INTRODUCTION:

Tuberculous meningitis (TBM) is a severe form of extrapulmonary infection caused by Mycobacterium tuberculosis with a high mortality and morbidity rate.

In developed countries, more than 50% of ME TB patients receive permanent neurological sequel, and more than two-thirds of patients experience disability complications with a ME TB mortality rate of 19.3-21.5%. Study in Saiful Anwar Hospital Malang Indonesia, ME TB revealed 42,9% mortality rate^1,2. The high mortality and disability rates are often caused by the difficulty of establishing the diagnosis of ME TB and the many complications that arise as well as some TB drugs that are difficult to penetrate the blood brain barrier¹^,³^,⁴.

Several methods for identifying tuberculosis meningitis have been developed, such as PCR and serological methods. In developing countries, PCR testing is still limited to certain hospitals, with high costs and varying sensitivity, so it is still necessary to develop serological tests that can be used to diagnose tuberculosis meningitis. Serological examination using antigens is considered promising measure because it is easy to do, cost-effective, and can be done in developing countries⁵. Several studies using antibodies for the diagnosis of ME TB and several antigens have been identified and are promising to be evaluated as a diagnosis of ME TB including antigen 5, p32 antigen, 30-kDa antigen, cord factor, 88-kDa antigen, ESAT-6, and others⁶^,⁷.

Antigen 38 (Ag38) sometimes called PstS-1 is a lipoprotein secreted by M.tb and is able to increase the response of B and T cells with high specificity, it is expected to be a candidate for a new biomarker for tuberculosis diagnosis⁸^,⁹. Epitopes are specific amino acids (peptides) on the surface of an antigen that are capable of causing an immune response, epitopes are able to bind to antibodies and/or antigen presenting cells (APCs). Previous study suggested that the antigen-recombinant epitope (RecAg38) was able to cause the formation of p201–220 and p230–249 antibodies in unimmunized samples. on the other hand, epitope studies using dead tubercles were able to stimulate the formation of antibodies exclusively in N-terminal peptides 1-20 peptides, the same thing happened when using live TB tubercle samples obtained the detection of non-continuous epitopes binding Ag38¹⁰^,¹¹^,¹².

Several studies have shown the detection of IgG, IgM and IgA after exposure to Ag38. Furthermore, it was shown that the sensitivity of IgA and Ig M was lower than that of IgG, with sensitivity rates between 16%-94% and specificity of 93-100% depending on status and selection of patient selection and antibody response found predominantly in patients with TB smear examination. Antigen levels were highest in the group and then stimulated strong antibody production and this is very good for confirmation of serological tests to determine TB¹³^,¹⁴.

Previous study, antigen 38 could be detected in Liquor cerebrospinal (LCS) tuberculosis meningitis in children patient with sensitivity 83, 93%, and specificity ranging from 92–96% with ELISA andPCR definite tuberculosis meningitis subject¹⁵^,¹⁶. However, the study of antigen 38 epitopes in central nervous system TB infection is still limited, including studies of antigen 38 and RecAg38 epitopes even though these antigens play a role in the TB infection process outside the CNS and also inside the CNS, so further studies need to be carried out to prove the role of both antigens in the CNS. The purpose of this study is to prove that recombinant protein antigen 38 and epitope antigen 38 can produce IgG and IgM antibodies in experimental animals as a test for the diagnosis of tuberculosis and tuberculosis meningitis.

MATERIAL AND METHODS:

This study is a part of long series of studies for the investigation of antigen 38 and epitope antigen 38 in tuberculosis meningitis using IgG and IgM antibodies made from initial research¹⁷^,¹⁸^,¹⁹^,²⁰. The process of making antibodies is carried out the following procedure:

Strains and plasmids:

E. coli BL21-(DE-3) strains used from the Central Laboratory of Basic Sciences, Brawijaya University, Malang, Indonesia.

Production of recombinant antigen 38:

To produce recombinant RecAg38, this E. coli BL21-(DE3)/pMBhis was cultured at 37°C in Luria Broth medium containing ampicillin (100µg/mL). Once cells reached an optical density (OD 600) of 0.6, IPTG isopropylthio-β-galactoside was added to induce subsequent transcription and translation. After 3 hours post-induction, cells were harvested by centrifugation at 8000 x g for 10 at 4 °C. The supernatant is discarded and the pellets are washed with phosphate buffer (pH 7.2) and frozen in liquid nitrogen before being stored at -20°C. The next day, the pellets are subjected to sonication treatment)¹⁷.

