Title of Invention

A METHOD FOR ISOLATION OF 30 KD PROTEIN ANTIGEN IN TUBERCULOUS MENINGITIS CEREBROSPINAL FLUID

Abstract

A method for isolating a 30 Kilodalton(KD) protein antigen in tuberculous meningitis cerebrospinal fluid comprising of step 1) isolation of 30 KD by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and treating said protein in buffer solution (0.15M phosphate buffered saline (PBS), pH-7.4) for attaining pre equiliburum stage, the said pre equilibrated protein is eluted by electroelution from the SDS-PAGE using whole gel eluter system for 1 hour, at 30 volts, checking the said eluted protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and confirming by two dimensional polyacrylamide gel electrophoresis and further confirming by western blotting method.

Full Text

FORM -2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION SECTION -10 TITLE A method for isolation of 30 KD protein antigen in tuberculous meningitis cerebrospinal fluid Applicant CENTRAL INDIA INSTITUTE OF MEDICAL SCIENCES BIOCHEMISTRY RESEARCH LABORATORY 88/2, Bajaj Nagar Nagpur-440010 (Maharashtra) India 'The following specification particularly described and ascertains the nature of this invention and the manner in which it is to be performed." Field of Invention:The invention disclosed in this specification relates to a method for isolation of 30 KD protein antigen in tuberculous meningitis cerebrospinal fluid. The disclosed is useful in detection of the Mycobacterium Tuberculosis (MTB) in cerebrospinal fluid (CSF) which is responsible for Tuberculous Meningitis (TBM). Background and prior art of the Invention: Tuberculous meningitis (TBM) remains a major health problem in the underdeveloped and developing countries. Nearly 20% of the tuberculosis patients have problem of TBM. It remains the most serious and with multiple central nervous system (GNS) complication. Even in the developed countries where a decade ago it was rare, it has made its appearance following HIV infection (Horner PJ 19991,Selwyn, 1991). Early diagnosis of TBM in adult is very difficult from whatever the resources are available to the physicians. Prompt diagnosis and treatment of TBM saves lives. However, about 30% of TBM patients die despite of anti tuberculosis chemotherapy. Delay in diagnosis and treatment are regarded as major contributing factors in the high mortality reported in many recent studies. Early diagnosis and early institution of treatment is must for satisfactory improvement. (Park SC et al, 1993; Daniel TM, 1987; Kennedy DH, 1979; Lincoln EM; 1963; Kilpatrick ME 1986; Traub MA, 1984; Thwaities GE 2002). The diagnosis of TBM relies on detection of Mycobacterium tuberculosis (MTB) in CSF, Director Ziehl Nelsons staining of the CSF for o Acid Fast Baciilis (AFB) remains the corner stone for rapid diagnosis, but these techniques lack sensitivity (only 5-10%) (Bal V, 1983; Kox F, 1995; Krambovities E, 1984; Udani PM, 1973; Udani PM, 1970). The sensitivity of direct AFB by Zeihl neelsen staining in the CSF is quite low (Molavi A, 1985; Kilpatric ME, 1986; Traub MA, 1984; Grance 1984;Miormer H). In many clinical studies less thatn 25% of specimens were found to be smear positive (Sulivan J L, 1977; Ocana, 1983). Culture of MTB is still the gold standard test for diagnosis of TBM (Gourie Devi M, 1989), but it takes very long time i.e. 6-8 weeks to demonstrate the growth of MTB (Glass Roth J, 1980; Gourie Devi M, 1989; Baig V, 1995; Srivastava, 1994;). Therefore laboratory cultivation of MTB is too slow and is of no use in clinical decision making (Kadival GV, 1994). The short term radiometric methods i.e. BACTEC culture is less sensitive for the isolation of MTB in CSF (Bonigton, 1998). Newer diagnostic techniques, such as those that use Polymerase Chain Reaction (PCR), have not been assessed completely. There are problems of sensitivity when dealing with CSF samples containing very small amount of MTB DNA which depends on the presence of MTB organism in the CSF, which is very rate (Nakajima H, 1995) and secondly this technique is not possible in most settings in the* developing world where majority cases of TBM are seen. The other currently developed immunological assays such as ELISA, RIA for TBM diagnosis remains inadequate or inaccessible in developing countries (Mathai A et al, 2003). Radioimmunoassay (RIA), Western blot analysis etc were used to assess the immunodomiant response and to delineate the specificity. However, all the aforementioned techniques have been reported to have problems with specificity (Li Z 1999; Radhakrishnan VV, 1991; Maniar P; 1990 Dana Niculesa, 1996; Sundravali N, 1979; Naidu AK, Brooks JB, 