Detection of salinomycin and lasalocid in chicken liver by icELISA based on functional bispecific single-chain antibody (scDb) and interpretation of molecular recognition mechanism.

Salinomycin (SAL) and lasalocid (LAS) are widely used as ionophore antibiotics for coccidiosis control. However, their common use as feed additives has led to the occurrence of feed cross-contamination, which has toxic effects on non-target animals. There have been few reports on multiple-residue detection for SAL and LAS in recent years. In this study, two single-chain antibody fragments (scFvs) capable of specifically recognizing SAL and LAS were constructed. Using LAS-scFv and SAL-scFv as parent antibodies, a complete bispecific single-chain diabody (scDb) against both LAS and SAL was built using splicing by overlap extension polymerase chain reaction (SOE-PCR). In addition, the key amino acid sites and interaction energy of antibody variable regions for small-molecule recognition were preliminarily studied by homology modeling and molecular docking. Finally, IC50 values of 12.9 and 8.6 ng/mL, with a linear range of 6.9-24.0 and 4.7-16.0 ng/mL, were obtained for LAS-scFv and SAL-scFv, respectively. An indirect competitive enzyme-linked immunosorbent assay (icELISA) method was established using scDb to obtain an IC50 of 3.5 ng/mL for LAS and 4.1 ng/mL for SAL, which showed better sensitivity and specificity than those of the parent scFv antibodies. The recoveries of LAS and SAL in chicken liver were 89.2-92.7%(CV<4.7%) and 88.6-90.2% (CV<6.8%)), respectively.

d-Glycero-ß-d-Manno-Heptose 1-Phosphate and d-Glycero-ß-d-Manno-Heptose 1,7-Biphosphate Are Both Innate Immune Agonists.

d-Glycero-ß-d-manno-heptose 1,7-biphosphate (ß-HBP) is a novel microbial-associated molecular pattern that triggers inflammation and thus has the potential to act as an immune modulator in many therapeutic contexts. To better understand the structure-activity relationship of this molecule, we chemically synthesized analogs of ß-HBP and tested their ability to induce canonical TIFA-dependent inflammation in human embryonic kidney cells (HEK 293T) and colonic epithelial cells (HCT 116). Of the analogs tested, only d-glycero-ß-d-manno-heptose 1-phosphate (ß-HMP) induced TIFA-dependent NF-κB activation and cytokine production in a manner similar to ß-HBP. This finding expands the spectrum of metabolites from the Gram-negative ADP-heptose biosynthesis pathway that can function as innate immune agonists and provides a more readily available agonist of the TIFA-dependent inflammatory pathway that can be easily produced by synthetic methods.

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors.

Okadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

Monoclonal antibody production and immunochemical detection of polyether antibiotics.

Polyether antibiotics such as monensin and salinomycin have been primarily used as coccidiostat and growth promoter. Since residues of these antibiotic in food may pose a health risk for sensitive individuals, their use should be carefully monitored. An immunochemical method was developed for the determination of polyether antibiotic using monoclonal antibody (Mab) produced by immunized mice. Conjugates of monensin, salinomycin and laidlomycin were prepared with bovine serum albumin (BSA), keyhole limpet haemocyanine (KLH) and ovalbumin (OVA) by mixed anhydride method and then used as immunogene to produce Mab. Eight hybridoma cell lines were isolated that produced Mabs that competed with polyether antibiotic-protein conjugates in BALB/c-SP2/0 fusion system. Two hybridoma with higher sensitivity, designated as 4G11F and 1C8F1F, were cultured for mass production and then purified from ascites fluid. Antibiotic-protein conjugates were quantitavely analyzed by using the purified Mabs through a competitive enzyme-linked immunosorbent assay (ELISA).

Production of polyclonal antibodies against territrem B and detection of territrem B in the conidia of Aspergillus terreus 23-1 by immunoelectron microscopy.

Territrem B, a fungal metabolite isolated from Aspergillums terreus 23-1, is a tremorgenic mycotoxin. Immunoelectron microscopy using anti-territrem B polyclonal antibody was used to detect territrem B in the fungal body of A. terreus 23-1 at different times of culture without shaking on potato dextrose (PD) agar medium. The anti-territrem B serum was produced by immunization of rabbits with 4beta-hydroxymethyl-4beta-demethylterritrem B-sccinate bound by a linker to keyhole limpet hemocyanin. This antiserum recognized territrems and immunoelectron microscopy using this antiserum, and colloidal gold-conjugated anti-rabbit IgG antibodies showed that territrem B was localized to the fungal body of A. terreus 23-1. Territrem B was first seen in the cytoplasm of the conidia after 4 days' culture on PD agar medium. Maximal territrem B production in the conidia was seen on the 14th day of culture; however, territrem B was not formed in the hyphae at any stage of culture. These results are consistent with the previous finding that the formation of territrems is related to fungal sporulation.

Development of an ultrasensitive immunoassay for rapid measurement of okadaic acid and its isomers.

This report highlights the characteristics of an okadaic acid immunoassay with limits of detection in the subfemtomole range. Two different immunoassay formats were investigated and their characteristics compared in relation to linear ranges, limits of detection, and cross-reactivity with other seafood toxins present in water and/or mussel samples. The developed ELISA system can be manipulated to quantitatively measure total diarrhetic shellfish poisoning (DSP) content or for okadaic acid and dinophysistoxin-1 individual concentrations by variation of the format of the immunoassay. Real mussel samples were validated in percentage recovery test. Calibration curves were established, and aliquots of real samples were tested. Very good recoveries were attained, highlighting the validity of the ELISA system to accurately determine the DSP concentration in mussel samples.

Specificity of mouse monoclonal anti-okadaic acid antibodies to okadaic acid and its analogs among diarrhetic shellfish toxins.

