle described by Catt and Tregear [Science 158, 1570-1572 (1967)] and is suitable for the detection of hybridoma antibodies (see also Stahli et al in J. Immunol.Methods 32, 297-304 (1980) as well as Kennet, R.H. in Current Topics in Microbiology and Immunology, Vol. 81, p. 77-91 (1978). Interferon preparations of HLI a, 0, r and 6 are individually diluted in polypropylene test tubes with phosphate-buffered sodium chloride solution (PBS) to an end concentration of approximately 0.2 to 0.5 pg inter20 feron/ml (approximately 1-3 pg total protein per ml; except for HLI δ where approximately 10-15 pg of total protein are present per ml), in order to coat therewith polyvinyl chloride microtitre plates (Cooke Laboratory Products Division, Dynatech Laboratories, Inc.) as in the case of Stahli et al. loc. cit.. 50 pi of the interferon solution is placed in each well and left for at least 4 hours at room temperature in a humid chamber or held at (sic) 4°C for several weeks. Before use the wells are filled with 3% bovine serum albumin (BSA) in PBS and left for 30 to 60 minutes in order to block all protein binding positions. The plates are then washed four times with PBS. 50 μΐ of supernatant of the hybridoma cultures are incubated in in each case two wells for at least 4 hours at room temperature.

Control experiments (unspecific binding) are carried out on plates which are coated only with 3% BSA. After four-fold washing with PBS, there is added to each well μΐ of sheep-anti-mouse Ig-antibody (purified by 125 affinity chromatography and labelled with I; 50 000 to 100 000 cpm per 20 to 30 ng of antibody in PBS containing 1% BSA) and it is incubatad at room temperature for at least 4 hours. The plates are washed four times to five times with PBS and then divided into the individual wells, which are then tested for radio20 activity in a gamma-scintillation spectrometer E) Results a) Initial screening by antibody binding (SABA) against partially purified HU fractions 152 of the 240 cultures show growth of hybridomas.

The supernatants are tested for interferon-specific antibodies in the SABA test, when the cultures are approximately 20-30% confluent (12-29 days after the fusion). Antibody binding is tested parallel with HLI a, HLI 0, HLI γ and HLI 6. Of the 152 cultures, 13 supernatants show high antibody binding against all four partially purified interferon preparations. The medium of four further hybridomas shows lower binding activity (3-4 times greater than unspecific binding) against all four interferon preparations. These cultures are discarded. The media of all other hybridomas (135 growing cultures) yield unspecific bindings against all four HLI preparations. Of the 13 specific hybridomas, 9 are characterized representative for the present invention.

Their designation is LI-1, LI-2, LI-3, LI-5, LI-6, LI-7, LI-8, LI-9 and LI-12.

As is evident from Figure 1, the antibody binding of the 9 specific hybridomas shows a different behaviour to the 4 partially purified interferon preparations.

LI-1, LI-2, LI-3, LI-5, LI-6, LI-7 and LI-8 show an identical characterization with the following binding sequence: HLI a < HLI δ < HLI γ < HLI 0. For LI-9 the binding order is HLI δ < HLI γ < HLI 0. LI-12 is different from all others. b) Antibody binding (SABA) against highly purified leukocyte interferon Six purified (a 80% pure) interferon species (see step 9 of Table 3 of DOS 29 47 134) are immobilized by adsorption on polyvinyl chloride microtitre plates. In this case there are concerned HLI a^, c^, β2» ύ2Ύ3 as well as hli-K of the cell line KG-1 [Koeffler, H.P. et al. Science 200, 1153-1154 (1978)], which is purified in analogy to the steps 1 to 9 of Table 3 of the mentioned DOS. Since no clear separation into individual species was to be observed in the steps 8 and 9, the main fraction with the interferon activity was pooled.

It accordingly represents a mixture of different species. The adsorption of interferon (50 μΐ/per depression) is carried out overnight with approximately 0.5 pg of interferon and 2 pg of BSA per ml of PBS followed by blocking off of the protein binding positions with 3% PBS as described previously.