Recombinant Antigen 38 Purification:

The Protino 2000 protein purification kit (Dureen, Germany) was prepared in a refrigerator temperature of 4ºC. The resin was wetted with 4 ml of LEW (washing) buffer. The supernatant from the extract after IPTG induction was added and collected (flow trough). Washing 1 x 4ml with LEW buffer. Elution with 3 x 3 ml elution buffer. The result is stored at 40°C.

Finally, all fractions were analyzed using 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following is the result of SDS-PAGE using Thermo Marker 26634. The presence of RecAg38 was confirmed using Coomassie blue staining.

Immunization using rec Ag38 and Epitope rec Ag38:

A total of male mice (n= 12), aged 6-8 weeks, were purchased from the Faculty of Veterinary Medicine, Brawijaya University, Malang. Acclimatization for 1 week, two antigens are used; recombinant antigen 38, and two epitopes, namely QGTIKTWDDPQIAALNPGVNLP and AGFASKTPANQAISMIDGPAPD. These two epitopes are the results of previous research obtained using in silico methods⁹. Two antigens were emulsified with compound froud adjuvant (CFA). Immunization was carried out by intraperitoneal injection at a dose of 50μg /0.3ml PBS. Boosters were carried out at weeks 2, 3 and 4 using two antigens emulsified with Incomplate Freud's Adjuvant (IFA) at a dose of 0.1ml. Serum is taken 1 week after the last booster⁹^,^21,22.

Antibody measurement procedure:

Serum from experimental animals was then diluted 1/1000 in additional dilution buffer (100μl/well) and plates were incubated at 37ºC for 1 hour. Anti-mouse Ig antibodies conjugated with alkaline phosphatase were diluted in 1/500 to, 1/1000 in dilution buffer and 100 PI volume was added to each well. 100μl of dissolved PNPP (p-Nitrophenyl phosphate) was mixed into each well and the plate was incubated at 37ºC for 30 minutes. A total of 50μl 2 M NaOH was mixed into each well as a blocking agent. Optical density was read at a wavelength of 405nm using an ELISA reader²¹.

ELISA Sandwich Procedure:

CSS samples from patients were centrifuge 4x30 minutes. Reagents and samples are prepared according to the guidelines of the kit. 50ul standard is added, then added 50ul biotinylated detection antibody in each well (except blank well). When the well is ready, it will be closed using a seal plate, then incubation is carried out 45 minutes with a temperature of 37℃. Washing 4x and added 100μl 100 HRP Conjuncate except blank well and closed blank with plate sealer then incubated for 30 minutes at 37℃. Washing again 5x and add 90μL of reagent substrate to each well and then cover again, then incubate for 15 minutes at 37℃. After the well color becomes blue, add 50μ stop solution in each well and again change the color to yellow.

Data analysis:

Specificity and sensitivity tests were analyzed using Friedman test using SPSS, while comparison tests using post-hoc ANOVA tests.

Ethical Approval:

This research has been approved by the Medical Faculty Brawijaya University ethics commission with no 196/EC/KEPK. S3/08/2022.

RESULT AND DISCUSSION:

This research is a continuation of previous research conducted in silico⁹. In silico studies are very useful in predicting epitopes, new drugs, antibiotic resistance and others^4,⁹^,19^-²⁷. Based on the results of SDS-PAGE using Marker Thermo 26634, the presence of RecAg38 was confirmed using Coomassie blue coloring (Fig 1). The results showed that the recombinant antigen marker 38 was at position 38 kDa. After the elution process 1, 2 and 3, a specific pure antigen 38 recommendation was obtained, namely at 38 kDa.

Fig 1. SDS-PAGE Recombinant Antigen 38 results.

Information: (1) IPTG Induction non induction pellets; (2) Flow trough (3) Washing 1 (4) Washing 2 (5) Elution 1 (6) Elution 2, (7) Elution 3 (8) Marker.

