Fluorescence immunoassay based on the inner-filter effect of carbon dots for highly sensitive amantadine detection in foodstuffs.

Immunoassays with ultra-high sensitivity for the rapid detection of chemical contaminants in food are urgently required. However, conventional enzyme-linked immunosorbent assay (ELISA) usually suffer from the moderate sensitivity. Herein, we aim to improve the sensitivity of conventional ELISA by employing the fluorescent carbon dots (CDs) as the signal probes based on the principle of inner filter effect (IFE). In this strategy, the enzymatically formed products of horseradish peroxidase/alkaline phosphatase efficiently quenched the CDs via the IFE. The absorption signal of the conventional ELISA was converted into the fluorescence signal. The fluorescent immunoassay was successfully developed and used to detect amantadine residues in chicken, achieving a limit of detection of 0.02 ng mL-1. The fluorescent immunoassay is a straightforward, extendable and general strategy and exhibits potential in detecting trace amounts of chemical contaminants in foodstuff.

Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken.

A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

Specific colorimetric ELISA method based on DNA hybridization reaction and non-crosslinking gold nanoparticles aggregation for the detection of amantadine.

Amantadine (AMD), a banned antiviral veterinary drug, is still being abused. This study developed a novel enzyme linked immunosorbent assay for the colorimetric detection of AMD involving DNA hybridization reaction and non-crosslinking gold nanoparticles (AuNPs) aggregation. Accordingly, the Primer 1-AuNPs-anti-AMD monoclonal antibody (mAb) could be captured by AMD artificial antigen on ELISA wells. Primer 2, which was complementary paired to Primer 1, was eventually added into the ELISA wells. After the hybridization reaction, the free Primer 2 in the supernatant was mixed with AuNPs and NaCl and induced a rapid color change of AuNPs. The lack of AMD in the sample resulted in capturing a substantial Primer 1-AuNPs-mAb complex and limited free Primer 2 in the supernatant. After adding NaCl, the color of AuNPs turned blue with limited Primer 2. This simple and visualized novel method had good sensitivity (0.033 µM) and exhibited a potential application for AMD screening on site.

Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver.

A monoclonal antibody (mAb) was produced in order to monitor the illegal use of amantadine and rimantadine in animals. The produced mAb 2G3 exhibited an IC50 value of 15.8µgL-1 for amantadine and exhibited cross-reactivity to both amantadine (100%) and rimantadine (70.6%). Standard curves ranged from 5 to 80µgL-1 for 2G3. The limits of detection of the developed indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) ranged from 5.0µgkg-1 to 5.4µgkg-1 in chicken muscle and liver. The recoveries were 81.3% to 98.1% with a coefficient of variation less than 15.7%. Good correlations were observed between the results of the ic-ELISA and liquid chromatography-mass spectrometry in the incurred tissues. These results suggest that ic-ELISA is a sensitive, accurate, and low-cost method that could be a useful tool for screening the residues of amantadine and rimantadine in chicken muscle and liver.

Immune modulator adamantylamide dipeptide stimulates efficient major histocompatibility complex class I-restricted responses in mice.

Adamantylamide L-alanyl-D-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8+ T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.

Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia.

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

Intranasal vaccination with recombinant outer membrane protein CD and adamantylamide dipeptide as the mucosal adjuvant enhances pulmonary clearance of Moraxella catarrhalis in an experimental murine model.

Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.

Adamantylamide dipeptide as effective immunoadjuvant in rabbits and mice.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

Effects of in vivo administration of adamantylamide dipeptide on bone marrow granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) and on ability of serum of the treated mice to stimulate GM-CFC colony formation in vitro: comparison with muramyl dipeptide and glucan.

Adamantylamide dipeptide (AdDP), muramyl dipeptide (MDP), and glucan were shown to increase significantly the numbers of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of mice. However, whereas the sera of mice given MDP or glucan were found to stimulate the growth of colonies from GM-CFC in vitro, i.e. to produce a colony-stimulating activity (CSA), administration of AdDP did not lead to this effect. Nevertheless, when serum of mice given AdDP was added to the cultures concomitantly with a suboptimal concentration of mouse interleukin-3 (mIL-3), a broad spectrum hemopoietic stimulator, counts of colonies from GM-CFC were significantly increased, and accelerated growth of the colonies was found as well. This property of AdDP, i.e. its ability to exhibit co-stimulating activity (CoSA) without being able to exhibit CSA, suggests that AdDP acts in hemopoietic tissues differently as compared with the two other immunomodulators studied. It can be hypothesized that the action of AdDP is more specific when compared with its natural related compound, MDP, as well as with glucan. Our findings prove the possibility to stimulate by AdDP the granulopoietic compartment of hemopoiesis and are in agreement with previous observations concerning the absence of systemic side effects of AdDP. Both these qualities of AdDP may be advantageous when pondering over contingent clinical utilization of AdDP as hemopoietic stimulator.

Adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from HIV-1 transmembrane region glycoprotein gp41.

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile- Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with coentrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.

Adverse reactions to amantadine prophylaxis of influenza in a retirement home.

OBJECTIVE: Controversy exists about the safety of following the recommendation of the Immunization Practice Committee of the Centers for Disease Control that nursing home residents be given amantadine prophylaxis during influenza outbreaks. This study was undertaken to define the incidence of adverse reactions to amantadine in the elderly and to identify risk factors for side effects. DESIGN: A retrospective cohort study. SETTING: A retirement home which offered amantadine prophylaxis to its residents during a presumed influenza outbreak. PARTICIPANTS: Of the 96 elderly residents, 79 accepted the offer of amantadine prophylaxis. MAIN OUTCOME MEASURES: Attributable adverse health outcomes as assessed by chart review. RESULTS: 41% of the people receiving amantadine had attributable adverse reactions, of which 22% were classified as severe. Severe adverse reactions were associated with residence in the assisted living section of the facility (P = 0.002), a greater number of underlying diagnoses (P = 0.009), congestive heart failure (P = 0.02), and high serum creatinine (P = 0.02). A person with none of these risk factors had a 7% chance of having a severe adverse outcome compared to a 70% chance for someone with all four risk factors. CONCLUSION: The findings raise concern that the prophylactic administration of amantadine to all elderly residents of nursing and retirement homes may be associated with a high incidence of unacceptable reactions, particularly among less healthy residents.

The immunomodulatory property of a novel synthetic compound adamantylamide dipeptide.

The adjuvant effect of a novel synthetic compound adamantylamide dipeptide (AdDP) was tested in a model of delayed hypersensitivity to ovalbumin in guinea pigs, and of immunostimulatory activity in the experiments with 3H thymidine where DNA biosynthesis was measured in several immunocompetent organs after administration of adamantylamide dipeptide. In both tests, the compound proved to be active since delayed hypersensitivity was potentiated and a marked increase in the utilization of 3H thymidine in liver, thymus and spleen has been observed. The novel compound appeared to be devoid of other effects of MDP such as pyrogenicity and arthritogenicity.