Published on March 4, 2014
Biochemical Studies on Mycobacterium Tuberculosis Antigen BY Mohamed Mostafa Omran ,Biotechnology Research Center New Damietta, Egypt
Mycobacterium Tuberculosis • One third of the world’s population are infected with M. Tuberculosis. • M. Tuberculosis lead to death of one person every 10 minutes. • M. tuberculosis is the causative agent of Tuberculosis (TB).
M. tuberculosis • Curved rods. • More resistant than other bacteria to acids. •Acid fast bacilli (AFB).
EPIDEMILOGY OF TB Range of rate per 100,000 0-9 10-24 25-49 50-99 %(Egypt (0.1-2.4 or more 100 No report
Transmission of TB Patient with TB .Inhalation of M. tuberculosis
Types of human TB • Pulmonary TB (~ 80%). • Extra-pulmonary TB (~ 20%).
Control of TB 1. Vaccination (BCG) 2. Effective therapy 3. Education 4. Early diagnosis
Diagnosis of TB Traditional methods: 1. Ziehl- Neelsen (ZN) stain. 2. Fluorescence microscope. X-ray. 3 .Tuberculin test. 4 Cultures. 5 New methods: 6. Serodiagnosis (Detection of TB antibody) 7. PCR (Detection of DNA of M. tuberculosis) 8. Detection of TB antigen.
Traditional methods ZN stain M tuberculosis . showing as red rods against blue background. M. tuberculosis showing as white Yellow rods against .the dark background
Traditional methods X-ray Tuberculin test
Traditional methods Cultures Culture is highly sensitive and specific test but require 6 to 8 weeks to achieve m axim sensitivity um
New methods Detection of TB antibody TB antibody do not differentiated between latent and active infection
Aim of the work 1. Identification of a TB antigen in different body fluids of Infected TB patients using monoclonal antibody. 2. Biochemical characterization of TB antigen. 3. Diagnosis of pulmonary and extra.pulmonary TB using TB antigen
Total number in the study (506 individuals) Patients (n= 389) : Pulmonary TB: 296 patients . Extra-Pulmonary TB : 93 patients. Controls (117): Healthy volunteers: 48 individuals Non-tuberculous diseases: 69 individuals
Patients with Pulmonary and extra-pulmonary TB 100 90 % 76 80 70 ) %(Percentag 60 50 % 24 40 30 20 10 0 Pulmonary TB Extra-Pulmonary TB
All samples were obtained from the Department of Chest Diseases at Sayd Galal University Hospital, Al-Azhar University and Abbasia fever Hospital, Cairo, Egypt. 1. Serum 2. Cerebrospinal fluid )CSF( 3. Ascetic fluid
Monoclonal Antibody ()TB-55 mAb
Sodium Dodocyl Sulphate- polyacrylamide Gel Electrophoresis )SDS-PAGE(
W estern blotting 1-Separation by electrophoresis 2-Blotting tank Peptides transferred to nitrocellulose sheet (blot) +ve 3-Blocking of free binding sites Bovine serum albumin -ve + + + + 5- Blot results 4- Immunostaining of the blot Substrate Antigen bands visualized Primary antibody (TB-55 mAb) Secondary antibody Enzyme - labeled
Purification of TB antigen 1. Preparative gel electrophoresis. 2. Electroelution.
TB Antigen detection using dot-ELISA Nitrocellulose membrane
Statistical analyses Sensitivity, specificity, efficiency, and positive predictive )PPV( and negative predictive )NPV( values were calculated as following Reference Test Evaluated test Total + ve - ve + ve True + ve (a) False –ve (c) a+c - ve False + ve (b) True -ve (d) b+d a+ = a / (ab+c) × 100. c + d Specificity = d / (b + d) ×100. Efficiency = (a + d)/(a + b + c + d) ×100. PPV = a / (a +b) ×100. NPV = d / (c +d) ×100. Total Sensitivity a+b+c+d
1. Identification of TB antigen in BCG, serum, CSF and ascites.
Identification of the TB-55 mAb target antigen in BCG SDS-PAGE and western blotting for BCG.
Identification of TB antigen ( 55 kDa) in serum samples of pulmonary TB (Mr.) : Molecular weight markers Lane (1-4): serum sample from non infected individuals. Lanes 5-9: serum sample from pulmonary TB patients.
Identification of TB antigen (55 kDa) in serum samples of extra- pulmonary TB ;Mr.) : Molecular weight markers( .Lane (1): serum sample from non infected individual Lane 2: peritonitis TB; Lane 3: meningitis TB Lane 4: lymph nodes TB; Lane 5: genitourinary tract TB ; ; Lane 6: potts disease TB; Lane 7: arthritis TB Lane 8: sinusitis TB; Lane 9: millary tuberculosis TB
Identification of TB antigen (55 kDa) in CSF (Mr.) : Molecular weight markers Lanes (1-3): CSF from nontuberculous diseases patients. Lanes (4-9): CSF from tuberculous meningitis patients.
Identification of TB antigen (55 kDa) in ascetic fluid (Mr.) : Molecular weight markers; Lanes (1-2): non-tuberculous ascites fluids. Lanes (3-9): tuberculous ascetic fluid from peritonitis TB patients.