ELISA Test on IgG Antibodies:

Antigen and antibody reactions were compared between the treatment (IgG peptide antibodies) and control (placebo) groups. Elisa Test showed that there is a significant difference (P< 0.001) between control group and treated group, indicating the treatment antibodies, namely IgG, can react better than the control group (Table 1).

Table 1: Result on the measurement of IgG Antibodies using ELISA

Sample Code	Cons. Ag (ug/mL)	Cons. Ab	OD
Control (P1)	1.25	1/1000	0.685
	2.5		0.658
	5		0.571
	10		0.675
P1	1.25	1/1000	0.677
	2.5		0.569
	5		0.606
	10		0.509
Control (P2)	1.25	1/1000	0.695
	2.5		0.68
	5		0.671
	10		0.675
P2	1.25	1/1000	1.038
	2.5		1.098
	5		1.137
	10		1.12
Control (P3)	1.25	1/1000	1.509
	2.5		1.634
	5		1.645
	10		1.755
P3 (Ag38 KDa)	1.25	1/1000	1.705
	2.5		1.963
	5		2.225
	10		3.508

The results of the ELISA test examination on IgG antibodies showed that in the IgG antibody treatment group after 38 kDa antigen injection, namely in the 38 kDa recombinant antigen P3 group, it had a relatively higher value when compared to the control group and all other antigen and antibody treatment groups. Based on these results, it can be concluded that protein and epitope recombinant antigen 38 can produce IgG antibodies in experimental animals as a marker of humoral immune response as an early sign of TB infection and a diagnosis test for tuberculosis meningitis. From the table it can be seen that the highest OD (Optical Density) value is 3,508 in the P3 group (Ag38 kDa) with an antibody concentration of 1/1000 and an antigen concentration of 10ug/mL, and in each concentration of antibody treatment which has a higher value compared to other concentrations, namely at a concentration of 1/1000, so it can be concluded that the group shows a greater amount of primary antibodies bound to the antigen, reflects higher concentrations of specific antibodies in the sample (Table 1).

To determine the best antibody harvest, The data that has been collected is carried out statistical analysis of data using Friedman's post-hoc 2-way with a value of p-value = <0.001, obtained the following results (Table 2):

Table 2. Post Hoc Test IgG Antibody Test

	K(P1)	P1	K(P2)	P2	K(P3)	P3 (Ag38)
Control (P1)		1.000	1.000	0.519	0.000	0.000
P1	1.000		0.011	0.004	0.000	0.000
Control (P2)	1.000	0.011		1.000	0.005	0.001
P2	0.519	0.004	1.000		0.015	0.002
Control (P3)	0.000	0.000	0.005	0.015		1.000
P3 (Ag38)	0.000	0.000	0.001	0.002	1.000

In the post-hoc test, the comparison results between groups K (P1) and K (P2) showed no significant difference (p = 1,000), while the comparison between groups K (P1) with K (P3) and K (P2) with K (P3) there was a significant difference, evidenced by the p-value values respectively, namely p = 0.000 and 0.001 (p-value <0.05). The results of the post-hoc test comparing the expression of group P1 with P2, and group P1 with P3 and group P2 with P3 obtained a value of p-value <0.05, namely p = 0.004, 0.000 and 0.002, it can be concluded that there are significant differences in expression between the three treatments.

ELISA Test on IgM Antibodies:

While the results of the ELISA test examination on IgM antibodies showed that in the IgM antibody treatment group after 38 kDa antigen injection, namely in the 38 kDa recombinant antigen P3 group, it had a relatively higher value when compared to the control group and all other antigen and antibody treatment groups. Based on these results, it can be concluded that protein and epitope recombinant antigen 38 can produce IgM antibodies in experimental animals as a test for the diagnosis of tuberculosis meningitis. From the table it can be seen that the highest OD (Optical Density) value is 1,368 in the P2 group with an antibody concentration of 1/5000 and an antigen concentration of 10 ug / mL, and in each concentration of antibody treatment which has a higher value compared to other concentrations, namely in the concentration of the P1: 1/1000 group; P2: 1/5000; and P3:1/2500, so it can be concluded that in the group showed a greater number of primary antibodies bound to the antigen, reflecting a higher concentration of specific antibodies in the sample (table 3).