The antibody binding with the culture medium of the 9 specific hybridomas against the 6 different, purified interferons is shown in Figure 2.. There can again., be differentiated various groups of antibodies with different characteristic binding behaviour. The group 1 embraces the antibodies LI-1 and LI-2, which have identical binding characteristics, but which do not rec52269 ognize HLI Y^. The group 2 embraces the antibodies LI-3, LI-5, LI-6, LI-7 and LI-8 with a possible differentiation of two sub-groups. Sub-group 2a (LI-3, LI-5 and LI-6) shows overall higher binding (Figure 1 and Figure 2) than sub-group 2b (LI-7 and LI-8), whereby the latter sub-group binds relatively stronger to purified ELI and HLI-κ than the first. According to the interferon binding behaviour of the remaining antibodies in accordance with Figure 2 and Figure 3, LI-9 has the most characteristics of sub-group 2b. LI-12 appears, in turn, to be unique.

Having regard to the fact of the high antibody binding to different purified interferons it is very unlikely that any of these antibodies was not directed against interferon, but against contaminants. This is indicated for most of these antibodies also by the capability of neutralizing the biological activity of interferon. LI-1, LI-2, LI-3, LI-5, LI-6, LI-7, LI-8 and LI-9 annul the inhibition of the viral CPE on MDBK cells induced by interferon. The interferon neutralization is tested with partially purified HLlY(a) and/or purified HLI-K, HLI γ^, HLI and crude leucocyte interferon. None of the antibodies was in a position to neutralize crude leucocyte interferon. LI-12 neutralized none of the tested interferons (see Table 1 for this). 2) Isotypes of the heavy and light chains of the monoclonal interferon antibodies Monoclonal antibodies of hybridoma culture medium are firstly bound to interferon-coated microtitre plates as has been described previously for the SABA test. After washing, they are incubated with isotype-specific rabbit anti-mouse Ig antiserum (Nordic). For the detection there is used goat anti-rabbit IgG, which is labelled with horseradish-peroxidase. As the enzyme substrate there are used 2,21-azino-di-(3-ethyl-benzothiazoline sulphate) and hydrogen peroxide. The results are compiled in Table 1, second column. Seven of the tested antibodies have the immunoglobulin chains Y^/k, one has Y2j3/k an8 one has μ/k. All seven γ^/k antibodies as well as also the Y2i/k neutralize interferon, whereas the μ/k does not neutralize interferon, as is likewise evident from Table 1.

The ability of the antibodies according to the present invention to bind with high affinity to interferons obtained by recombinant DNA technology is evident from 2o Table 6.

IG 3) Cloning and stabilization of the interferon-specific hybridomas For the preparation of stable hybridoma lines, all 9 specific hybridomas are cloned by limiting dilution in microtitre plates with mouse-peritoneal cells as the nutritive layer [Hengartner H. et al. in Current Topics in Microbiology and Immunology, Vol. 81, 92-99 (1978)].

The cloning was begun at the point in time of the transfer of the original cultures into flasks or shortly there10 after. The clones were tested against HLI 0 with the SABA test. The results of this first cloning of the 9 hybridomas is shown in Table 2.

From each hybridoma there are intensively cultivated two to four strongly positive clones and not only in15 jected i.p. into mice for ascites production but also frozen. In most cases the selected clones of a hybridoma are pooled for simplification and reduction of work.

