Noninvasive Urine Biomarker Lateral Flow Immunoassay for Monitoring Active Onchocerciasis.

The parasitic disease onchocerciasis is the second leading cause of preventable blindness, afflicting more than 18 million people worldwide. Despite an available treatment, ivermectin, and control efforts by the World Health Organization, onchocerciasis remains a burden in many regions. With an estimated 120 million people living in areas at risk of infection, efforts are now shifting from prevention to surveillance and elimination. The lack of a robust, point-of-care diagnostic for an active Onchocerca infection has been a limiting factor in these efforts. Previously, we reported the discovery of the biomarker N-acetyl-tyramine- O-glucuronide (NATOG) in human urine samples and its ability to track treatment progression between medicated patients relative to placebo; we also established its capability to monitor disease burden in a jird model. NATOG is a human-produced metabolite of tyramine, which itself is produced as a nematode neurotransmitter. The ability of NATOG to distinguish between active and past infection overcomes the limitations of antibody biomarkers and PCR methodologies. Lateral flow immunoassay (LFIA) diagnostics offer the versatility and simplicity to be employed in the field and are inexpensive enough to be utilized in large-scale screening efforts. Herein, we report the development and assessment of a NATOG-based urine LFIA for onchocerciasis, which accurately identified 85% of analyzed patient samples ( N = 27).

Induced accumulation of tyramine, serotonin, and related amines in response to Bipolaris sorokiniana infection in barley.

The inducible metabolites were analyzed in barley leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of barley. HPLC analysis revealed that B. sorokiniana-infected leaves accumulated 4 hydrophilic compounds. They were purified by ODS column chromatography and preparative HPLC. Spectroscopic analyses revealed that they were tyramine (1), 3-(2-aminoethyl)-3-hydroxyindolin-2-one (2), serotonin (3), and 5,5'-dihydroxy-2,4'-bitryptamine (4). Among these, 2 and 4 have not been reported as natural products. They showed antifungal activity in an assay of inhibition of B. sorokiniana conidia germination, suggesting that they play a role in the chemical defense of barley as phytoalexins. The accumulation of 1-4 was examined also in the leaves of rice and foxtail millet. Rice leaves accumulated 2, 3, and 4, whereas foxtail millet leaves accumulated 3 and 4 in response to pathogen attack, suggesting the generality of accumulation of 3 and 4 in the Poaceae species.

Tyramine Pathways in Citrus Plant Defense: Glycoconjugates of Tyramine and Its N-Methylated Derivatives.

Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to occur in Citrus genus plants. The compounds tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and N,N-dimethyltyramine-O-ß-d-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon, mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-ß-d-glucoside was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-ß-d-glucoside, N-methyltyramine-O-ß-d-glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-ß-glucoside. However, the latter tyramine metabolite, never described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine, supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.

The major cockroach allergen Bla g 4 binds tyramine and octopamine.

Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and isothermal titration calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands. Accession numbers: The atomic coordinates and the structure factors have been deposited to the Protein Data Band under accession codes: 4N7C for native Bla g 4 and 4N7D for the Se-Met Bla g 4 structure.

A filtration method for rapid preparation of conjugates for immunoassay.

Modification of protein and other biopolymers by labeling them with small or macromolecules has become a very powerful research tool in biochemistry, molecular biology, diagnostics, and therapeutics. However, current methodologies available for their preparations are not straightforward and take several hours of incubation time. In this paper, we describe a new filtration-assisted technique for covalent conjugation between the reactive functional groups of two different molecules (small or macromolecules). Compared to the current method, this new approach significantly reduces the total reaction time from several hours to just a few minutes. The technique has been used for the preparation of conjugates of a small molecule to a protein such as biotin-BSA conjugate or small molecules to a small molecule such as biotin-tyramine conjugate or protein-protein conjugation such as antibody-horseradish peroxidase conjugate. The procedure consists of filtering the reaction mixture multiple times through membrane micropores with the help of two syringes, which make the cross filtration process less laborious. The method saves time, allows conjugation of less than 1mg protein and produces conjugates better than those obtained by the current methods. Although the present technique has been applied on some common conjugation methods, it provides a potentially general method, and may further be expanded for the synthesis of several other macromolecular conjugates.

Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy.

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.

Tyramide signal amplification in brain immunocytochemistry: adaptation to electron microscopy.

The tyramide signal amplification (TSA) technique is well-established in light microscopic immunohistochemistry and in situ hybridization to improve the signal-to-noise ratio. The present study deals with its adaptation to the electron microscopic level using the pre-embedding technique and a modified protocol. The outcome of immunolabeling of most of the antigens tested in brain tissue, including endothelial and neuronal nitric oxide synthase, glial fibrillary acidic protein, and isolectin B4, was greatly improved. If signal amplification is required, the TSA-technique proved to be reliable with high specificity and good ultrastructural resolution.

Production and characterization of monoclonal and polyclonal antibodies against thyrotropin-releasing hormone.

