COMPOSITION AND ITS PREPARATION PROCESS USING ANTIGEN CONJUGATED TO ENZYMATIC ACTIVITY FOR IMMUNOLOGICAL DIAGNOSIS AND CHAGAS' DISEASE IMMUNOLOGICAL DIAGNOSIS KITS, FOR INDIVIDUAL AND EPIDEMIOLOGICAL APPLICATION, BASED ON THAT COMPOSITION

Abstract: This patent deals with the use of antigens conjugated to an enzymatic activity for the detection of immune-complexes against antigenic mixtures containing conjugated antigen, hence allowing the detection of antibodies even with low titre, through the development of a color reaction. The method is based on the specificity of antibodies and can be used for the diagnosis of any infectious disease with the condition of using the appropriate conjugated antigen. The diagnosis kits for Chagas' disease make use of the recombinant Trypanosoma cruzi antigens described in the patent application PCT/BR/91/00003, (Brazilian application number 9001479, of MAR, 30, 1990), and are indicated for the immunological diagnosis of Chagas' disease using either ELISA or the antigens immobilized to solid supports such as nitrocellulose, nylon, polyvinyl, polycarbonate and polyallomer.

Description: "C0MP0SITI0N AND ITS PREPARATION PROCESS USING ANTIGEN CONJUGATED TO ENZYflATIC ACTIVITY FOR IMMUNOLOGICAL DIAGNOSIS AND CHAGAS'DISEASE IMMUNOLOGICAL DIAGNOSIS KITS, FOR INDIVIDUAL AND EPIDEMIOLOGICAL APPLICATION, BASED ON THAT COMPOSITION". BACKGROUND OF THE INVENTION This tent request refers to the use of antige s conjugated to an enz i mat i c activity (detectable by color re ction).. i di osti ass ys or' Chag s' dise se nd other i ectious diseases. The Metho s generall used for the serologi l iagno is of i fe tious iseases. rel o the es e of antibodies against the pathogen, i the sera of the in ected patient. But these tec niques can p esent some l imitations i their speci icit whe crude antigen preparations are use . On the other hand, the use of purified antigens can present problems i terms of cost a d large scale apl ic bil ity. However, the de elopment of recowbinant DNA techniques paved the way to the large scale obtention of purified a tigens with lo er cos a d safer conditions. Accordingly, our work with Trypanoso_»a cruzi ( the e hiologica ' l agent of Chagas' disease) , resulted in the cloning and expression in bacteria of antigens fro t e arasite th t re s e ifi l recognized by chagasic sera. The ELISA test (enz me l inked im unoensim tic assa ) is frequentl used for serological diag osis. This me hod allows the Ide tifica io a d quantificatio of a ige s or antibodies in biological fluids. The . onvent i onal ELISA consists i n the detection of the complex an ibody-antigen by a second antibody (against the antibody that reacts with the antigen) conjugated to an enzimatic activity (peroxϊdase, alkaline phosphat se and others). Alternatively, the i mmune-comp 1ex can be also detected using Staphylσcoccus aureus protein-A or protein-G conjugated to the enzimatic activity. However, this conventional ELISA does not detec low t iter antibo ies, resulting in the failure to distinguish among positive and negative sera. In the case of the use of recombinant antigens, this problem can be ampl ified in virtue of the cross-reac ivity with bacterial antigens that can eventually contaminate the preparation. A method based on the detection of highly specific antibodies (irrespective to their titre) should avoid the limitations of the ELISA test described above. This can be performed by using the antigen to sensitize the ELISA support and to develop the immune-com leκ through its conjugation to the enzymatic activity or radioactive label l ing. This patent application refers to the development of a direct ELISA and its use in the diagnosis of Chagas ' disease. This method relies on the use of the antigen for sensitizing the plate and for the detection of the fmmune- coβplexes. The method, which is graphically represented in the scheme of Figure 1, is fast and allows the visual reading of the result. Some results using the direct ELISA and the comparison with a conventional ELISA are d scribed belo . The esults presented in figure 2 compare the reactivit of hum n chagasic sera (lane P), non-chagasi sera rom endeM i c areas (lane N-i) and non-chagasic sera from blood bank ( -2) using a conventional ELISA (Fig. i-A) and the direct ELISA (Fig. i-B). It can be observed that the cut¬ off l i e is much more cle r i the case of th direct th n i the case of the i direct ELISA. The results presented in Table I compare the reactivit of t e direct an the indirect ELISA ith different hum n ser . The direct ELISA is much more specific a d sensible than the conve ional ELISA since no false positive or