A Bispecific Antibody Based Assay Shows Potential for Detecting Tuberculosis in Resource Constrained Laboratory Settings

The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings

Effect of cupric-isonicotinic acid hydrazide complex on avian myeloblastosis virus reverse transcriptase and its associated enzyme activities

Cupric complex of isonicotinic acid hydrazide inhibits DNA synthesis by avian myloblastosis virus reverse transcriptase. This inhibition occurs in the presence of either ribonucleotide or deoxyribonucleotide templates. The inhibition of reverse transcriptase by cupric-INH complex is considerably reduced when stored or proteolytically cleaved enzyme was used in the reaction. The complex also inhibits the reverse transciptase-associated RNase H activity. The cupric-isonicotinic acid hydrazide complex cleaves pBR 322 from I DNA into smaller molecules in the presence or absence of reverse transcriptase-associated endonuclease. However, in the presence of the enzyme the DNA is cleaved to a greater extent

Inhibition of avian myeloblastosis virus reverse transcriptase and virus inactivation by metal complexes of isonicotinic acid hydrazide

The cupric and ferric complexes of isonicotinic acid hydrazide (INH) inhibit the DNA synthesis catalysed by avian myeloblastosis virus (AMV) reverse transcriptase. The inhibition was to the extent of 95% by 50 μM of cupric-INH complex and 55% by 100 μM of ferric-INH complex. These complexes have been found to bind preferentially to the enzyme than to the template-primer. Kinetic analysis showed that the cupric-INH complex is a non-competitive inhibitor with respect to dTTP. The time course of inhibition has revealed that the complexes are inhibitory even after the initiation of polynucleotide synthesis. In vivo toxicity studies in 1-day-old chicks have shown that the complexes are not toxic up to a concentration of 500 μg per chick. Infection of the 1-day-old chicks with AMV pretreated with 150 μg of either of the complexes prevented symptoms of leukemia due to virus inactivation

Antiviral activity of liposome-encapsulated cupric complex of isonicotinic acid hydrazide against avian myeloblastosis virus infection

Cupric complex of isonicotinic acid hydrazide (cupric-INH complex) has been shown to inactivate avian myeloblastosis virus (AMV). The complex did not have any demonstrable chemotherapeutic activity against AMV infection in chicks. However, when encapsulated in positive charged liposomes the complex partially protected the chicks from AMV infection. The levels and stability of the complex in various tissues particularly in bone marrow, the target tissue for AMV multiplication, were higher in liposome-encapsulated cupric-INH complex injected than in chicks injected with unencapsulated complex

Effect of cupric-isonicotinic acid hydrazide complex on normal and avian myeloblastosis virus-infected cells cultivated in vitro

The cupric complex of isonicotinic acid hydrazide was found to be nontoxic to normal yolk sac macrophages upto a concentration of 100 ΜM. At this concentration the complex did not significantly inhibit DNA, RNA or protein synthesis in these cells. The complex inhibited the avian myeloblastosis virus multiplication in these cells when added 0–4 h post-infection as demonstrated by the inhibition of both focus formation and expression of viral specific antigens. This inhibition was not observed when the complex was added 8 and 16 h after avian myeloblastosis virus infection. The studies carried out on avian myeloblastosis virus-transformed myeloblasts indicated that the complex had no effect on the colony (focus) formation. The results suggest that the complex inhibits the virus multiplication by interfering in an early event of viral growth cycle, possibly the process of reverse transcription

Studies on the Interaction of Cupric Isonicotinohydrazide with DNA

The interaction of cupric isonicotinohydrazide $(Cu^{II}INH)$, an antiviral compound, with calf thymus DNA was investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR). Gel electrophoresis of DNA incubated with $Cu^{II}INH$ showed cleavage of DNA to various extents. This cleavage was found to be time and concentration dependent. In the presence of $Cu^{II}INH$ the positive CD band at 274 nm disappeared and the negative band at 246 nm showed a decrease in the mean residual ellipticity value, indicating binding of $Cu^{II}INH$ to DNA. $^{31}P$ NMR studies indicated that the binding of copper in $Cu^{II}INH$ is to the phosphate oxygen of the DNA backbone. The binding of $Cu^{II}INH$ was also found to be reversible. Addition of ethylenediaminetetraacetic acid to the $Cu^{II}INH-DNA$ complex resulted in breaking of the complex and restoring the original structural features of the B family of DNA in the resulting fragments. At the concentration level of $Cu^{II}INH$ employed, both $CuS0_4$ and INH independently did not show any interaction with DNA