Table 3. ELISA Test Results on IgM Antibodies

Sample Code	Cons. Ag (ug/mL)	Cons. Ab	OD
K (P1)	1.25	1/1000	1.022
	2.5		0.843
	5		0.772
	10		0.912
P1	1.25	1/1000	1.291
	2.5		1.268
	5		1.282
	10		1.296
	1.25	1/5000	0.705
	2.5		0.699
	5		0.635
	10		0.726
	1.25	1/5000	1.2
	2.5		1.286
	5		1.327
	10		1.368
	1.25	1/2500	0.123
	2.5		0.182
	5		0.178
	10		0.2
P3 (Ag 38KDa)	1.25	1/1000	0.196
	2.5		0.174
	5		0.196
	10		0.327
	1.25	1/2500	0.184
	2.5		0.218
	5		0.243
	10		0.246

To determine the best antibody harvest,. The data that has been collected is carried out statistical analysis of data using Friedman's post-hoc 2-way ANOVA Pairwise Comparisons test with a value of p-value = <0.001, obtained the following results (Table 4):

Table 4. Post Hoc Test IgM Antibody Test

	K(P1)	P1	K(P2)	P2	K(P3)	P3 (Ag38)
K(P1)		0.945	1.000	0.000	0.001	0.337
P1	0.945		1.000	0.269	0.000	0.001
K(P2)	1.000	1.000		0.168	0.000	0.001
P2	0.000	0.269	0.168		0.000	0.000
K(P3)	0.001	0.000	0.000	0.000		1.000
P3 (Ag38)	0.337	0.001	0.001	0.000	1.000

Post-hoc test showed that there was no significant difference between groups K(P1) and K(P2) and groups K(P1) and K(P3) so that it could be interpreted that the concentration of control antibodies in weeks 2 and 3 and week 2 and 4 was no difference (p=1.000 and 0.337), while group K(P2) with K(P3) there was a significant difference, This is evidenced by the p-value of p = 0.000. The expression P1 and P2 obtained a p-value of p = 0.269, so it can be concluded that there is no significant difference in expression between the two treatments. Then there is a significant difference between the expressions P1 and P3 and P2 and P3, this is evidenced by the p-value value respectively, namely p = 0.001 and 0.000.

Research on antigen 38 with media states that Lowenstein-Jensen (LJ) culture showed growth only in the group given bacterial infection, while in other groups there was no growth. These findings suggest that recombinant protein antibodies Ag38kDa, Rifampicin, and their combination can eradicate bacteria in the treatment group. There was no growth in LJ culture media. Combining recombinant antibodies with Rifampicin can develop anti-M.tb (Mycobacterium tuberculosis) treatment by providing passive vaccine candidates to accelerate the recovery of tuberculosis patients. This study provides interesting results in ex-vivo administration of recombinant Ag38kDa antibodies can inhibit infection with the M.tb pathogen, thereby reducing cellular immune responses¹³^,²⁸. In accordance with this theory, our results show that recombinant protein antigen 38 detected in the SDS-PAGE assay using Coomassie blue staining at marker markers 38 kDa elution 1,2 and 3 obtained specific pure antigen 38 recommendations. These results can be used as a diagnostic test for tuberculosis and can be developed as a new marker of tuberculosis meningitis.

The Ag38 kDa protein contains two M.tb-specific B cell epitopes, the most potent immunogen of which it prosses high specificity and sensitivity. Therefore, this study developed the recombinant protein Ag38kDa injected into mice to produce the recombinant protein Ag38kDa antibody. The use of Ag38kDa recombinant protein antibodies combined with Rifampicin is expected to be an additional treatment in increasing the effectiveness of therapy and shortening the duration of TB treatment⁹^,²⁸^,²⁹.

