METHOD TO DETECT TUMOR NECROSIS FACTOR (TNF) AS BIOACTIVE FORM

Abstract: Disclosed is a method to detect oligomeric, biologically active TNF, which method uses reactants only capable of reacting with TNF as oligomeric form, and incapable of reacting with TNF as complexed form onto receptor, or with TNF as monomeric form. A kit for carrying out the method is disclosed as well.

Description: πcThOD Tu DC I EL I I UΠOK cuROaia r«. ιuκ t IMr _ Hi BIOACTIVE FORM" The present invention relates to a method to determine Tumor Necrosis Factor (TNF) as b oact ve form. More part cularly, the present invention relates to a method to detect oligomeric, bioactive T F, also in the presence of monomeric TNF, or T F as complexed form with receptors, both biologically inactive. Alpha-TNF, also designated "cachectin", is a cytok ne mainly secreted by activated macrophages. TNF was initially identified in serum of mice infected with "Calmette Guerin" baci llus (BCG), owing to its capab lity of inducing the haemorrhag c necros s of some tumors in experimental animal models. At a later t e, it was discovered that TNF is among the main inflammation mediators. In particular circumstances of acute and/or chronic product on, TNF can cause patholog c conditions, such as septic shock and lipid dismetabolis (cachex a), typical of patients affected by infective chronic and neoplastic pathologies. It was furthermore verified that TNF plays a role, together with other cytokines and lymphokiπes, in immunoregulation, angiogenesis, cellular differentiation and growth processes (Beutler B. and Cerami A., 1939, Ann. Rev. Immunol. 7, 025-655). The marked cytotoxicity of T F for some tumoral cell lines and its capabi lity of inducing nechrosis of some tumors in_yjvo suggest potential pharmacological applications in ant -neoplastic therapies. Furthermore, the development of compounds capable of neutralizing the toxic effects induced by endogenic TNF under hypei— production conditions, such as, e.g., is septic shock, are equally well potentiaIly -useable for therapeutical purposes. TNF is thought to perfom its pleiotropic effects through receptors present on the membrane of target cells. Actually, two different receptor types have been purified and characterized (Smith CA. et al., 1990, Science 248, 1019-1023; Schall T.J. et al., 1990, Cell 61., 361-370). Furthermore, soluble forms of such molecules, corresponding to their extra-cellular portions, displaying powerful TNF inhibitory activity, were purified from urines of normal subjects and, in larger amount, from urines of feverish patients affected by chronic idney failure, or from serum of patients affected by neoplastic diseases (Nophar Y. et al., 1990 EHB0 J. 9, 3269-3278). A large number of experimental evidences suggest that the man festation of its various biological activities by TNF, at cellular or tissuta Level, is regulated by the modulation of the expression of membrane receptors and release of soluble forms. Besides the different molecular forms of receptors, also the existence was described of different molecular forms of TNF. TridimensioπaI structure analysis showed that TNF is a molecule composed by three identical sub-units of 17.5 KD, strictly associated with one another. Studies of analytical ultracentrifugation, gel electrophoresis, cross-l n ng, X ray dispersion and gel filtration showed that T F is in oligomeric form also in solution (Wingfield P, Pain R.K. and Craig S., 1987, FEBS Lett. 211, 179-184; Arakawa T. and Yphatis D.A., 1987, J. Biol. Chem. 262, 7484-7435; Lam K.S., Scuderi P. and Salomon S.E., 1988, J. Biol. Response Hod. 7, 267-275; Lew t-Benteley A. et al., 1988, J. Mol. Biol. 199, 389- 392). The oligomeric structure, i.e., the bioactive form, seems however to be unstable at bioactive Cpicomolar) concentrations, th monomeric TNF being partially formed, which is character zed by poor or null capab lity of b nding the receptor and hence of performing b ological act vity, n terms of cytotoxicity. It was consequently hypothesized that changes in quaternary TNF structure, consequent