Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody.

Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg(2+) was developed. A new bifunctional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immunogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg(2+). Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg(2+) was constructed in concentration range of 0.1-100 ng mL(-1). The values of IC(50) and LOD of the assay were found to be 1.12 and 0.08 ng mL(-1). The cross-reactivity was lower than 2% with MNA, CH(3)Hg, and CH(3)Hg-MNA and was 11.5% and 4.4% for Hg(+) and Au(3+), respectively. No cross-reactivity was found with other metal ions such as Cu(2+), Sn(2+), Ni(2+), Mn(2+), Pb(2+), Zn(2+), Cd(2+), Fe(2+), Co(2+), Mg(2+), Ca(2+), and anions such as Cl(-), NO(3)(-), NO(2)(-), HCO(3)(-), F(-), and SO(4)(2-), indicating that the assay displays not only high sensitivity but also high selectivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg(2+) and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0-113.0% and coefficients of variation of 1.9-18.6% were obtained. A linear relationship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coefficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a feasible analytical method for highly sensitive and specific, fast, simple, and accurate determination of uncomplexed trace Hg(2+) in environmental and food samples.

Influenza viral neuraminidase: the forgotten antigen.

Influenza is the most common cause of vaccine-preventable morbidity and mortality despite the availability of the conventional trivalent inactivated vaccine and the live-attenuated influenza vaccine. These vaccines induce an immunity dominated by the response to hemagglutinin (HA) and are most effective when there is sufficient antigenic relatedness between the vaccine strain and the HA of the circulating wild-type virus. Vaccine strategies against influenza may benefit from inclusion of other viral antigens in addition to HA. Epidemiologic evidence and studies in animals and humans indicate that anti-neuraminidase (NA) immunity will provide protection against severe illness or death in the event of a significant antigenic change in the HA component of the vaccine. However, there is little NA immunity induced by trivalent inactivated vaccine and live-attenuated influenza vaccine. The quantity of NA in influenza vaccines is not standardized and varies significantly among manufacturers, production lots and tested strains. The activity and stability of the NA enzyme is influenced by concentration of divalent cations. If immunity against NA is desirable, a better understanding of how the enzymatic properties affect the immunogenicity is needed.

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

The uptake by blood-borne phagocytes of monosodium urate is dependent on heat-labile serum factor(s) and divalent cations.

Accumulation in tissues of post-apoptotic cells is a feature frequently observed in patients with systemic lupus erythematosus and in murine models of systemic autoimmune diseases. One of the endogenous danger molecules released by secondarily necrotic cells is monosodium urate (MSU), which is already established to be the causative agent of gout. Here, we show that MSU is taken up by eosinophils, neutrophils and monocytes in a process involving (a) heat-labile serum factor(s) and divalent cations. The uptake induces the release of the pro-inflammatory cytokines IL-1beta/IL-18/TNFalpha and IL-6/IL-8 by monocytes and PMN, respectively.

Production of metal ion-dependent monoclonal antibodies against peptides in bovine prothrombin fragment 1.

Bovine prothrombin fragment 1 (F-1: the amino-terminal 156 residues of prothrombin) is used as a model to study the Ca(II) and phospholipid binding of prothrombin. The 35-46 segment in F-1 posses an alpha-helical region and three aromatic residues, conserved in several vitamin K-dependent blood coagulation factors. These residues are believed to have a specific function and to be important in the phospholipid binding of F-1. The 47-62 region, a disulfide loop, is believed to stabilize the gamma-carboxyglutamic acid domain of the protein. Goals of this research were to produce monoclonal antibodies against the above two sequences, for later functional studies. Antibodies S9-32.8 and S9-5.5 were produced against the 35-46 sequence; antibody S11-23.4 was raised against the 47-62 region. Both S9-32.8 and S9-5.5 bound to F-1 immobilized on ELISA plates in the presence of 10 mM Ca(II) with higher affinity than to F-1 coated in the presence of 10 mM Mg(II) or in the absence of metal ions. S11-23.4 showed greatest binding to F-1 coated in the presence of 10 mM Mg(II). Thus, the epitopes of the antibodies are metal ion-dependent and are developed by Ca(II) binding to F-1.

Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.

Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors.

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 microg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 microM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i. e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.

Alveolar macrophages bind and phagocytose allergen-containing pollen starch granules via C-type lectin and integrin receptors: implications for airway inflammatory disease.

Recent studies suggest that IgE-independent mechanisms of airway inflammation contribute significantly to the pathophysiology of allergic airway inflammatory diseases such as asthma. Such mechanisms may involve direct interactions between inhaled allergens and cells of the respiratory tract such as macrophages, dendritic cells, and epithelial cells. In this study, we investigated receptor-mediated interactions occurring between alveolar macrophages and allergen-containing pollen starch granules (PSG). We report here that PSG are released from a range of grass species and are rapidly bound and phagocytosed by alveolar macrophages. Human monocyte-derived dendritic cells also bound PSG but no internalization was observed. Phagocytosis of PSG was dependent on Mg2+ and Ca2+ and was inhibited by neo-glycoproteins such as galactose-BSA and N-acetylgalactose-BSA. Partial inhibition of phagocytosis was also seen with the Arg-Gly-Asp-Ser (RGDS) motif and with an anti-CD18 mAb (OX42). The combination of both neo-glycoprotein and anti-CD18 achieved the greatest degree of inhibition (>90%). Together, these data suggest a role for both C-type lectins and beta2-integrins in the binding and internalization of PSG. The consequences of this interaction included a rapid up-regulation of inducible NO synthase mRNA and subsequent release of NO by alveolar macrophages. Thus, receptor-mediated recognition of inhaled allergenic particles by alveolar macrophages may represent a potential mechanism for modulating the inflammatory response associated with allergic airway diseases such as asthma.

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.