Our IgG and IgM antibody ELISA test results fit with the theory explaining that IgG antibody levels are higher in the most advanced and extensive forms of the disease. Patients with active TB usually show a strong IgG response but poor IgM and IgA responses. While the function of anti-M.tbantibodies in providing protective immunity is still under investigation, it has been proposed that they could be used as diagnostic markers of active disease. Previous studies analyzing IgG antibodies showed that anti-M.tbIgG antibodies were elevated in patients with active disease. In our results, IgG antibody levels were higher in the most active TB patients with or without anti-TB chemotherapy compared to health control patients, and the IgG positivity rate was the highest among the three isotypes, indicating that IgG antibodies were the highest. Broad antibody isotypes³⁰^,³¹.

High levels of antibodies to Ag38 are thought to be due to the presence of HLA phenotypes in TB that are more epitopically focused and can be recognized as antigens or also these antigens can trigger a pro-inflammatory response (Th17). Another specific epitope is Ag19 which contributes to increasing antibodies after antigen 38 with epitope-specific with TB23 antibody (TB23 Mab). AB 23 is often used for the detection of extrapulmonary TB¹⁴^,³².

Other studies have also shown that the function of M.tb antigen detection in cerebrospinal fluid (CSF) is for rapid and accurate diagnosis of TBM. This study is the first study to quantify simultaneously the M. TB antigen protein and DNA in the CSF for the diagnosis of TBM. The detection of HspX ‘dormancy antigen’ in CSF from patients with active disease was striking because its expression is believed to be induced upon bacterial exposure to dormancy-associated signals. In contrast, anti-HspX antibodies were detected in asymptomatic TB contacts rather than in those with active disease. Moreover, the occurrence of mycobacterial heterogeneity and dormant bacterial sub-populations in active TB patients and animal models has been reported. Therefore the efficient detection of HspX antigen suggests that the bacteria may be exposed to dormancy-inducing signals in the CSF milieu¹⁵^,³³.

The detection of other antigens in CSF is also noteworthy. GlcB and MPT51 are believed to be expressed very early during infection, while PstS1 and Ag85B are associated primarily with multibacillary or advanced disease. Interestingly, the arrest of M. tb multiplication in mouse lung is accompanied by a significant decrease in the levels of Ag85 complex- and PstS1- encoding mRNAs. The detection of ‘dormancy’ antigen on one hand and secretory proteins and those involved in cell wall synthesis, cell adhesion and metabolism on the other hand in the CSF suggests that bacteria of varying and diverse physiological states and phenotypes may coexist in the CSF environment¹⁵^,³⁴.

Based on this theory, it can be explained that in our study the highest OD (Optical Density) value indicates a greater amountof primary antibodies bound to the antigen, reflecting a higher concentration of specific antibodies in the sample. Then our results also prove that recombinant protein and epitope antigen 38 can produce IgG, IgM antibodies in experimental animals as a tuberculosis diagnosis test and subsequently can help detect TB and tuberculosis meningitis.

CONCLUSION:

Recombinant protein antigen 38 and epitope antigen 38 can produce IgG, IgM antibodies in experimental animals as a marker test for tuberculosis and candidate marker for tuberculosis meningitis.

ACKNOWLEDGEMENT:

Wisnu Barlianto as Dean Medical Faculty Brawijaya University, Loeky as head of Biomedical Science Program, Faculty of Medicine, Brawijaya University, Malang Indonesia. Suci and Yuda, from Laboratorium Biomedik Medical faculty Brawijaya University

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Nguyen DT. Agarwal S. Graviss EA. Trends of Tuberculosis Meningitis and Associated Mortality in Texas, 2010-2017, a Large Population-Based Analysis. PLoS One. 2019; 14(2): 1-11. doi:10.1371/journal.pone.0212729.

2. Munir B. Rianawati SB. Sutanto H. Agus SU. Mortality of Central Nervous System Infection Adult Patients in Indonesia (Descriptive Study In A Tertier Referral Hospital). J Neurol Sci. 2017; 381. doi:10.1016/j.jns.2017.08.3383.

3. Wilkinson RJ. et al.Tuberculous meningitis. Nat. Rev. Neurol.2017; 13(10): 581–598. doi: 10.1038/nrneurol.2017.120.

4. Mohammed MS. Sultana GD. Utilization of Bedaquiline among Drug Resistant-Tuberculosis patients. Asian J Pharm Res. 2022; 12(1): 132-136.doi:10.52711/2231-5691.2022.00020

5. Ssebambulidde K. Gakuru J. Ellis J. Cresswell FV.Bahr NC. Improving Technology to Diagnose Tuberculous Meningitis: Are We There Yet? Front Neurol. 2022; 13(May): 4–11.