to changes in concentration, might perform an important role in the regulation of physio-pathological processes induced by such a molecule (Smith R.A. and Baglioni . , 1987, J. Biol. Chem. 262, 6951-6954). In some pathologic situations, such as septic shock ( 'aage et al., 1987, Lancet j, 355), rejection of renal transplantations (Haury et aI., 1987, J. Exp. Med. iόό, 1132), parasitic infections ( Scuderi et al., 1986, Lancet ϋ, 1364) and various neoplasiae (Balkwill et al., 1987, Lancet ϋ, 1229), the plasmatic Levels of TNF display a considerable ncrease. The development of fast and s mple analytical methods for iQ_vi , tro measuring T F levels from biological fluids is hence of considerable importance from clinic and diagnostic view poiπts. TNF leveLs in biological fluids under normal and pathologic conditions are commonly determined by means of traditional immunologic determinations or biological assays. The low corre la ion - leveI between the results obtained from both assay types on pathologic sera suggests the existence of bioinactive antigenic forms CBalkwiLl et al., 1937, Lancet i.i, 1229), probably constituted by monomeric TNF, or solubLe-receptor- complexed TNF. The level of TNF as bioactive form is usually determined in biological fluids by means of bioassays based on the cytotoxic effect displayed by TNF on sensible celLs te.g,> L929 cells (Kramer et al., 1986, J. Immunol. Heth. 93, 210) and WEHI 164 cells (Espevik et al., 1986, J. Immunol. Heth. 95, 99)3. The presently used i munoassays make it possible total antigenic TNF to be determined -- which is potentially constituted by free oligomeric TNF, which is bioactive, and monomeric TNF, or soLuble-receptor- GompLexed THF, both biologically inactive. It was demonstrated that under pathologic conditions, TNF antigen can reach naπo oLar Levels CBalkwiLl et al., 1987, Lancet i.i., 1229). The biological determination of TNF on sensible cells shows a series of problems connected with execution d fficulties: such tests require accurate specificity checks, special equipment for cellular cultures, keeping cellular lines in culture for Long time periods, and a specialistic experience in cell cultivation, all the above not always being avai lable. Furthermore, execution of assay normally requires from 3 to 4 days; it is decidedly Long and laborious and does not make it possible the analysis of a large number of samples to be carried out. As a consequence, such a methodology is decidedly inadequate for routine analyses in clinic laboratories. Furthermore, the execution of the biological assay requires that the sample is di luted in a suitable medium for cellular growth, normally containing, serum, or other factors produced by the same cells and therefore uncontrollable, which may alter the actual concentration of T F as bioactive form in the analysed sample . Therefore, the need results evident for having available a method to in_y_i__.ro detect TNF, which discriminates the bioactive form (i.e., oligomeric, not i nhi bi tor-comp Lexed, TNF) from the bioinactive form (monomeric TNF and/or so lub le- receptor-co p Lexed TNF), easy to be used also by non-specialized staff, and which does not require cell cultivations. Therefore, an Object of the present invention is a method to detect TNF as bioactive form comprising the steps of: - immobi lizing on a substrate, a capturing reactant capable of only react ng with TNF molecules as oligomeric form, but not with TNF as complexed form with the receptor or TNF as monomer c form; - incubating said capturing reactant with a sample; - incubating said sample with a detector reactant; o. - wasmng; - performing the measurement, by means of a reaction of said detector reactant. Preferably, said detector reactant is capable of only reacting with molecules of TNF in oligomeric form, but not with TNF as complexed form with a receptor or TNF as monomeric form conjugated with a detection system; said capturing reactant has an affinity constant for oligomeric TNF higher than 10 9 H - x and an affinity constant for receptor-complexed TNF