false negative results were observed using this method. DESCRIPTION OF THE INVENTION The proced res of the metho are as follows. The antigen or antigenic mixture, in a concentration ranging fro* 0.01 to 1 mg/fitl, depending on the antigen, is immobil ized in the sol id support. The antigen is diluted in 50«(t carbonate buffer, phosphate buffer or any other- suitable buffer for immobi ization to the support, which can be pol carbonate, polyallomer, pol ypro i 1 ene , polyvinyl, nylon and ni rocellulose. The sol id phase sensitized with the antigen is washed with phosphate buffered sal ine pK 7.0-7.5 containinig 0,32 detergent, and dried out. The sera to be analysed are appl ied onto the support containing the antigen and the ensemble is incubated at 25-37 C for times varying from 30 minutes to 120 /ainutes. Following this? the solid phase is washed with phosphate buffered saline or any other suitable buffer. The r ffiune-complej-.es are developed with the total or fractions of the antigenic mixture used to sensitize the support, conjugated to an enzimatic activity or radi oac i el ly labelled. The conjugated antigen is diluted in phosphate buffered saline, or any suitable buffer, containing a blocking agent such as gelatin, bovine sera, BSA, ailk, etc., in order to avoid unespecific binding to the support. After incubation at 37" C, the immune- cofflplexεs are developed according to the enzimatic reaction suitable for the conjugated enzyme or radioactive label 1 ing. Two kits for the diagnosis of Chagas' disease were developed based on the procedures described above: a direct ELISA kit and a kit using a membrane (nylon or ni rocellulose) as support. The Direct ELISA kit is composed of a micro-titre plate coated with the antigens (described in the Interna ional Application under the Patent Cooperation Treaty PCT/BR 91/00003, Brazilian Application number 9001479) in a concentration of O.lmg/ml, a flask containing the conjugated antigen, three tubes containing control sera that consist of negative, positive and cut-off sera, a flask with washing buffer (buffered saline solution, pH7,5, containing 0 .27. Tween - -20), washing buffer containing foetal bovine sera 52 and the solutions necessary for. .developing the enzimatic reaction. The kit also contains an explanatory note on how to proceed. Briefl , the sera to be test d (undiluted or diluted until 1:200) in a volume of 50 ^1 are put in the well and incubated for 30-ό0 minutes at 37* C and then removed. The wells are washed three times with shing solution as described above and then, the antigen co jugated to the enz matic activity dilute 1:1000 is added in a final volume of 50*ul and incubated at 37* C for 60 minutes. The wells are washed three times with wash ing buffer and then the substrate of the enzyme is added (3,3', 5,5'- tetra ethy1 benz i d i ne 0,01 m /m1 in DHS0 and 0 , 35_ h drogen peroxide in the case of pero idaεe). Following 10 minutes of incubation at room temperatu e, the reaction is stopped b the additio of 4P. sulphuric acid. The results can be re d visually or i the spectrophotometer b the absorbance at 450nm. I the case of the kit using a sol id sopport (nitrocellulose, nylon, pol allo er, pol carbonate, polyvi l, etc.), the antigens are spre d onto the membrane and their position is signaled. The strips are then placed inside a tube that is the reaction vessel. The kit cont ins 20 tubes with strips, the conjugate solution, co tro sera and washing buffer consisting of phosphate buffered sal ine solution (pH 7.5) contai ing 0.3_ Tweεn - -20. In dditio , the kit contains a dilution solution (washing buffer contai ing 52 bovine serum) and the reagents necessar for developing the enzy atic reactio , as desc ibed abov . The serum sa le to be tested is diluted 1:100 in washing solution and 2u.l of this solution are put inside the reaction tube containing the membrane. After incubation at 37° C for 60 minutes, the serufή is removed and the strip is washed three times with 2Λ.1 of washing solution. Following this, the conjugate in a final volume of 2ml and i n a dilution of 1:1000 is added to the reaction vessel containing the strip, and incubated for additional 60 minutes at 37° C. The conjugate is removed, the strip is washed twice with washing buffer and the substrate of the enzyme is added. The reaction proceeds for 10 minutes and is then stopped with water. The result of the reaction is determined by the intensity of the color- respective to the controls. These kits are stable for at least six months at 4° C and for one month at 37* C. The conjugate is kept in hy iolo ical saline solution containing 20.2 glycerol and proteinase inhibitors.

