A highly sensitive test system, based on the immuno-PCR method, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was to obtain supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were to be used as DNA-tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not lower than 10 pg/mL.

Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.

We studied the effect of combined treatment with cisplatin, glucosaminylmuramyl dipeptide, and TNF-α on viability of MCF-7, U-937, B16, and L-929 tumor cells, Ehrlich ascites carcinoma cells, and normal cells (human peripheral blood lymphocytes, peritoneal macrophages, and mouse bone marrow cells). Glucosaminylmuramyl dipeptide was nontoxic for normal and tumor cells, but promoted death of tumor cells after administration in combination with cisplatin and/or TNF-α. At the same time, glucosaminylmuramyl dipeptide did not modulate the cytotoxic effect of individual or combined treatment with cisplatin and TNF-α on normal cells. Administration of glucosaminylmuramyl dipeptide to cultured MCF-7 cells 20 h before the study increased the potentiating effect of muramyl peptide.

As shown in this work, the synthetic immunomodulator glucosaminylmuramyldipeptide (GMDP) can be included into acellular pertussis vaccine (APV). The optimal doses of GMDP, ranging from 0.001 to 0.0001 microg, have been found. These doses enhance the protective activity of APV, especially its low-active doses. GMDP decrease the manifestations of toxic, anaphylactogenic and pyrogenic properties of APV, which may lead to the decrease of the antigenic load of APV on the body of the vaccines and thus to lessening the side-effects of vaccination. GMDP has been shown to considerably increase, in comparison with common pertussis vaccine and APV, the percentage of phagocytizing leukocytes by day 14. The immunization of mice with APV with and without GMDP in doses of 0.01 and 0.001 microg leads to a change in T-lymphocyte/B-lymphocyte ratio in the population of spleen lymphocytes.

Flow cytometry was used to demonstrate that cultured human melanoma BRO cells expressed membrane‐bound tumour necrosis factor‐alpha (TNF‐α) and were able to release TNF‐α upon treatment with glucosaminylmuramyl dipeptide (GMDP). The released TNF‐α was shown to prime melanoma cells, previously unable to respond to GMDP by increasing expression of melanoma‐associated antigens, making them sensitive to GMDP treatment.

A significant difference in the set of surface antigens was found for melanoma sublines of different metastatic potential. On the one hand, cells of a subline with a higher metastatic potential lost antigens involved in immunological recognition (HLA-DR, ICAM-1) and development of antitumor immunity (IL-1 beta); on the other hand, they acquired new antigenic determinants LAMP-1 and SiaLe(x) promoting ligand interactions with receptors in active metastasis. At the same time, tumor-associated antigens MUG-18 and MCA-C1 as well as ICAM-3 adhesion molecules were found on the cell surface of both sublines. Glucosaminylmuramyl dipeptide selectively regulated expression of these antigens; namely, its modulating effect appeared only if the antigen was initially present on the cell surface.

In this study flow cytometry was used to show that macrophages were the major population of murine peritoneal exudate cells (MPEC), increasing Ia expression upon treatment with N-acetylglucosaminyl-beta-1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Modulation of Ia expression resulted from direct action of GMDP on macrophages, rather than from effect of cytokines released by T-cells. The effect of GMDP on two populations of macrophages, namely, slow and rapid responding, was studied in detail. Rapid responding cells were represented by Ia-positive macrophages: GMDP augmented their Ia expression. In contrast, slow responding subpopulation was represented by initially Ia-negative macrophages, in which GMDP induced de novo synthesis of Ia-antigens. The ability to induce Ia expression was also characteristic for other adjuvant-active N-acetylglucosamine-containing muramyl peptides (GMPs). Macrophages were shown to engulf GMPs by endocytosis. Activation of macrophages by GMDP resulted in an increase in their phagocytic activity.

The muramylpeptide N-acetylglucosaminyl-beta 1----4-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) was shown by flow cytometry to produce a dose-dependent increase in the expression of Ia-antigens by mouse peritoneal macrophages in vitro. Muramyldipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) had a similar, but weaker, effect. GMDP also induced expression of Ia-antigens by murine peritoneal macrophages in vivo. GMDP acted directly on the macrophages because Ia-antigen expression by cells of the cloned mouse myelomonocytic line WEHI-3 was also induced. Expression of the interleukin-2 receptor on the surface of the macrophages was also stimulated by GMDP, indicating that GMDP may influence development of the immune response through this mechanism.