1979). Treatment of TBM is time sensitive and if delayed, leads to complications such as hydrocephalus, vasculitis, optic atrophy etc. It is therefore imperative that pr9oper specific treatment should be started at the earliest and for this at present early definitive diagnosis is limiting factor. Various MTB antigen markers such as Lipoarabinomannan, purified protein derivatives (PPD) Heat shock protein of 62 Kd and 14 Kd, GroE, Ag 85 complex, 31 Kd secretary antigen etc have now been recognized as a potential marker for diagnosis of TBM and Pulmonary tuberculosis (Wiker HG et al, 1992; Nair RE et al, 2001; Park SC et al, 1993, Friedon TR et al, 2003 Drobnieoski FA et al, 2003). However their presence remains questionable and many of these antigens are reported in the blood only but not in the CSF and thus question the veracity of the various diagnostic techniques currently under use for the diagnosis of TBM. Although CT and Magnetic Resonance Imaging (MRI) provide crucial assistance, but observations noted are often non specific. Moreover these equipments are not available in many health centers and often prove to costly to the patients. A retrospective study was done to correlate culture of MTB and detection of antigen in CSF by Inhibition ELISA (Radhakrishnan VV et al, 1991). hi another study CSF from TBM and various other neurological disorders were studied for the presence of IgG and mycobacterial antibodies by using capturing ELISA (by anti BCG) and multilayered ELISA (using M tuberculosis soluble extract) respectively (Cho.T Y, 1995). Some workers have conducted prospective clinical study to evaluate the reliability of the antigen and antibodies of PPD in CSF samples of TBM patients (Winter W D et el, 1981; Mathai A et al, 2001). However, the entire reported antigen, which is used for diagnosis of TBM in CSF have problems with specificity and sensitivity. Summary of Invention: in order to obviate above-mentioned drawback so to develop early and rapid diagnosis of TBM, we investigated to find a specific protein marker in CSF, which will be useful for the diagnosis of TBM. We have observed the presence of 30 Kd protein antigen in CSF of proven and suspected TBM patients. Antibodies against 30 Kd protein was raised and used for the detection of 30 Kd protein antigen in CSF of TBM and other infectious and non infectious neurological disorders by using various immunological techniques such as Subgle Radial Immunodiffusion (SRID), Dot ELISA, Antigen capturing ELISA etc kd antibodies when reacted with the antigen 85 complex shows positive reaction indicates that the selected 30 Kd protein band carries the antigen 85 complex, which is specific to M tuberculosis and could be considered as diagnostic marker for TBM. The presence of antigen 85 complex in CSF was also confirmed by LC-MS/MS, TB Fasta and 2 dimension electrophoresis. Antigen 85 complex is 30-31 kd protein group of three closely related components Viz Antigen 85a, Antigen 85bd and Antigen 85c which has been identified in the blood and is useful for the diagnosis of pulmonary tuberculosis. To confirm the presence of Antigen 85 complex in the CSDF, we performed following experiments by using the standard available antibodies against Antigen 85 complex. Dot immunobinding assay Antigen capturing ELISA Affinity capturing ELISA Western blot assay Using the said methodology it was demonstrated that the CSF samples collected from patients suffering from TBM carrying the Antigen 85 complex antigen of M tuberculosis. We report here for the first time presence of Antigen 85 complex in CSF even though it has been shown in the serum of patients with pulmonary tuberculosis. Antigen 85 complex is the secreted protein of M tuberculosis and will be a very useful diagnostic diagnostic kit is made for detection of antigen 85 complex in CSF of TBM patients which has been demonstrated with 90% efficiency using 200 samples collected from patients. The antigen 85 complex also interacts with the immune system at an early stage of infectious process and induce both humoral and cell mediated immune response in M tuberculosis infected patients, Antigen 85 alone or in combination with other M Tuberculosis secreted protein, as well as antigens 85 based DNA vaccine, induce strong cell mediated immune responses and protective immunity in animal models. Thus it is also possible to design peptides that could be tested for their protective role during mycobacterial infection of central nervous system. Methods: Collection of CSF samples from patients: The patients comprised of two groups, a control group of non TM neurological disorders such as pyogenic meningitis, viral meningitis, Gullian