From all 9 hybridomas there is obtained ascites liquor for the production of antibodies on a large scale. Ascites cells are likewise transferred into-cultures and frozen. 4) Grouping of the antibodies according to their interactions with HLI The main group of the seven hybridomas, which produce γ^/Χ immunoglobulin, can be divided into two groups on the basis of their antibody interaction with purified interferons. The first group (LI-1 and LI-2) does not recognize purified HLI y3, whereas the second group (LI-3 and LI-5 to LI-8) recognizes HLly^. The relative, quantitative binding of the monoclonal antibodies to six different purified interferons (see Figure 2) and the difference between neutralizing and non-neutralizing antibodies yields information for the differentiation of different epitopes (antigenic determinants) of HLI and for the demonstration of structural differences between different purified interferons. From the binding behaviour of the antibodies (see Figure 2) ±t is clear that LI-1 and LI-2 recognize a different structure than all other antibodies and that HLI is structurally different from all other tested interferons, since it does not exhibit the epitope recognized by LI-1 and LI-2. LI-12, a non-neutralizing antibody, recognizes with approximately the same, although perhaps lower affinity, a less variable determinant which is present in all tested interferons. The results in respect of the antibody binding and of the interferon neutralization indicate that at least three different epitopes of HLI can be recognized and defined by the collection of the monoclonal antibodies.

Exanple 2 Determination of Interferon 1) Selection of a suitable pair of monoclonal antibodies For the differentiation between conpetitive binding of two antibodies to the same epitope and additive binding to various epitopes, the binding of the antibodies is tested alone and in pairs and in all possible combinations to immobilized interferon. Supernatants of confluent hybridoma cultures are used without further purification as the antibody source. Partially purified leucocyte interferon (20-30% pure HLi β ) is added in defined amounts (0.1 to 0.2 pg interferon/ml) to polyvinyl chloride ndcrotitre plates as is described in Exanple 1 for the SABA. The blocking off of all protein binding positions with 3% BSA follows. 50 yl of antibody solution consisting of in each case 25 yl of two culture supernatants or 2 x 25 yl of the same antibody are Incubated at room tenperature for 7 hours, The plates are washed with FES (0.01 M potassium phosphate, pH 7.3; 0.14 M sodium 125 chloride) and incubated for 5 hours with an excess of I ' labelled sheep-anti-mouse-Ig iimunoglobulin in a volume of 50 yl per well In order to measure the amount of monoclonal antibodies which are bound to interferon (Table 3). The binding of two antibodies is considered to be additive when the binding of these antibodies exceeds that with 50 yl of the higher binding individual antibody. On the basis of this criterion it was found that LI-1 binds additively with almost all other antibodies and that LI-9 gives the best additive binding with LI-1.

LI-1 and LI-9 from ascites liquor are purified according to standard methods, i.e. by ammonium sulphate precipitation (50% saturation) and DEAE-cellulose chromatography, up to at least 90%, as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. 20 125 pg of each antibody are labelled with I according to the chloramine-T method to a specific activity of approximately 20 pCi/pg. Free iodine is separated from labelled antibody by gel filtration on Sephadex*G-25 in the presence of PBS and 1% BSA.