The specificity of five mouse monoclonal antibodies to okadaic acid was studied for use in an enzyme-linked immunosorbent assay of okadaic acid and its analogs. OA8-2 and OA22-22 antibodies (IgG2a-kappa), which bind more strongly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 than to okaic acid or 7-O-palmitoyl-okadaic acid in 50% aqueous methanol, were useful in the detection of dinophysistoxins-1 and -3. OA10-8 (IgG1-kappa), which binds more strongly to 7-O-palmitoyl-okadaic acid and 7-O-palmitoyl-dinophysistoxin-1 than to okadaic or dinophysistoxin-1 in 50% aqueous methanol, was useful in the detection of dinophysistoxin-3. OA423-3 (IgG1-kappa), which binds weakly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 in 20% aqueous methanol, was useful in the selective detection of okadaic acid. OA958-2 (IgG1-kappa), which binds with equal strength to each of the four toxins in methanol, was useful in the detection of all okadaic acid analogs, and the minimum detectable concentration was 30 ng/ml. OA423-3 and OA958-2 retained their binding ability in 50% acetone, ethyl ether, or benzene in methanol.

Binding competition of okadaic acid derivatives to anti-okadaic acid antibody.

The serologic activities of structurally related okadaic acid derivatives have been determined. Binding of [3H]okadaic acid to rabbit anti-okadaic acid is inhibited with equal effectiveness by okadaic acid, dinophysistoxin-1, acanthifolicin, okadaic acid tetramethyl ether, and okadaic acid spiroketal II. Okadaic acid spiroketal I, which lacks the F- and G-rings of okadaic acid, inhibits serologic binding about 60 times less effectively. The F- and G-rings of okadaic acid may comprise part of the epitopes recognized by some of the polyclonal antibodies.

Production of monoclonal antibodies to salinomycin.

Protein conjugates of salinomycin were prepared and utilized to produce murine monoclonal antibodies (MAbs) in the BALB/c-P3X63.Ag 8.653 fusion system. Several antibodies were cloned, stabilized, and evaluated for cross-reactivity to other ionophorous antibiotics. Selected antibodies were used to develop quantitative assays for salinomycin by means of an enzyme-linked immunosorbent assay (ELISA). These assays were sensitive to 50 ng of salinomycin.

Anticoccidial activity of narasin in broiler chickens reared in floor pens.

The anticoccidial activity of the ionophore narasin was tested in 3 floor-pen experiments. Narasin was tested at levels of 40, 60, 80, 100, or 120 ppm and compared against feeding ration containing 80, 100, or 121 ppm monensin or no medication. Feeding at all levels of narasin significantly prevented coccidiosis-induced mortality and improved weight gains and feed conversion ratios compared with the same parameters in groups given unmedicated feed. Protection with narasin was equal to or slightly greater than the protection obtained with monensin. The reduction in intestinal lesion scores was greater with narasin medication than with monensin. Analysis of pooled data from these trials indicated that a level between 48 and 96 ppm narasin would provide the optimum, depending on whether maximum weight gain or optimum feed conversion ratio was desired. Testing for coccidial immunity using the immunity challenge technique indicated that, based on the parameters of weight gain and lesion scores, even levels as low as 40 ppm narasin had enough efficacy to significantly reduce the amount of immunity which developed in medicated birds compared with the level of immunity developed in birds receiving unmedicated feed.

Macrophage activation and mobilization in nude mice by Corynebacterium parvum and pyran: a functional and histologic study.

Peritoneal macrophages from both congenitally athymic ("nude") mice and heterozygous littermates were activated by pyran copolymer or by Corynebacterium parvum vaccine. C parvum did not produce an increase in the number of peritoneal macrophages in nude mice, although it did produce a typical splenomegaly. Pyran produced an even greater influx of macrophages in the peritoneum of nude mice, when compared to normal mice, but did not produce splenomegaly in nude mice. Pyran- and C parvum-induced splenomegaly were accompanied by an increase in the apparent T-cell population of germinal centers. These experiments indicate that: 1) Macrophage activation, per se, by either C parvum, is a thymus-independent event; 2) Macrophage mobilization, as determined by organomegaly or PEC number, does not have an obligatory requirement for T-cells (depending on the agent used); 3) Macrophage activation may not always correlate with mobilization; and 4) Mechanisms for attracting and sequestering macrophages in the peritoneum may be different from those of the spleen.

Expression of a new cell surface antigen on activated murine macrophages.

A macrophage cell-surface antigen associated with pyran and Corynebacterium parvum-activated macrophages and P388D1 cells but not detectable on normal or glycogen and thioglycollate-elicited murine macrophages has been described. The antigen was demonstrated both by complement-mediated cytotoxicity and immunofluorescence, using an appropriately absorbed rabbit antiserum to P388D1. This antiserum should enable the characterization of activated macrophage cell populations on an individual cell basis and should be a useful probe to study the interactions of macrophages with tumor cells and the relationship of activation to cell-surface changes.

Specific potentiation of L1210 vaccine by pyran copolymer.

Pyran copolymer (NSC 46015) was found to potentiate strongly the immune response of C57BL/6J X DBA/2 F1 mice to 10(4) live L1210 tumor cells following suboptimal vaccination with 10(7) radiation-inactivated L1210 cells. Optimal immunity to challenge was produced by concomitant i.p administration of pyran and L1210 vaccine, and activity was dependent upon both pyran and vaccine dosages. In addition, this immunopotentiation seemed to be related to the intrinsic viscosity of different pyran preparations tested, although all the pyran compounds had significant activity. Furthermore, the increased immunity of subsequent live tumor challenge appeared to be specific for the vaccinating cell type.