Claims: CLAIflS 1 ) Compositio for i muno1 og i ca1 diagnosis usin a tige co jugat d to enzymatic activity, contai ing the enzyme, h t y be erox i d se , l l i e phos at se or ease , conjugated to the dequate purifye antigen to antibodies capture, added to sodium carbonate solution 1,5 f , pH 9,5 and borohydrate sol tion 0,2 mg/ml. 2) Preparation process of antigen conjugated to enzymatic ctivity, comprising the following steps: a) Immobil ization of the antigen to a sol id support using the antigen in a optimal standard condition. b ) The sol id support is washed in the appropri te solution containing detergent? c) Appl ication of the samples to be tested onto the sol id phase a i ubation at ideal times and temperature. d) Washi g of the sol id phase with washing buffer at the end of the incubation period. e) Preparation of the enzyme to be conjugated. f) Addition of the antigen and of the enzyme, followed by the ddition of 0.0015M sodium carbonate pH 9.5 and incubation at appropri te temperature and time. g) Addition of borohydrate solution a d incub tio at appropriate times and temperature. h) Dyalisis ag inst 0 , 01fl phosphate buffered sal ine, PH i) Applic tion f the enzy e-conj ted antigen diluted in washing buffer and containing a competitive blocking agent. j) Incubation at the adequate temperature for adequate time. k) Washing of the solid support with washing buffer and dry ing. 1) Development of the enzymatic reaction with the appropriate substrate. m) Addition of the adequate blocking agent for stopping the reaction. 3) Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step "a" the antigen is a purified one, diluted in 0.051-i carbonate buffer, that should be a phosphate buffer, when a solid support like nitrocellulose, nylon, polyallomer, polycarbonate or polyvinyl, is used for the immob i 1 ization. 4> Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step "b" the washing solution consists of I fl phosphate buffered saline pH 7.0, containing 0.3% detergent. 5> Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step "c" the incubation is done for 45 minutes at 25-37" C. 6) Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step "e", the enzyme is prepared by treatment with 0.02.". metaper i odate and stirring for 20 in at room temperature following dyalisis against 1 mPl acetate buffer pH 4.4. 7> Preparation process of antigen conjugated to enzymatic acti ity according to claim 1, wherein step "f" the incubation is performed at room temperature for 2 hours. 8 ) Preparation process of antigen conjugated to enzy atic acti ity according to claim 1, wherein step "g" 0.2 -tig/ml boroh drate solution e dded followed b 2 hours incubation at 4 C C . 9) Prepar tion process of antige conjugat d to enzymatic activity according to claim 1, wherein step "h" the materials are diluted for 24 hours at 4 β C. 10) Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step the blocking agent can be foetal serum, bovine serum albumin, gelatin, non fat milk a d others. 11) Preparation process of antigen conjugated to enzymatic acti it according to claim 1, wherein step "j" the incubation is done at 25* C to 37" C for 30 minutes. 12) Preparation process of antigen conjugate to enzy atic activity according to claim 1, wherein step "k" in the case of using perox i dase conjugated to the antigen, the employed buffer is O.Olfl itrate/phosphate containing 3,3', 5, 5' tetramethy1 -benzyd i ne I g/ml d i ethy -sul ox i de a d h ogen peroxide- 0.3Z. 13) Preparation process of antigen conjugated to enzymatic activity according to claim 1, wherein step "1" the blocking agent is diluted sulfuric acid. 14) Prepar tion process of antigen conju ated to enz matic activity according to claim 1, wherei step " tn " no aci is use in the cases where the support consists of n lo . itrocellulose, polyallomer, polycarbonate or polyvynil. 