Barre syndrome, seizure, fungal meningitis, viral meningitis, epilepsy, tumors, ischeamia, headache etc and experimental group of clinically suspected and confirmed cases of TBM. CSF samples were taken from patients admitted in Central India Institute of Medical Sciences (CIIMS). The patients comprised of two groups, a control group of non TM neurological disorders such as pyogenic meningitis, viral meningitis, Gullian Barre syndrome, seizure, fungal meningitis, viral meningitis, epilepsy, tumors, ischeamia, headache etc and experimental group of clinically suspected and confirmed cases of TBM. CSF samples were taken from patients admitted in Central India Institute of Medical Sciences (CIIMS). The age of the patients ranged between 2 to 60 years (both male and female patients), CSF samples were stored at -20°C until used. Criteria for case selection: TBM was clinically suspected on the basis of the following criteria and history- 1. Sub acute onset of fever, headache and signs of meningeal irritation. 2. Elevated protein and lymphocytes, decreased sugar level in CSF and abnormal CSF :blood glucose ratio. 3. Good clinical response to anti TB medication. Demonstration of 30kd protein band in the CSF of TBM patients by Sodium dodecyl sulphate Polyacryiamide Gel Electrophoresis (SDS-PAGE): CSF samples obtained from patients of TBM, non tuberculous meningitis and other neurological disorders were subjected to Sodium dodecyl sulphate polyacryiamide gel electrophoresis (SDS-PAGE). SDS PAGE was performed with vertical slab gel electrophoresis system (BROVIGA, INDIA) by using standard Laemmali method (Laemmali UK, 1970). 4% stacking gel and 10% running gel were used. The electrophoresis wjas carried out at 250 volts at 50 m Amps. The gel was developed by staining with coomassie brilliant blue GR-250 and the band patterns w?ere studied. The band size (molecular weight) was measured by comparing with molecular weight marker run alongside the sample obtained Genei Baiiglore India. A study shows the presence of 30Kd and 14 Kd protein band in CSF of suspected and confirmed cases of TBM. These bands were markedly absent in the patients of non-tuberculous meningitis and other neurological disorders (control group) except in few cases. 30Kd protein band was detected in 84/92 of TBM cases (91%, while 4/54 in non-tuberculous meningitis (all pyogenic meningitis) cases (7%), and 18/358 in other neurological disorders (5%). The 14 Kd protein band however was not of much diagnostic importance since it was found in 45% of cases. The presence of this band was also noted in 12% cases of non tuberculous meningitis, but only 7/358(2%) in other neurological disorders. The above observation also agrees well with the biochemical and pathological findings observed in the CSF of TMB patients. Electrocution: After the separation of protein from CSF patients with TBM by SDS-PAGE, the 30KD protein band was sliced from the gel and pre-equilibriated in the elution buffer (0.15M phosphate buffered saline (PBS), pH-7.4). The gel was electroeluted in a whole gel eluter system (Biotech, India) for 1 hr at 30 volts, and harvested from the unit and dialyzed against PBS and the protein content, was measured by Bio Lab KIT. Running native PAGE was used to check the purity of protein. Immunization (Antibodies against 30 Kd protein antigen) New Zealand white rabbit was immunized intramuscularly with 1 ml of 30Kd protein antigen solution containing 2mg of protein in 0.14 M NaCL, Buffered to pH 7.3 with 0.02 M sodium phosphate (phosphate buffered saline) after it has been emulisified with an equal volume of complete Freunds adjuvant. Immunizations were repeated on days 10th, 20th and 30* after the primary immunization. Sera were collected from the marginal ear vein two weeks after each boost. Serum was obtained by allowing the blood to stand for 3-5 lirs at room temperature followed by overnight cold storage(4°C) to form a clot. The clot was gently removed with the aid of a wooden stick and the serum was centrifuged for 10 min at 2500xg at 4°C. The supernatant serum was removed and stored at -10°C in 1ml portions, (titre 1:5000). Single radial immunodiffusion (Mancini technique); A method of Single Radial Immunodiffusion (SRID) or Mancini technique was adopted to detect 30Kd protein antigen in CSF of TBM patients. 