Unlabelled antibodies LI-1 and LI-9 to approximately 20 pg per ml of PBS are adsorbed overnight on PVC microtitre plates (Cooke Laboratory, Products Division, Dynatech Laboratories, Inc.) (100 pl/depression). During 30 to 60 minutes all protein binding positions are blocked with 3% BSA in PBS. Purified leucocyte interferon of the KG-1 myeloid cell line at a concentration of 10 units/100 pi of PBS and 1% BSA are incubated together with 125 5 one of the I labelled antibodies (1.3 x 10 cpm) in antibody-coated wells. Control wells contain labelled antibody, but no interferon. After incubation for 3 hours at room temperature, the liquor is removed and the wells of the plates are washed four times with PBS and then tested for radioactivity in a gamma-scintillation spectrometer. The results are shown in Table 4. The combination of LI-1 with LI-9 gives interferon52269 -dependent sandwich-like antibody binding. The better combination is immobilized LI-9 with labelled LI-1 in solution. For this reason the further experiments have been carried out with immobilized LI-9 and labelled LI-1. a) One-step incubation procedure Each well of a PVC microtitre plate is coated with 100 μΐ of LI-9 (15 μg/ml in PBS) at room temperature overnight or at 4°C for days or weeks in a humid chamber. Before use the solution of LI-9 is removed and the wells are filled with 3% BSA for 30 to 60 minutes and then washed four times with PBS. Approximately 140 125 000 cpm of I labelled LI-1 (approximately 4 ng, specific activity approximately 20 μCi/μg) in 0.1 ml of PBS containing 1% BSA and 100 to 150 pg of human IgG per ml are added to each 'well. Correspondingly diluted interferon solutions (1-20 μΐ) are added to the well and mixed by brief shaking. After 2 to 3 hour incubation at room temperature, the liquor is poured on to absorption paper. The plate is washed four times to five times with PBS and the individual wells are tested for radioactivity in a gamma-scintillation spectrometer. In the case of high interferon concentrations (> ca. 5000-6000 ϋ/ml) a partial inhibition occurs The results obtained are reproduced in Figure 3 and Table 5. b) Two-step incubation procedure LI-9 coated and BSA-saturated PVC microtitre plates are prepared as described previously. 50 μΐ per well of “ interferon solution in PBS containing 18 BSA are incubated at room temperature for 30 minutes, whereupon the plates are washed four times with PBS. 125 Thereupon, 50 μΐ of I LI-1 (approximately 150 000 cpm/ 4 ng) PBS containing 1% BSA and 150 μg of human IgG per ml are added to each well, whereupon the plates are held at room temperature for 2 hours and then washed four times with PBS and tested for radioactivity as mentioned above. The results obtained are reproduced in Figure 4. In contrast to the one-step procedure no partial inhibition at high interferon concentration is seen. c) Eetasnination of antiviral inactivated inbarfeim Heat Inactivaticn of Intsteran A saluticn of Irtefercn A (Gydfel et al. teture Uol. 290, Itoh 1981) in File's minirtBl essential medium aontainirg W fetal bovine saun was diluted 1:480 in jhogiiatefcuffered saline (0.14 M Nsd, 8 πΜ tte/ΐΌ^, 1.5 nMΚΗΙΌ,, 3 nM id, 0.9 πΜ t«l 0.5 πΜ ί«12) attaining 1 mg/inl of bovine serun altunin. The diluted solution aontainad Interferon A at a oaooantraticn or 5 rg/M.. After heating at 65°C fcr the tutes noted, sanples of 0.14 ml were claoed m an icE bath until assayed. Both antiviral and irniuoological were perfemsd cn the sane tfev. Fran each 0.14-ml saipLe, 0.10 ml was used for the radio52269 9. Ρ. jimunassEy ad 10 yl far antiviral ass^ in duplicate. The radio innus assy is psrfcniEd acmrdirg to the twa-st^j inabatarn pxcedro nentjcred «da: b). The dstamiraticn of the antiviral· activity is paiczned acmrdiig to the CPE-Shihiticn fast. The resits ate givei in figure 6. Eton this exatple ere cai gather that the antibody pair 11-141-9 can distirgui^i tetaeai antiviral· active ad antiviral inactive infcerfercn. firm figjre 6 thans is furthamze evitfett that the antiviral ad the irnitne activity decrease similaisously. d) Highly sensitive xnuerferon determination in human plasma or serum (radioimmune procedure) A single incubation of 0.1 ml of 40% human plasma or serum (neutral pH) containing approximately 250 000 cpm l (1.5 ng) of I LI-1 (specific activity 100 to 110 yCi/yg LI-1) is carried out at room temperature for 14 to 16 hours in PVC microtitre plates, which are coated with LI-9 as described previously. After four-fold washing with PBS, the wells are numbered as mentioned previously.