Lower than 105 H- x . According to a preferred form of practical embodiment, said capturing reactant and said detector reactant comprise the same reactant molecule and oligomeric TNF contains at least two binding sites for said reactant molecule. Still according to the invention, said capturing reactant and said detector reactant comprise polycloπal or monoclonal anti-TNF antibodies, which preferably are πeutralizers of TNF/receptor bond, still more preferably, monoclonal Hab-78 antibody (GB patent application No. 9028123.1; Barbanti P. et al. "2nd ι nt . Congress on Trauma, Shock and Sepsis: Hechanisms and Therapeutic Approaches", 1991, Munich). According to an alternative form of practical embodiment, said capturing reactant and said detector reactant comprise TNF receptors or synthetic molecules or recombinaπt o lecules. According to preferred forms of practical embodiment, said substrate comprises titratioπ microslabs or polystyrene tubes or poly-Cvinyl chloride) tubes, and said detection system comprises enzymatic molecules of fluorescent compounds or chemoLuminescent compounds or rad oactive compounds or colloidal metals, or coloured latex particles. Said enzymatic molecules are preferably comprised within the group consisting of pero idase, alkaline phosphatase, urease, beta-galactosidase and said colloidal metals are comprised within the group consisting of gold, si lver, copper, platinum, copper hydroxide, aluminum hydroxide, vanadium oxide, manganese hydroxide. Sti ll according to the present invention, said measurement is carried out by causing said detector reactant to react with chromogenic or fluorogenic substrates, and reading the signal. A further Object of the present invention is a kit to detect, from a sample, TNF as bioactive form, which kit comprises: - a capturing reactant immobi lized on a substrate, capable of only reacting with TNF molecules in oligomer c form, but not with receptor-complexed TNF or TNF n monomeric form; - solutions to incubate said sample; - a detector reactant capable of only reacting with TNF molecules in oligomeric form, but not with receptor- complexed TNF or TNF in monomeric form, conjugated with a detection system; - a wash solution; - a reference sample. Preferably, said capturing reactant and said detector reactant comprise Hab-7δ antibody, and said kit also comprises a detection system for total TNF. The present invention is disclosed now by means of application examples thereof, in which reference is made to Figure 1, which shows the assay calibration line. Examβj.e__ E_____E__.____ . _- . i2__._2i_£._!.___soild__hase_for_carr__πg_out_ he assay__for_TNF_in_o_igome£ic_form The monoclonal antibody, endowed with TNF neutralizing activity, was immobilized on poly-Cvinyl chloride) slabs, as follows: to each well of 9ό-well slabs, 50 pi of 50 mπ sodium phosphate buffer pH 7.3, containing 0.15 H sodium chloride (PBS) and 10 j _.g/ml of monoclonal Mab-73 antibody (GB patent application No. 9028123.1; Barbanti P. et al. "2 nd Int. Congress on Trauma, Shock and Sepsis: Mechanisms and Therapeutic Approaches", 1991, Munich) were added. The slabs were then incubated at room temperature- (r.t.) for 2 hours and were then blocked with a 3% calf serum albumine solution, for 2 hours at r.t.. The slabs prepared in that way can be stored some weeks at 4°C. E x a π]2_ e_2 Eι___-2__.j_≤£i2D_2_f __■!_= i 2 12 ______ -Si UO____ 2i._2u r-_.iD2_.2i_i-_.ili_: 3§sa^_ f o r_ TNF _in_o_igome ri c_ f o rm MonocLonal Mab-t8 antibody was biotinylated as follows: a solution containing 1 mg of Mab-7δ/ml, 600 ul, was mixed with ό j-,1 of N-hydroxysuccinimidic ester of D-biotinyl-ό-ami no-caproi c acid, 10 mg/ml in dimethyl-suIfoxide, and was incubated for a 4-hour time . at r.t.. Then 60 ill of a 1 M lysine solution was added. The mixture was further incubated for 1 hout at r.t., and was then dialyzed overnight against P3S. The product was di luted to 3 ml with PBS, and was stored at -20 < =C. Examg_e_3 De_ermination_of_TNF_in_o_igomeric_form The slabs sensibi lized with Mab-8 antibody as disclosed in Example 