15> Kits for the i mmunolog i al diagnosis of Chagas' disease based on antigens conjugated to enzymatic activity, wherein Trypanosona cruzireco b inant antigens described i n the interna ional application PCT/BP. 91/00003 (Brazilian application number 9001479, of PIAR., 30, 1990), are used. lό) Kits for the i mmunol og i al diagnosis of Chagas' disease, according to claim 15, wherein there are two its, based, one, in the ELISA test and another with ni rocellulose, nylon, polyallomer, pol carbonate or polyvinyl as support. 17) its for the i munolo ical diagnosis of Chagas' disease, according to claim 16, wherein the kits based in the ELISA test consists of a 96 micro-well plate coated with the appropriate antigens, a vial containing antigen(s) conjugated to the enzymatic activity, three vials containing control sera (negative, positive and cut- off), washing buffer, dilution buffer and the enzymatic substrate, IS) Kits for the i mmunolo ical diagnosis of Chagas' disease, according to claim 17, wherein the antigen concentration is 100 .9 *l? the washing buffer consists of buffered saline (pH 7.2) containing 0.3 Tween-20 and the dilution buffer consists of washing buffer added to 2Z bovine serum. 19) Kits for the i mmunolo ical diagnosis of Chagas' disease, according to claim 16, wherein the kits which use other so id supports (such as n lon, nitrocellulose, etc.) consists of 20 t bes contai ing the membr es w ere the ntigen(ε) have been spotted. 20) Kits for the immunological di gnosis of Chagas * ' disease, according to claim 16, wherein the ELISA proced re is as follows: a) The teste sera are dropped in the wells of the se sitized micro-well plates, incubated for the ppropri te time and temperature, and then removed. b ) The substrate of the enzyme is added afte shi g throughl the wells. c) The reaction is stopped by the addition of 4Pi sulfuric A id. d) The re ding of the plates is performed acco dingly. 21) Kits for the immunolo i al diagnosis of Chagas' disease, according to claim 20, wherein step "a" the sera are appl ied to the wells undiluted or diluted up to 1:200 and the incubation is done at 37" C for 30-6 minutes? step "b" each well is washed three times? step "c" the process takes place for 60 minutes at room temperature and step "d" the result can be obtained by optical reading and comparison with the control sera or spectrophotomet icall t 450nm . 22) Kits for the immunological diagnosis of Chagas' disease, according to Claim 16, wherein the immobil ization is done at nitrocellulose, n lon, polyallomer, polycarbonate o polyvi yl, the procedures shall be as ol lows: a) The tested serum is adequately diluted. b) Incubation for the appropriate time and te perature, re oval of the serum and washing of the stri (support). c) Addition of the conjugate at an appropriate dilution and incubation at established temperature for the convenient time. d> Removal of the conjugate and washing of the strip. e) Addition of the solution containing the substrate of the enzyme, incubation for the convenient time a the established temperature, and stopping of the reaction by the removal of the substrate and addition of 2 ml water. f) Evaluation of the result as in Claim 21. 23) Kits for the i munolo ical diagnosis of Chagas' disease, according to Claim 22, wherein step "a" the serum is diluted at 1:100 in washing solution and are added in a final volume of 2ml to a tube containing the strip where the capture antigen is immobilized; step "b" the incubation is done at 37° C for 60 min and the strip is washed three times with 2ml of washing solution? step "c" the conjugate is diluted 1:1000 and the incubation is done at 37* C for 60 minutes? step "d" the strip is washed three times with 2ml of washing solution. 24) Kits for the immunological diagnosis of Chagas' disease, according to Claim 15, wherein they are stable for at least six months at 4° C and at least one month at temperatures around 30 β C.

Inventors: CERQUEIRA DE ALMEIDA ELZA CARM, GOLDENBERG SAMUEL, KRIEGER MARCO AURELIO, CERQUEIRA DE ALMEIDA, ELZA, CARMEN, GOLDENBERG, SAMUEL, KRIEGER, MARCO, AURELIO
Applicants: FUNDAC O OSWALDO CRUZ FIOCRUZ, FUNDACO OSWALDO CRUZ (FIOCRUZ) - SUPERINTENDENCIA
Application year: 1991
Main classification: C12R1
IPC codes: G01N33