One experiment is carried out with interferon, which is added to citrate plasma of a healthy volunteer. Each 0.1 ml of PBS test mixture contains 40 yl of plasma, 1.3 ng of 20 I LI-1 (256 000 cpm) and different amounts of crude human leucocyte interferon (approximately 0.1% pure) from chronic myologous leukaemia cells. The results are reproduced in Figure 5, where the amount of interferon is plotted as the abscissa. The-incubation medium in accordance with step 1 of Table 3 of DOS No. 29 47 134 is used as the crude leucocyte interferon. π '1 ., ο Example 3 Highly sensitive determination of interferon in serum of patients (enzyme immune procedure) Into the corresponding number of test tubes (10 x 75 mm) there are in each case pipetted 0.05 ml of test solution (0.1 mol/1 of sodium phosphate, pH 6.5 with 10 g/l of BSA and 0.5 yg/ml of monoclonal LI-1 anti-interferon-peroxidase conjugate in interferon-free human serum) 0.2 ml of the patient:'s plasma to be analyzed or of the interferon standard (0 U/ml, 12.5 U/ml, 25 U/ml and 50 U/ml of interferon) and of the interferon control serum (e.g. 30 U/ml of interferon) is admixed, in each case there is added a polystyrene bead (0 = 6.5 mm) coated with monoclonal LI-9 anti-interferon and incubated at room temperature (18-2O°C) for 16 hours. Subsequently, the polystyrene bead are washed three times with in each case 2 to 5 ml of distilled water, transferred into in each case 0.5 ml of substrate buffer for the determination of the activity of the peroxidase (0.1 mol/1 of potassium citrate buffer of pH 5.0 with 6 mmol/1 of HjOj ^0 1 of o-phenylenediamine) and incubated at room temperature (18-26°C) for 30 minutes. In order to stop the peroxidative activity as well as to intensify the light absorption, 0.5 ml of 4 N HC1 are admixed and within 30 minutes the extinction is measured photometrically at the wavelength. 492 nm. In the Table there are presented the values of an interferon determination and compared with the values which have been obtained with the interferon standard. Crude leucocyte interferon (incubation medium in accordance with step 1 of Table 3 of DOS 29 47 134) is used as the interferon standard.

Table Sample materialΔΕ492 nm/RT/30 min. Interferon standard 0 U/ml interferon 0.065 / 0.070 12.5 U/ml 0.245 / 0.235 25 O/ml 0.425 / 0.430 50 U/ml 0.775 0.790 Control serum (30 U/ml interferon) 0.505 / 0.490 Patient's plasma No 8327 0 U/ml interferon No 8328 0 U/ml interferon No 8336 9 O/ml interferon No 8338 0 U/ml interferon No 8339 5 U/ml interferon No 8363 0 U/ml interferon No 8341 2 U/ml interferon No 8344 16 U/ml interferon 5286® Table 1 Epitope Monoclonal antibody Isotypes Chains Neutralization LI-1 > γχΑ Yes I LI-2 Y-jA Yes LI-3 y^/k Yes LI-5 y±/k Yes LI-6 ύ-,Α Yes IX LI-7 :gG γχΑ Yes LI-8 Υ-,Α Yes LI-9Y2b/k Yes III ί LI-12 IgM μ A No 53369 Table 2 Culture Cells per 96 wells” Tested clones or wells Number positive Number negative LI-1 100 6 6 0 LI-2 loo 11 6 5 LI-3 100 7 7 0 LI-5 100 13 13 0 LI-6 100 12 12 0 LI-7 100 10 8 2 LI-8 100 9 8 1 LI-9 100 10 8 2 LI-12 100 4 0 4 LI-12 300 48 6 42 U1 LI-12 r w 1 \D r* H 1 03 j LI-7 1 LI-6 ] t- M 1 tn r< Μ 1 GJ LI-2 LI-1 “I f r tn w co 1 o h- o Η l·-. r f-* Ok H CO tO 1 co co to ο o Η Η Η* Γ W tn -4 K ςη Η σ» 1 Ο tO to u> ο ο o κ )-» 5—* r ω GJ ςη •4 H σι A sj 4 J Ok Ο». tn GJ cn ο ο· ο o κ W Η* K r* -J Η ο Ok tn K gj cn GJ tn to 1 o to tO *4 tn σι o ο ο ο o K H Η* Η Η H* f ft) o ω Η σι 4 M o to ιο tO Ok σι 1 to tO w ο 0» *4 4 o O ο ο ο o W 1-^ W , r-1 C1 O· GJ o Ok Ιο cn •4 y-, 4 Ok CO •4 to !_» tO 1 o CO O ft) •4 tn «4 co o o o ο ο ο o Η· f—' r· H-* ο cn e> h-1 Ok o CO σ» Η •4 ' to VI 1 tO f4 c. G*> co tn cn to o *4 to λ. ίθ ο ο O o o o o ο M w H' W 1—' κ !—· c- tO c -4 O* tO Ct G1 G1 co Ηί \O co c\ c> t4 >4 Ο. Ok fO 1 •. -j co ·. ►—* Ο Γ\ Ct b-c /-·> ο Ο o • 4 σι SO an > 3 Ρ Ρ Π* Η rt Η Η Η Η ιΗ· ! 1 σ ο. σ ΙΟ Η 0 0 »< *r Ρ η Φ fi) Η 3 to rt σι ex < Ω 0 3 σ Η· 3 Ρ rt Η· 0 Ρ Ρ Η· Η 3 Η Η σ 1 1 0 Η Η κο α & < cr φ Η W Φ Cb S Η· cr 125 Ν) ~ΪΓ Η £* ΙΟ Η Ο to interferon wi O' U) inte W σι ίΟ abelled antibo to λ κ σ α σι lU Hi y < Η ο Φ 0 H C 0 rt 5 cr ω Η-Ό binding (c Μ 3 Φ •σ tO Λ» α ο s ο co Η· Η· CD tO 5 Ηι (£ ΗΩ Table Table 5 Interferon species Interferon units per determination CPM[125I]LI-1 bindingal 100 2,202 °2 100 1,63301 100 82202 100 8,13503 100 70Y1 100 7,036Y2 100 4,804Y3 100 101Y4 100 198Y5 100 121 δ 100 5,498 Table 6 Antibody Interferon LI-1 LI-3 LI-5 LI-6 LI-7 LI-8 LI-9 A + + + + + + + 5 B + - - - - - - D - + + + + + F - - - - - Legend: The interferons designated as A-F are those obtained by the 10 DNA recombinant technology and described in the publication of David V. Goeddel et al. in Nature Vol. 290, March 1981.