1, were washed by fi lling the wells with PBS, and. emptying them (3 times) . The wells were then fi lled wi h the samples to be analysed, suitably - di luted with PBS containing 1% BSA (PBS-B). The slab was incubated again overnight at 4° C and was washed with PBS containing 0.05% Tween 20 (PBS-T). At the end, to each well 50 jλl of biotinylated Nab-Neu, diluted in the ratio of 1 :200 with PBS-B containing 0.05% Tween 20 (PBS-BT) was added. After a 2-hour incubation at 37°C, the slabs were washed with PBS-T, were f lled with 50 LCI /well of a solution of streptavidine-peroxidase, ava lable from the market (Sigma Chemical Co.), diluted in the ratio of 1 :1000 with PBS-BT, and were then incubated again for 1 hour at 37°C. After being washed (8 times) with PBS-T, the slab was stained with a chromogenic solution containing ABTS C2,2 ' -azi no-di - (3-ethy l-benzy Lthi azo li ne sulfon- ate)_l, avai lable from the market (from Kirkegaard and Perry Laborator es Inc.). In Figure 1, the assay calibration Line is reported, which was obtained with recombinant human TNF purchased from Esquire Chemie AG (Zurich, Switzerland). cx_mg_e__ __:Si_t£_θiQ__Ii20_2J__ES£iii£_£_2._._____.__3_£__2£ 2U92!D__I_i2_lE.£ Oligomeric and monomeric TNF were prepared by dissociation of recombinant DNF with 0.1% Triton X-100, as described in Literature CSmith R.A., Baglioni C. (1987), J. Biol. Chem. 262, 0951-09543 and were separated by HPLC gel-filtrat on on TSK3000SW chromatograpπi c column (LKB-Pharmacia) (flow rate: 0.5 ml/minute). The chromatographi c fractions corresponding to monomeric TNF resulted to be totally inactive at the assay for oligomeric TNF. The presence of monomeric TNF in such fractions was checked by means of a conventional assay performed with polyclonal ant-TNF antibodies as described in Example 6. Exam£_e_5 E£§El£2ii2__2________.2.Li___E{_.__.I£_f2£________£__}__i21 Polyclonal aπti-TNF Hab-78 antibody was prepared by immunization of Balb/c mice and was purified according to known methods from literature (Bring an et al., 1987, Hybridoma 5, 489). Purified immunoglobul ns were immobilized on poly-Cvinyl chloride) slabs, as follows: to each well of 9ό-welL slabs, 50 wl of 50 mπ sodium phosphate buffer pH 7.3, containing 0.15 M sodium chloride (PBS) and 10 jug/ml of immuπog lobuli ns were added. The slabs were then incubated 2 hours at r.t. and were then blocked with a 3% calf serum albumine soLut on, for 2 hours at r.t.. The slabs prepared in that way can be stored some weeks at 4° C. I I . cx amg_e_o D_termjn__joπ_of_tota__anti9eni__TNF The slabs sensibi lized with mouse anti-TNF immuno- globulins as described in Example 5, were washed by filling the wells with PBS, and emptying them (3 times). The wells were then fi lled with the samples to be analysed, suitably d luted with PBS containing 1% B3A (PBS-B) . The slab was incubated again, overnight, at 4°C, and was washed ith PBS conta n ng 0.05% Tween 20 (PBS-T) . At the end, to each well 50 ul was added of IP 300 rabb t anti-TNF antiserum (Genzyme Corporation) di luted 1 :2000 with PBS-B containing 0.05% Tween 20 (PBS-BT). After 2 hours of incubation at 37°C, the slabs were washed with PBS-T, were fi lled with 50 ul/well, of a solution, available from the market (Sigma Chemical Co.), of goat anti-rabbit- mmunog Lobu I i n globulins conjugated with peroxidase, di luted 1 : 1000 with PBS-BT, and were incubated again, for 1 hour at 37° C. After being washed (8 times) ith PBS-T, the slab was stained with a chromogenic solution containing ABTS C2,2 ! -az i no-di - (3-e t hy l-benzy I h i azo l- ine sulfonate)},- avai lable from the market (from Kirke- gaard and Perry Laboratories Inc.). The present inven ion has been disclosed with specific reference to some of its preferred forms of practical embodi ent, but t must be understood that variations and/or modifications aiming at reducing the necessary steps for the assay, and at increasing the sensibi lity thereof may be supplied by those ski lled n the art without thereby departing from the scope of protection of the same invention.