6. Pukazhvanthen P. Anbarasu D. Basirudeen SAK. Raja A. Singh M. Assessing Humoral Immune Response of 4 Recombinant Antigens for Serodiagnosis of Tuberculosis. Tuberculosis. 2014; 94(6): 622-633. doi:10.1016/j.tube.2014.09.006.

7. Woldeamanuel YW. Girma B. A 43-year systematic review and meta-analysis: Case-fatality and risk of death among adults with tuberculous meningitis in Africa. J. Neurol. 2014; 261(5): 851–865. doi: 10.1007/S00415-013-7060-6/FIGURES/7.

8. Singh S. Singh J. Kumar S. et al. Poor Performance of Serological Tests in the Diagnosis of Pulmonary Tuberculosis: Evidence From a Contact Tracing Field Study. PLoS One. 2012; 7(7): 1-9. doi:10.1371/journal.pone.0040213.

9. Munir B. Yuni D. Hidayati N. NazwarTA. RarasTM. PrawiroSR. The Ag38-rec Mycobacterium tuberculosis Antigen as a New Candidate Marker for The Diagnostic of Tuberculosis Meningitis : In Silico Approach. Research J. Pharm. and Tech. 2023; 16(12): 1–7.

10. VordermeierHM. HarrisD P.Moreno C. Singh M. Ivanyi J. The nature of the immunogen determines the specificity of antibodies and T cells to selected peptides of the 38 kDa mycobacterial antigen.International Immunology. 1995; 7(4): 559–566. doi: 10.1093/intimm/7.4.559.

11. Hao X. et al.Characterization of antibody response against 16kD and 38kD of M. tuberculosis in the assisted diagnosis of active pulmonary tuberculosis. Ann. Transl. Med. 2020; 8(15): 945–945. doi: 10.21037/ATM-20-5476.

12. Sai PP.Ramesh MP. Recombinant Peroxidase: Production and its Prospective Applications- A Review. Res J Pharm Tech. 2018; 11(7): 3186-3196.doi:10.5958/0974-360X.2018.00586.3

13. Verma RK. Jain A. Antibodies to mycobacterial antigens for diagnosis of tuberculosis. FEMS Immunol. Med. Microbiol. 2007; 51(3): 453–461.doi: 10.1111/j.1574-695X.2007.00302.x.

14. BothamleyGH.Epitope-specific antibody levels in tuberculosis: biomarkers of protection, disease, and response to treatment.Front. Immunol. 2014; 5(JUN). doi: 10.3389/FIMMU.2014.00243.

15. Haldar S.et al.Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devR DNA Impacts the Rapid Diagnosis of Tuberculous Meningitis in Children.PLoS ONE. 2012; 7(9). doi: 10.1371/journal.pone.0044630.

16. Daniel TM. JanickiBW.Mycobacterial antigens: a review of their isolation, chemistry, and immunological properties.Microbiol. Rev.1978; 42(1): 84–113. doi: 10.1128/MR.42.1.84-113.1978.

17. RarasTYM. Sholeh G. Lyrawati D. Salivary sIg-A response against the recombinant Ag38 antigen of Mycobacterium tuberculosis Indonesian strain.International Journal of Clinical and Experimental Medicine. 2014; 7(1): 129–135.

18. Nurhidayati DY. Yuliati Y. Fahrinda A. Raras TYM. Sujuti H. Prawiro SR. Effect of Ag38kDa recombinant protein antibodies of Mycobacterium tuberculosis and Rifampicin on CD4 and CD8 lymphocytes: Ex vivo study. In: AIP Conf Proc. Indonesia: AIP Publishing; 2023.

19. Budaya TN. Permatasari HK. Widodo N. Prawiro SR. The Evolution of Polyclonal Antibody from Specific Epitope 47kDA for Detection of Bladder Cancer. Asian Pacific J Cancer Prev. 2023; 24(9): 3155–64.