A + designates that a given interferon was completely absorbed, a + partially absorbed and a - not absorbed by the columns loaded with the respective antibody. 53269 φ 3 Ρ Ο b3 0 Ρ φ sr 0 Φ 3 φ 0 0 rt (η ρ ft ρ (X Ρ 0 TJ Ρ 0 3 Φ □ Τ5 Φ Ω Ό SX Οι Ρ ft 0 η Φ Ρι Ρ 0 φ Φ Ω ft W Ρ Ρ Ρ» Φ 5* ω Ρ Ω 0 φ rr rt Ρ Ρ Π & • < 3 Φ Ρ 1 in Ρ Η tO 3 3 Ω Φ Φ φ 0 Ρ 0 CQ Ρ tn η C Φ 0 £ Ρ π Φ 0 Ο> rt 3 Φ Φ Ρ· 3 ρ rt Φ Ω 3 Η» Φ Ρ 0 ft 3 Ό Φ ε Φ Ό Ρ Ρ 0. Φ 0) rt ΓΓ sr Φ Ρ 3* Ω ω Φ W Ω rt Ρ 0 Οι 0 Ρ 0 Ρ’ Οι Cr Β C ί Ρ 3 Φ 3 (0 Ό 0 Φ ο. 0 rt ρ Οι Οι 0 0 W φ ο 0 Ω rt rt Ρ 3* ρ Ό Η Φ 0 φ rt ρ· Μ η Ρ 0 C Ρ 3 Ρ Η η Τ Ρ tr rt Φ 3 0 Φ ft tr 3 Ό Φ ρ 0 1 σ ιθ Ρ ΤΙ ρ Φ ω 0 rt 3 Η P‘ Φ 0 0 0 < Φ 3 C Φ Ω ω Ρ η Τ » Ρ W Η 0 ο Φ Ω 0*> 3 Φ Ρ* Οι Οι C C φ Ρ ρ φ (0 φ Ρ φ σι Ω ? in to ι ι Antibody column pool Ammonium sulphate precipitation Step < *4 ~ 0 to ΙΟ ο 3 Η ο ο ο Ρ 3 ►3 0 10 ft »4 φ —- Ρ Ρ 3 to ω ο ώ V ο ο ο ** Ρ rr φ ρ 3 φ *4 ο — 3 1-9 ρ η ο X X 3 ft ft Ρ Ρ Φ Ρ* Ρ * ΓΤ < Η ο ο W Ρ νρ <45 Ρ —' rt ο W to to —»Φ co β Ω Τ3 X 10 X 3 rt Φ Ρ Ρ Ω Ρ X Ρ <+ <ρ κ ο ο φ ρ ρ 00 Ρ σι rr ρ ο Β>< 0 co (0 Ι-η >0 Ρ Η Φ C σι Ρ» Ω Ρ ο σι Η ft Ρ ο ο ο fi or Ω Φ ft Ρ 0 *$ Ρ Ρ © \£ ο ύθ Φ Η in C Ρ Οι

