Serological Cross-Reaction between Six Thiadiazine by Indirect ELISA Test and Their Antimicrobial Activity

Abstract

Malaria is a parasitic infection caused by a protozoon of the genus Plasmodium, transmitted to humans by female biting mosquitoes of the genus Anopheles. Chloroquine and its derivates have caused the parasite to develop drug resistance in endemic areas. For this reason, new anti-malarial drugs as treatments are crucial. This work aimed to evaluate the humoral response. with hyper-immune sera, of mice immunized with six derivatives of tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) by indirect ELISA test. The cross-reactivity between the compounds as antigens and their microbial activity on Gram-positive and Gram-negative bacteria was evaluated. The results of the humoral evaluation by indirect ELISA show that three bis-THTTs react with almost all of the above. Besides, three compounds used as antigens stimulate the BALB/c mice's immune system. The best combination of two antigens as a combined therapy displays similar absorbances between the antigens in the mixture, showing similar recognition by antibodies and their compounds. In addition, our results showed that different bis-THTT presented antimicrobial activity on Gram-positive bacteria, mainly on Staphylococcus aureus strains, and no inhibitory activity was observed on the Gram-negative bacteria tested.

Keywords: antimicrobial activity; cross-reaction; indirect ELISA; malaria; thiadiazine.

Figure 1

Chemical structure of bis-THTT (JH-1 to JH-6), and for Chloroquine (CQ).

Figure 2

FT-IR absorption spectrum of 2-(5-{6-[5-(Carboxymethyl)-2-thioxo-1,3,5-thiadiazinan-3-yl]butyl}-6-thioxo-1,3,5-thiadiazinan-3-yl)-ethanico acid (JH1). On the X-axis are represented the wavenumber (cm⁻¹) vs. transmittance on the Y-axis.

Figure 3

UV/Vis absorption spectrum of 2-(5-{6-[5-(Carboxymethyl)-2-thioxo-1,3,5-thiadiazinan-3-yl]butyl}-6-thioxo-1,3,5-thiadiazinan-3-yl)-ethanico acid (JH1). On the X-axis is represented the wavelength (nm) vs. absorbance on the Y-axis.

Figure 4

Results of indirect ELISA (Panels A1, A2, A3, A4, A5, A6 and A7), with 10 µg/mL concentration of antigens (CQ, JH1, JH2, JH3, JH4, JH5 and JH6). On the X-axis are represented the 1:200 dilutions of the hyperimmune sera vs. absorbance at 405 nm on the Y-axis.

Figure 5

In vitro antimicrobial activity of bis-THTTs (JH1, JH2, JH4, JH5 and JH6) on Gram-positive bacteria. (A): Staphylococcus aureus ATCC 25923, (B): Listeria monocytogenes CVCM 446. CQ was used as the positive control.

Figure 6

In vitro antimicrobial activity of bis-THTTs (JH1, JH2, JH4, JH5 and JH6) on Gram-negative bacteria. (A): Salmonella enteritidis CVCM 497, (B): Escherichia coli ATCC 25922, (C): Pseudomonas aeruginosa ATCC 10145. CQ was used as the positive control.

Figure 7

Antimicrobial activity test of bis-THTTs (JH1, JH2, JH4, JH5 and JH6) on different strains of Staphylococcus aureus. (A): S. aureus ATCC25923, (B): Enterotoxigenic S. aureus (SEA), (C): S. aureus ORSA. CQ was used as the positive control.