20. Raras TYM. Lyrawati D. Cloning and expression of pab gene of M. tuberculosis isolated from pulmonary TB patient in E.coli DH5α. Med J Indones. 2011; 20(4): 247–54.

21. Hidayat R. Wulandari P. Enzyme Linked Immunosorbent Assay (ELISA) Technique Guideline. Biosci. Med. J. Biomed. Transl. Res. 2021; 5(5): 447–453. doi: 10.32539/bsm.v5i5.228.

22. Fleri W. Paul S. Dhanda SK. Mahajan S. Xu X. Peters B.et al.The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design. Front. Immunol. 2017; 8 (MAR). doi: 10.3389/FIMMU.2017.00278.

23. Manojkumar R. Patle SHG. In-Silico Inhibition Studies of Phenothiazine Based Compounds on Quinolinic Acid Phosphoribosyltransferase (1QPQ) Enzyme as A Potent Anti-Tuberculosis Agent. Asian J Res Chem. 2011; 4(6): 990-996.

24. Sriroopreddy R. Raghuraman P. Reena R. Modeling and Docking Study of GABA-AT Protein in Mycobacterium tuberculosis - A Computational Approach. Res J Pharm Tech. 2018; 11(4): 1283-1288. doi:10.5958/0974-360X.2018.00238.X.

25. Chikoti SP. Venkataswamy M. Gandla H. Pindrathi P. Sandhya MJB. Medicinal Plants used for the Treatment of Bacterial Meningitis. Res J Pharm Dos Forms Technol. 2019; 11(3): 239-244.doi:10.5958/0975-4377.2019.00040.5.

26. Subramaniam S. Sangeetha D. Identification of New Inhibitor against Mycobacterium tuberculosis using Structure Based Drug Designing and Docking Studies. Res J Pharmacogn Phytochem. 2017; 9(3): 173-176. doi:10.5958/0975-4385.2017.00032.2.

27. Khoirul A. Bobi P. Meike TK. Yuliati. Winarsih S. Raras TYM. Prawiro SR. Multi Epitopes Potential on Surface SARS-CoV-2 Protein as a Covid-19 Vaccine Candidate. Res J Pharm Technol. 2022; 15(4): 1437-2. doi:10.52711/0974-360X.2022.00238.

28. Fan X. et al.Construction and immunogenicity of a T cell epitope-based subunit vaccine candidate against Mycobacterium tuberculosis. Vaccine. 2021; 39(47): 6860–6865. doi: 10.1016/j.vaccine.2021.10.034.

29. Morishita M. et al. Recent advances in immunological diagnosis for tuberculosis.Kekkaku. 1994; 69(2): 97–106.

30. Raras TYM. Fahrinda A. Yuliati Y. Nurhidayati DY. Sujuti H. Prawiro SR. Immune Response Upon the Administration of Recombinant Protein Antibodies Ag-38 kDa Mycobacterium tuberculosisand Rifampicin ex-vivo.African J. Infect. Dis. 2022; 16(2):71. doi: 10.21010/AJID.V16I2.8.

31. Amicosante M.et al.Evaluation of a novel tuberculosis complex-specific 34 kDa protein in the serological diagnosis of tuberculosis. Eur. Respir. J. 1995; 8(12): 2008–2014. doi: 10.1183/09031936.95.08122008.

32. El-Masry S. El-Kady I. ZaghloulMH.Al-BadraweyMK.Rapid and simple detection of a Mycobacterium circulating antigen in serum of pulmonary tuberculosis patients by using a monoclonal antibody and Fast-Dot-ELISA.Clin. Biochem. 2008. 41(3): 145–151. doi: 10.1016/J.CLINBIOCHEM.2007.11.008.

33. Hur Y. G.et al.Evaluation of antigen-specific immunoglobulin g responses in pulmonary tuberculosis patients and contacts. J. Clin. Microbiol. 53(3): 904–909. doi: 10.1128/JCM.03050-14.

34. Dinnes J.et al.A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol. Assess. 2007; 11(3): 1–178. doi: 10.3310/HTA11030.

Received on 05.10.2023 Modified on 24.02.2024

Research J. Pharm. and Tech 2024; 17(9):4337-4342.

DOI: 10.52711/0974-360X.2024.00670