2% agarose was prepared in phosphate buffered saline (PBS),pH 7.4 and was transferred in petridish. The polyclonal serum containing antibodies against 30Kd protein antigen was then added to the agarose gel. Wells were cut and CSF of control and test samples (suspected TBM) in different dilutions were loaded in the Wells Gel Plates were incubated in a moist chamber for 24 to 48 hrs, the gel was stained with coomasive brilliant blue GR 250 for 30min and then destained with a destaining solution containing 5:1:5 methanol, acetic acid and water. The developed protocol was tested with CSF of clinically suspected and confirmed Patients of TBM, Non tuberculous meningitis and other neurological disorders patients. Detection of 30 Kd protein antigen in CSF of patients in various disorders using plate seeded with polycioncai sera against culture filtrate antigen of M tuberculosis H37Rv. Wells b,d, f contained CSF samples which were obtained from patients of Multiple Sclerosis headache and Pyogenic meningitis, while wells a,c, e contained CSF obtained from clinically suspected TBM patients. Study indicates the results of 315 CSF samples (65 of TBM (including two confirmed cases of TBM) 47 of non tuberculous meningitis and 203 of other neurological disorders. The precipitin ring was present (i.e. test is positive for antigen detection)in both the confirmed cases of TBM. Out of 63 clinically suspected TBM patients the precipitin ring was present in 57 cases (90%). Thi test was positive in only 3 cases of Pyogenic meningitis out of 47 cases of non tuberculous meningitis (Pyogenic meningitis 25, fungal meningitis 16 and viral meningitis-6). This test was negative in 187 out of 203 cases of all other neurological disorders (having no sign and past history of tuberculosis) and gave a false positive test in 16 CSF samples (8%). Western Blotting: Eluted 30 kd protein was loaded and separated by SDS PAGE and electroblotted onto Nitrocellulose paper NCP) as per standard protocol described elsewhere. They were probed with 1:1000 dilutions of polyvalent antibodies to antigen 85 complex for 2 hrs at room temperature, followed by-secondary antibodies (Goat anti rabbit IgG-HRPO). The NCP was then treated with Tetramethyl benzindine/hydrogen peroxide (TMB/HO) substrate solution. Antigen 85 complex was used as a positive control in the experiment. Capturing ELISA: Antibody capturing ELISA : The flat bottom microliter wells were coated with 100 ill of Antigen 85 complex (lOugm/ml) and incubated overnight at 4°C. The wells were then washed with PBS solution, pH 7.4 and then non specific sites of coated antigen were blocked by the addition of lOOul of 2.5% BSA/PBS at 37°C for 1 hr. The blocking solution was then removed from the wells. 1 OOul of CSF samples (1:10) of TBM patients (30kd protein positive) was added to the wells and incubated at 37°C for 60minutes. The wells were again washed with the PBS. 1 OOul of affinity purified horseradish peroxidase conjugated antihuman IgG (Bangalore Genei, India) diluted 1:10,000 in PBS was added into the wells and incubated at 37°C for 60 min. After another washing with PBS, 100ul of TMB/H202 substrate solution was added into the wells and incubated at room temperature for about 20 mini The reaction was then stopped with 100 ul of 2.5N sulphuric acid and the resulting color was read at 450nm24. The same procedure was used for detection of antibodies to Antigen 85a, Antigen 85b, Antigen 85c in CSF samples of TBM by using their respective antigens. Antigen capturing ELISA: 100 ul of CSF samples (1:10) of TBM patients (30 ltD) protein positive) and non TBM patients (30 kD protein negative) were added to the wells blocked and incubated at 37°C for 2 hrs. After washing with PBS polyvalent antibody against antigen 85 complex was added and incubated at 37C for Ihr. After incubation secondary antibody (goat anti rabbit IgG-HRPO) was added and the solution was incubated for Ihr at 37°C. 100 ul of substrate solution was then added into the wells and incubated at room temperature for about 20min. The reaction was then stopped with lOOu of 2.5 N sulphuric acid and resulting colour was read at 450nm. The same procedure was used for detection of antigen 85 complex and its components. We Claim 1. A method for isolating a 30 Kilodalton(KD) protein antigen in tuberculous meningitis cerebrospinal fluid comprising of step 1) isolation of 30 KD by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and treating said protein in buffer solution (0.15M phosphate buffered saline (PBS), pH-7.4) for attaining pre equiliburum stage, the said pre equilibrated protein is eluted by electroelution from the SDS-PAGE using whole gel eluter system for 1 hour, at 30 volts, checking the said eluted protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and confirming by two dimensional polyacrylamide gel electrophoresis and further confirming by western blotting method. Dated this 29th day of January 2004

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