Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 52, Num. 3, 2004, pp. 359-362

Neurology India, Vol. 52, No. 3, July-September, 2004, pp. 359-362

Original Article

Demonstration of IgG antibodies to 30 Kd protein antigen in CSF for diagnosis of tuberculous meningitis by antibody-capturing ELISA

Kashyap RS, Kainthla RP, Satpute RM, Chandak NH, Purohit HJ, Taori GM, Daginawala HF

Biochemistry Research Laboratory, Central India Institute of Medical Sciences, Nagpur - 440 010
Correspondence Address:Biochemistry Research Laboratory, Central India Institute of Medical Sciences, Nagpur - 440 010 hfd_ciims@rediffmail.com

Code Number: ni04116

ABSTRACT

KEY WORDS: Tuberculous meningitis, mycobacterium tuberculosis, tubercular culture filtrate protein, 30 Kd protein antigen, cerebrospinal fluid, ELISA

INTRODUCTION

Materials and Methods

The patient population included all the patients admitted to the Neurology wards, Central India Institute of Medical Sciences (CIIMS) between January 2003 and July 2003, in whom a diagnostic lumbar puncture had been performed. CSF examination included total and differential cell counts, biochemistry, and smear for Gram′s, India ink, and acid-fast bacilli (AFB) staining. Patients were grouped as follows:

I. Tuberculous Meningitis:
Diagnosis of TBM was based on the clinical features, which included subacute or chronic fever, signs of meningeal irritation with or without other features of CNS involvement and CSF findings. CSF findings suggestive of TBM were increased proteins, decreased glucose (CSF to blood glucose ratio < 0.5), and pleocytosis with lymphocyte predominance. Patients in whom AFB was demonstrated either by smear or culture, were considered "definite" cases of TBM. In the remaining cases response to antituberculous drugs was considered as a criterion for diagnosis and these patients were considered as "suspected/probable" cases of TBM.

II. Pyogenic meningitis:
Pyogenic meningitis was suspected in patients with acute high-grade fever with features of meningitis and often with associated altered sensorium. CSF findings suggestive of pyogenic meningitis included increased proteins, very low sugar (CSF to blood glucose ratio < 0.2) and pleocytosis with polymorphonuclear predominance. However, CSF findings in partially treated cases of pyogenic meningitis can be very similar to TBM. Response to appropriate antibiotics was also considered as one of the diagnostic criteria.

III. Viral meningitis:
Viral meningitis was suspected in patients who presented with acute onset of fever and symptoms and signs of meningeal irritation. In such patients CSF showed mild increase in protein, glucose often normal (CSF to blood glucose ratio > 0.5) and pleocytosis, predominantly lymphocytic. None of them had clinical evidence of extracranial tuberculosis. Those with clinical suspicion of herpes encephalitis were treated with acyclovir and other viral meningitides were treated symptomatically.

IV. Non-infectious neurological disorders:
All other patients who had no clinical features of meningitis and had no evidence of CNS or extra-CNS tuberculosis were grouped in the non-infectious /control group.

Laboratory studies
About 2-3 ml of CSF was collected in sterile glass tubes in all the patients and was stored at -80^oC.

Antigens: After the separation of protein from the CSF of patients with TBM by SDS-PAGE, the 30Kd protein bands were sliced from the gels 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 h, at 30 volts,[14] and harvested from the unit and dialyzed against PBS, and the protein content was measured by Bio Lab Kit. Running native PAGE checked the purity of protein. CFP isolated from M. tuberculosis H₃₇ Rv Strain, basically are of bacterial origin (Colorado State University, USA). These are the proteins which are filtered after growing the culture of M. tuberculosis bacilli. These proteins (antigens) were used for antibody-capturing Enzyme Linked Immunosorbent Assay (ELISA).

Antibodies: A polyvalent antibody to 30Kd protein was raised in rabbits [1:1000] (Bangalore Genei, Bangalore), and H₃₇R_V Culture filtrate protein antibodies [1:5000] (rabbit polyclonal sera C-193) were obtained from Colorado State University, USA.

Antibody-capturing ELISA
Antibody-capturing ELISA described by Voller et al[15] was employed with minor modification. The assay was first standardized by reacting 30Kdpa or CFP antigen with respective antibodies with different dilutions prior to analysis of patients′ samples. The flat-bottom microliter wells were coated with 100 µl of 30Kdpa (15µgm/ml) or CFP antigen (10µgm/ml) and incubated overnight at 40C. 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 100 µl of 0.5% BSA/PBS at 37°C for 1 hour. The blocking solution was then removed from the wells. 100 µl of diluted CSF 1:10 in PBS was added to the wells and incubated at 37°C for 60 minutes. The wells were again washed with the PBS. 100 µl of affinity-purified horseradish peroxides conjugated anti-human 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, 100µl of Tetramethyl benzindine/hydrogen peroxide substrate solution was added into the wells and incubated at room temperature for about 20 min. The reaction was then stopped with 100 µl of 2.5 N sulphuric acid.

Results

The absorbance value of IgG to 30 Kdpa and CFP antigen in CSF are presented in [Figure - 1]. The cut-off values (OD=450nm) for positivity to 30Kdpa and CFP antigen in the control CSF were 0.046 and 0.150 respectively. In the patients with definite, probable or suspected TBM, the CSF positivity for 30 Kdpa was 80% (16/20) and for CFP it was 85% (17/20) [Table - 2]. While the CSF positivity for patients with pyogenic meningitis was 12.5% (1/8) for 30 Kdpa and 25% (2/8) for CFP. In the control group, 2 patients had CSF positivity for CFP and none for 30 Kdpa.

The sensitivity and specificity of IgG antibodies for the diagnosis of suspected patients of TBM using 30 Kdpa was 80% and 91% respectively. However, the sensitivity and specificity in the two confirmed cases of TBM was 100%. The sensitivity and specificity of the IgG antibodies using CFP antigen was 85% and 94% respectively. No significant difference was observed in the positivity to 30 Kdpa and CFP antigen in the CSF of TBM patients.

DISCUSSION

In this study antibody-capturing ELISA has been used to detect 30Kdpa antibodies in the CSF of TBM and control patients. Antibody-capturing ELISA is most commonly used for antibody detection in TBM because it is technically very simple with high sensitivity and specificity.[17] Hernandz et al[18] by using antibody-capturing ELISA demonstrated either IgG or/and IgM antibodies to BCG in CSF from all the patients with TBM and none in the control subjects, indicating that both the sensitivity and specificity of antibody detection by capturing ELISA were 100%. This data strongly suggests that capturing ELISA of CSF IgG antibodies is a promising tool for the diagnosis of TBM

Earlier many workers have attempted the use of various MTB antigen markers such as lipoarabinomannan, purified protein derivatives, heat shock protein of 62 Kd and 14Kd, GroE, Ag 85 complex, 38Kd antigen, etc[3],[7],[11] which have now been recognized as potential markers for the diagnosis of TBM and pulmonary tuberculosis. 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.

Acknowledgement

REFERENCES

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