Claims: C_JL_a_i_m_s 1. Method to detect TNF as b oactive form comprising the steps of: - immobilizing, on a substrate, a capturing reactant capable of only reacting, ith TNF molecules as oligomeric form, but not with TNF as complexed form with the receptor or TNF as monomeric form; - ncubating said capturing reactant with a sample; - incubating said sample with a detector reactant; - wash ng; - performing the measurement, by means of a react on of said detector reactant. 2. Method to detect TNF as bioacti e form according to claim 1, characterized in that said detector reactant is capable of only reacting with molecules of TNF in oligomeric form, but not with receptor-complexed TNF or TNF in monomeric form conjugated with a detection system. 3. Method to detect TNF as bioactive form accord ng to any of preceding claims, characterized n that said capturing reactant has an affinity constant for oligomeric TNF higher than 10 9 M - l and an affinity constant for receptor-complexed TNF lower than 10 5 M- * . 4. Method to detect TNF as bioactive form accord ng to any of preceding claims, characterized n that said capturing reactant and said detector reactant comprise the same reactant molecule and oligomeric TNF contains at least two binding sites for said reactant molecule. 5. Method to detect TNF as bioactive form according to any of preceding clai s, characterized in that said capturing reactant and said detector reactant comprise polyclonal or monoclonal anti-TNF antibodies, ό. Method to detect TNF as bioactive form according to claim 5, character zed in that said ant - TNF antibodies are neutralizers of TNF/receptor bond. 7. Method to detect TNF as bioactive form according to claim ό, characterized in that said anti- TNF πeutralizer antibody is Mab-78 antibody. 8. Method to detect TNF as bioacti e form according to any of claims from 1 to 4, characterized in that said capturing reactant and sa d detector reactant comprise TNF receptors or synthetic molecules or recombinant molecules. 9. Method to detect TNF as bioactive form according to any of preceding claims, character zed in that said substrate comprises titration microslabs or polystyrene tubes or poly-Cvinyl chloride) tubes and said detection system comprises enzymatic molecules of fluorescent compounds or chemoluminescent compounds or radioactive compounds or colloidal metals, or coloured latex parti cles . 10. Method to detect TNF as bioactive form according to claim 9, characterized in that said enzymatic molecules are comprised within the group consisting of peroxidase, alkaline phosphatase, urease, beta-gaLactosidase and said colloidal metals are comprised within the group consisting of gold, silver, copper, platinum, copper hydroxide, aluminum hydroxide, vanadium oxide, manganese hydroxide. i _ ) . 11. Method to detect TNF as bioactive form accord ng to claim 10, character zed in that said measurement is carried out by causing said detector reactant to react w th chromogenic or fluorogenic substrates, and reading the signal. 12. Kit to detect, from a sample, TNF as bioacti e form, character zed in that said kit comprises: - a capturing reactant immobi lized on a substrate, capable of only reacting with TNF molecules in oligomeric form, but not w th receptor-complexed TNF or T F in monomeric form; - solutions to incubate said sample; - a detector reactant capable of only reacting with TNF molecules in oligomeric form, but not with receptor- complexed TNF or TNF in monomeric form, conjugated with a detection system; - a wash so lut ion; - a reference sample. 13. Kit to detect, from a sample, TNF as bioactive form according to claim 12, characterized in that said capturing reactant and said detector reactant comprise Mab-78 antibody and said kit also comprises a detection system for total TNF.

Inventors: CORTI ANGELO, CORTI, ANGELO
Applicants: TECNOGEN SCPA
Application year: 1992
Main classification: C12P21
IPC codes: C07K16, C12P21, G01N33