2. Purification of TB antigen from serum, CSF and ascites
SDS-PAGE of TB 55 kDa antigen from serum of patients with TB Pulmonary (Mr.) Extra-Pulmonary : Molecular weight markers; Lane 1: Crud serum sample. .Lane 2:The TCA precipitate fraction of TB purified antigen .Lane 3: The TCA supernatant fraction of TB purified antigen
SDS-PAGE of purified 55 kDa antigens from CSF and ascites CSF (Mr.) Ascites : Molecular weight markers; Lane 1: Crud sample (CSF or ascites). .Lane 2:The TCA precipitate fraction of TB purified antigen .Lane 3: The TCA supernatant fraction of TB purified antigen
OD at 200 nm Capillary electrophoresis for purified TB antigens Serum .)Time (min OD at 200 nm .)Time (min Serum .)Time (min .)Time (min
3. Biochemical Characterization of TB antigen from serum, CSF and ascites.
Partial biochemical characterization of purified TB antigen. Reactivity of purified antigen using dotELISA Treatment Concentration Incubation time Treated Untreated Acid 0.2 M HCl 1 hour -Ve + Ve Base 0.2 M NaOH 1 hour -Ve + Ve Type of reagents Trichloroacetic acid + Ve 40% 15 min. a. Precipitate + Ve b. Supernatant -Ve 20 mM 18 hours + Ve + Ve Mercaptoethanol 180 M 1 hour -Ve + Ve Protease enzyme 1 mg/ml 45 min. -Ve + Ve Pepsin enzyme 1 mg/ml 45 min. -Ve + Ve Periodate
Amino acid analysis of TB antigen using HPLC Name Concentration (nmol/mg protein) Leucine Isoleucine Valine Proline Methionine Tyrosine Alanine 47.14 49.28 81.42 59.28 98 94.28 113.14 Hydrophilic amino acids Glycine Serine Therionine 755.71 214.28 55.71 46.4 % Basic amino acids Lysine Arginine Histidine 162.85 85.71 111.42 % 16.3 Acidic amino acids Glutamic acid Aspartic acid 135.71 142.85 Type Hydrophobic amino acids % 24.6% 12.7
Amino acid analysis of 55-kDa using HPLC 46.4 50 (%) 40 30 24.6 20 16.3 12.7 10 0 Hydrophobic Hydrophilic Basic Acidic The 55- kDa antigen is a basic polypeptide .chain with a hydrophilic nature
4. Diagnostic potential of TB antigen in pulmonary and extra-pulmonary TB
Detection of circulating 55-kDa antigen using Dot- ELISA (+ve) control : Serum sample of infected individual. (- ve) control : Serum sample of non-infected individual : Strong positive test +).Serum samples with high antigen level (3+, 4 : Weak positive test +). Serum samples with low antigen level (1+,2
Advantages of detection TB 55-kDa antigen using Dot-ELISA in serum samples of pulmonary TB TB-Ag detection (Clinical diagnosis (gold standard + . Pulmonary tuberculosis patients 257 (a) Patients with respiratory diseases other than TB and Healthy controls ( b )4 Total Sensitivity = 87 % %, %PPV = 98 %, %,Efficiency = 90 261 Total 39 (c) 296 ( d )113 117 152 413 Specificity = 97 NPV = 74
Advantages of detection TB 55-kDa antigen using Dot-ELISA in serum samples of Extra-pulmonary TB Clinical diagnosis (gold (standard TB-Ag detection Total + - .Extra-pulmonary tuberculosis (a )84 patients Patients with respiratory ( b )4 diseases other than TB and Healthy controls Total Sensitivity = 90 %, PPV = 95 %, 88 (c )9 93 113 ( d) 117 122 210 Specificity = 97 %, NPV = 93 % Efficiency = 94 %,
Overall Advantages of detection TB 55-kDa antigen using Dot-ELISA in 506 serum samples (Clinical diagnosis (gold standard TB-Ag detection Total + - TB patients (pulmonary and extra-pulmonary) (a )341 (c )48 389 Patients with respiratory diseases other than TB and Healthy controls ( b )4 ( d )113 117 345 161 506 Total %, Sensitivity = 88 %, PPV = 99 %, %,Efficiency = 90 Specificity = 97 NPV = 70%
Conclusion 1. 55–kDa TB antigen was identified in serum, CSF and ascites fluid of TB infected patients using westen blot. 2. The purified TB antigen showed a single band at 55–kDa in SDS-PAGE and one peak when analyzed by Capillary electrophoresis at 11 minutes. 3. The reactive epitopes of the purified antigen from serum, CSF and ascites have the same biochemical characteristics. The 55- kDa antigen is a basic polypeptide. 4 .chain with a hydrophilic nature
5. The TB antigen was detected using a simple and rapid dot-ELISA in 87% of individuals with pulmonary TB and 90% of individuals with extra-pulmonary TB. 6. TB antigen detection has sensitivity (88%), specificity (97%), and efficiency (90%) in 506 individuals.
WHO Recommendations To replace the “gold standard”, For TB diagnosis, a serological test must has sensitivity of over 80% and specificity of over 95%. So, the detection of 55-kDa TB antigen using dot-ELISA could replace the WHO gold standard for TB diagnosis.
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