In this article, two mouse monoclonal antibodies (83-7B5-A1 and 83-6B6-A10) and three rabbit polyclonal antibodies (1118, 8572, and 8577) directed against thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2) are described. The anti-TRH antibodies were raised by immunization with a TRH-bovine serum albumin conjugate obtained by coupling of the CO2H group of pGlu-His-Pro-OH to NH2 groups in the protein. The monoclonal antibodies were produced by hybridoma clones obtained by the fusion of SP2/0 myeloma cells with spleen cells of an immunized BALB/c mouse. Both monoclonal antibodies were of the IgG1 (kappa) subclass. Characterization of the anti-TRH antibodies showed that in general they are specific for the pGlu-His moiety. The cross-reactivities for the TRH-like peptides [Glu1]TRH, [Glu2]TRH, and [Phe2]TRH are low, while alterations at the Pro-NH2 moiety of TRH are recognized to varying extents. The specificities of these antibodies are markedly different from those previously obtained using TRH coupled through the histidine residue to protein as the immunogen.

Highly specific characterization of tyrosine phosphoproteins in tumor cells based on monoclonal antibodies defined by conjugated phosphotyramine.

Phosphotyrosine proteins of four different tumor cell lines were characterized by monoclonal antibodies exhibiting high affinity binding to phosphotyrosine. For the preparation of the antibody-producing mouse hybridoma cell lines we used a novel kind of immunizing antigen with phosphotyramine conjugated directly to carboxylic groups of carrier proteins. Screening for high affinity binding antibodies was based on their selective reactivity in immunoprecipitation, affinity chromatography and immunofluorescence. By means of affinity chromatography we established a one-step purification of phosphotyrosine proteins yielding substantial quantities of highly pure 170kDa EGF receptor from A431 tumor cells, 210kDa bcr-abl gene product from K562 tumor cells and 120 kDa transforming protein of the Abelson murine leukemia virus from TK tumor cells. Cross-reactivity with phosphoproteins containing no phosphotyrosine was not observed.

Antisera against catecholamines: specificity studies and physicochemical data for anti-dopamine and anti-p-tyramine antibodies.

Antibodies against dopamine and p-tyramine were raised in rabbits. The two catecholamines were conjugated to albumin by glutaraldehyde. The specificity of the antibodies was established by equilibrium dialysis competition experiments using an immunoreactive tritiated derivative synthesized by coupling dopamine or p-tyramine to N-alpha-acetyl-L-lysine N-methylamide with glutaraldehyde. Hence, these radiolabelled ligands mimicked the antigenic determinant of conjugated immunogens. A comparison of the data obtained showed the high specificity of each antiserum for its hapten coupled by glutaraldehyde. The anti-dopamine antibodies recognized dopamine-glutaraldehyde but not p-tyramine-glutaraldehyde. The opposite occurred for the anti-p-tyramine antibodies. A slight modification of the molecular structure provided the opportunity for a specific response against that molecule. But this difference was more important when related to the hapten region where the antibody affinity was maximal. The cross-reactivity was observed to be more important dopamine and p-tyramine than between dopamine and noradrenaline on the one hand and between p-tyramine and dopamine than p-tyramine and octopamine on the other hand.

Antibodies to catecholamines.

The haptens p-tyramine and synephrine were conjugated to bovine serum albumin by means of the formaldehyde condensation reaction. These conjugates were emulsified in adjuvant and injected into rabbits. Antiserum was harvested at 10-day intervals after booster injections. The anti-sera were screened by immunodiffusion. After detecting the presence of antibody, an antiserum was further characterized by antiserum dilution curves, standard curves, and cross-reactivity studies using labeled p-tyramine or labeled metanephrine. The cross-reactivity studies indicated that the antisera to either hapten had low affinity to compounds with structural deviations on the side chain or ring other than at the position ortho to the phenolic hydroxyl group. The antisera were more sensitive to 3-methyoxylated compounds (3-methody-tyramine and metanephrine) than to 3-hydroxy or unsubstituted compounds.

Determination of plasma and tissue levels of tyramine by radioimmunoassay.

Antibodies were prepared against tyramine. The antigen was prepared as follows: p-Aminohippuric acid was coupled to mBSA using a carbodiimide reagent. The amino group was diazotized an attached to the aromatif ring of TYR. The immunogen in Freund's complete adjuvant was injected into rabbits. The specificity of the resulting antibody was determined by radioimmunoassay. Using random-labeled TYR-3H, TYR, its metabolites, phenethylamine analogs, catecholamines, and certain amino acids were evaluated by a competitive binding assay method. With this technique 4 ng of TYR inhibited the binding of TYR-3H by 50%. The radioimmunoassay of TYR was used to measure the plasma, urine, and tissue levels of TYR in rabbits. The plasma disappearance curve of TYR revealed a biphasic pattern with t1/2 of 2 min and 54 min. The highest concentration of TYR was found in adrenals and spleen. The factthat the major metabolites of TYR and a series of pharmacologically important sympathomimetics and catecholamines did not interfere, makes the radioimmunoassay of TYR a useful, simple, sensitive, and spedific method for the direct analysis of TYR in biological meterials.