ABSTRACT
Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods-biological tests on animals or cell cultures as well as ELISA-is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins-enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)-in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.
Subject(s)
Exotoxins/analysis , Immunoassay/methods , Staphylococcus aureus/metabolism , Culture Media/analysis , Exotoxins/metabolism , Staphylococcus aureus/chemistry , Staphylococcus aureus/growth & developmentABSTRACT
xMAP technology was used for simultaneous identification of six protein toxins (staphylococcal enterotoxins A and B, cholera toxin, ricin, botulinum toxin A, and heat labile toxin of E. coli). Monoclonal antibody-conjugated xMAP microspheres and biotinilated monoclonal antibodies were used to detect the toxins in a sandwich immunoassay format. The detection limits were found to be 0.01 ng/mL for staphylococcal enterotoxin A, cholera toxin, botulinum toxin A, and ricin in model buffer (PBS-BSA) and 0.1 ng/mL for staphylococcal enterotoxin B and LT. In a complex matrix, such as cow milk, the limits of detection for staphylococcal enterotoxins A and B, cholera toxin, botulinum toxin A, and ricin increased 2- to 5-fold, while for LT the detection limit increased 30-fold in comparison with the same analysis in PBS-BSA. In the both PBS-BSA and milk samples, the xMAP test system was 3-200 times (depending on the toxin) more sensitive than ELISA systems with the same pairs of monoclonal antibodies used. The time required for a simultaneous analysis of six toxins using the xMAP system did not exceed the time required for ELISA to analyze one toxin. In the future, the assay may be used in clinical diagnostics and for food and environmental monitoring.
Subject(s)
Bacterial Toxins/analysis , Enzyme-Linked Immunosorbent Assay , Ricin/analysis , Animals , Antibodies, Monoclonal/immunology , Botulinum Toxins, Type A/analysis , Cattle , Enterotoxins/analysis , Milk/metabolismABSTRACT
We have shown that glucosaminyl muramyl dipeptide (GMDP) has been augmented the antitumor action of chemotherapy drug cisplatin and tumor necrosis factor-alpha (TNFalpha) on the Ehrlich ascites carcinoma and melanoma B-16 mouse tumor models. The doses of cisplatin, TNFalpha and GMDP and also the conditions of the drugs combination injection provided 100% survival of mice with Ehrlich ascites carcinoma were found. Furthermore, it was shown first that GMDP has been decreased toxicity of the cisplatin/TNFalpha combination and normalized the changes in the experimental mice hematological parameters which were produced by the CP/TNFalpha combination.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/administration & dosage , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/toxicity , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/toxicity , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/toxicity , Carcinoma, Ehrlich Tumor/drug therapy , Cisplatin/administration & dosage , Cisplatin/toxicity , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Synergism , Female , Humans , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Survival , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/toxicityABSTRACT
We have demonstrated that biologically active muramyl peptides, in particular, glucosaminylmuramyl dipeptide (GMDP), augmented in vitro cytotoxic activity of tumor necrosis factor-alpha (TNF-alpha) against murine fibrosarcoma L929 cells. The introduction of GMDP resulted in cytotoxic effect characteristic for substantially higher dose of cytokine. Even more potent was the combination of GMDP, TNF-alpha and Actinomycin D (ActD). According to clonogenic and MTT assays 100% L929 cells could be killed in culture with low doses of TNF-alpha and ActD if GMDP was present. When cisplatin was substituted for ActD similar results were obtained. GMDP also enhanced cytotoxicity of TNF-alpha and cisplatin against human breast carcinoma MCF7 and histiocytic lymphoma U937 cells. Normal cells, namely human peripheral blood leucocytes and murine peritoneal macrophages, were resistant to selected doses of TNF-alpha/cisplatin/GMDP.