Claims (6)

1. A pair of monoclonal antibodies which are directed against natural or recombinant human leukocyte interferon (HLI), and in which: 5 each antibody binds to a different epitope of HLI each antibody is of IgG isotype; one of the antibodies has a if heavy chain and does not recognise HLI$,; and the other of the antibodies has a^jb heav y chain 10 2. The pair of monoclonal antibodies of claim 1, wherein the antibody with the 6 heavy chain is labelled and the antibody with the £ 2b heavy chain is immobilised. 3. The pair of monoclonal antibodies of claim 2, 15 wherein the antibody with the 2S j heavy chain is labelled with radioactivity or an enzyme. 4. A solid phase sandwich test for-HLI which uses the pair of monoclonal antibodies of claim 2 or claim 3. 20 5. The solid phase sandwich test of claim 4 in which the pair of antibodies are incubated from the beginning together and only once. 6. A solid phase sandwich test for HLI according to claim 4, substantially as hereinbefore described in Exanples 2 and 3 F.R. KELLY & CO., AGENTS FOR THE APPLICANTS. F. HOFFMANN-LA ROCHE & CO AKTIENGESELLSCHAFT sheet 1 LI-1 LI-2 LI-3 LI-5 LI-6 LI-7 LI-8 LI-9 LI-12 1 = IFL a

2. - IFL β

3. = IFL Y

4. = IFL δ cpm - specific binding per test (after deduction of 300-400 cpm of non-specific binding against BSA; LI-12 non-specific binding about 1200 cpm) Figure 1 F. R. Kelly & Co. F. HOFFMANN-LA ROCHE S CO AKTIENGESELLSCHAFT sheet 2 IFL a. 4 = IFL γ. 2 = IFL a.

5. = IFL Y3 = IFL β.

6. = IFL K cpm (see Figure 1) Figure 2 F. R. Kelly & Co. F. HOFFMANN-LA ROCHE & CO AKTIENGESELLSCHAFT sheet 3 125 cpm: I-LI-1 bound per test; interferon concentration (abscissa) is plotted logarithmic for the main curve and plotted linear for the curve inserted on the loft. Figure 3 F. R. Kelly & Co F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT sheet 4 125 cpm: I—LI—1 bound per test; interferon concentration (abscissa) is plotted logarithmic for the main curve and plotted linear for the curve inserted on the left. Figure 4 F. R. Kelly & Co. F. HOFFMANN-LA ROCHE & CO. 52269 AKTIENGESELLSCHAFT sheet 5 CPM cpm: see Figure 3, inserted curve figure 5 F. R. Kelly & Co. F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT sheet 6 percent of control Figure 6 j legend· - O = radio immuno assay 0 = CPE inhibition test F. R. Kelly & Cn.