Development of a New Monoclonal Antibody against Brevetoxins in Oyster Samples Based on the Indirect Competitive Enzyme-Linked Immunosorbent Assay

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples.

A Polyol-Polyol Super-Carbon-Chain Compound Containing Thirty-Six Carbon Stereocenters from the Dinoflagellate Amphidinium gibbosum: Absolute Configuration and Multi-Segment Modification

A super-carbon-chain compound, named gibbosol C, featuring a polyoxygenated C70-linear-carbon-chain backbone encompassing two acyclic polyol chains, was obtained from the South China Sea dinoflagellate Amphidinium gibbosum. Its planar structure was elucidated by extensive NMR investigations, whereas its absolute configurations, featuring the presence of 36 carbon stereocenters and 30 hydroxy groups, were successfully established by comparison of NMR data of the ozonolyzed products with those of gibbosol A, combined with J-based configuration analysis, Kishi’s universal NMR database, and the modified Mosher’s MTPA ester method. Multi-segment modification was revealed as the smart biosynthetic strategy for the dinoflagellate to create remarkable super-carbon-chain compounds with structural diversity.

Pyrenosetins A–C, New Decalinoylspirotetramic Acid Derivatives Isolated by Bioactivity-Based Molecular Networking from the Seaweed-Derived Fungus Pyrenochaetopsis sp. FVE-001

Marine algae represent a prolific source of filamentous fungi for bioprospecting. In continuation of our search for new anticancer leads from fungi derived from the brown alga Fucus vesiculosus, an endophytic Pyrenochaetopsis sp. FVE-001 was selected for an in-depth chemical analysis. The crude fungal extract inhibited several cancer cell lines in vitro, and the highest anticancer activity was tracked to its CHCl3–soluble portion. A bioactivity-based molecular networking approach was applied to C18-SPE fractions of the CHCl3 subextract to predict the bioactivity scores of metabolites in the fractions and to aid targeted purification of anticancer metabolites. This approach led to a rapid isolation of three new decalinoylspirotetramic acid derivatives, pyrenosetins A–C (1–3) and the known decalin tetramic acid phomasetin (4). The structures of the compounds were elucidated by extensive NMR, HR-ESIMS, FT-IR spectroscopy, [α]D and Mosher’s ester method. Compounds 1 and 2 showed high anticancer activity against malignant melanoma cell line A-375 (IC50 values 2.8 and 6.3 μM, respectively), in line with the bioactivity predictions. This is the first study focusing on secondary metabolites of a marine-derived Pyrenochaetopsis sp. and the second investigation performed on the member of the genus Pyrenochaetopsis.

New Chromones from a Marine-Derived Fungus, Arthrinium sp., and Their Biological Activity

Five new chromone derivatives, arthones A–E (1–5), together with eight known biogenetically related cometabolites (6–13), were isolated from a deep-sea-derived fungus Arthrinium sp. UJNMF0008. Their structures were assigned by detailed analyses of spectroscopic data, while the absolute configurations of 1 and 5 were established by electronic circular dichroism (ECD) calculations and that of 2 was determined by modified Mosher ester method. Compounds 3 and 8 exhibited potent antioxidant property with DPPH and ABTS radical scavenging activities, with IC50 values ranging from 16.9 to 18.7 μM. Meanwhile, no compounds indicated obvious bioactivity in our antimicrobial and anti-inflammatory assays at 50.0 μM.

Delitpyrones: α-Pyrone Derivatives from a Freshwater Delitschia sp.

In research focused on the discovery of new chemical diversity from freshwater fungi, a peak library was built and evaluated against a prostate cancer cell line, E006AA-hT, which was derived from an African American, as this population is disproportionately affected by prostate cancer. The chemical study of the bioactive sample accessioned as G858 (Delitschia sp.) led to the isolation of eight new α-pyrone derivatives (1 – 7, and 11), as well as the new 3S*,4S*-7-ethyl-4,8-dihydroxy-3,6-dimethoxy-3,4-dihydronaphthalen-1(2H)-one (15). In addition, the known compounds 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (8), 5-(3-oxobutyl)-4-methoxy-6-methyl-2H-pyran-2-one (9), pyrenocine I (10), 5-butyl-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (12), sporidesmin A (13), 6-ethyl-2,7-dimethoxyjuglone (14), artrichitin (16), and lipopeptide 15G256ε (17) were also obtained. The structures of the new compounds were elucidated using a set of spectroscopic (NMR) and spectrometric (HRMS) methods. The absolute configuration of the most abundant member of each subclass of compounds was assigned through a modified Mosherʼs ester method. For 15, the relative configuration was assigned based on analysis of 3 J values. Compounds 1, 2, 5 – 14, 16, and 17 were evaluated against the cancer cell line E006AA-hT under hypoxic conditions, where compound 13 inhibited cell proliferation at a concentration of 2.5 µM.

Sintesis reagen imunokimia untuk deteksi okratoksin dengan metode imunokromatografik nanopartikel emas Synthesis of immunochemical reagent for ochratoxin detection using gold nanoparticle immunochromatographic method

The quality of Indonesiam coffee andcocoa products has been declined due to the contamination of fungi producing ochratoxin, a serious human mycotoxin. Therefore, develop-ment of fast, accurate and simple method for early detection of ochratoxin contamination is required. As a part of research attempted to develop early detection technique of ochratoxin in severall commodities, especially agricultural products, the objective of this study was to produce immunochemical reagent for ochratoxin detectionusing immunoglobulin Y (IgY) based on immunochromatographic method. Results showed that OTA-OVA could be synthesized using active ester method with addition of N-hydroxy-succiimide and dicyclocarboimide. The inter-mediate compound produced showed C=O stretching vibrational band at 1600 cm-1 and C-O stretching vibrational band at 1300-1000 cm-1. Antibody-gold nanoparticle conjugate was optimally produced at pH 9 and with antibody dilution of 1:7.5 (v/v). There was 50 nm absorb-tion shift in visible absorbtion after the antibody was conjugated with gold nanoparticle. Even though the test strip did not show clear visual-ization, the cut off of ochratoxin concentrationis obviously determined at 10 ppb. This results suggest thatthe technique could be used to detect ochratoxin contamination. Abstrak Komoditas kopi dan kakao Indonesia ter-kendala masalah mutu produk yang rendah akibat kontaminasi jamur yang menghasilkan okra-toksin. Okratoksin merupakan mikotoksin yang membahayakan kesehatan manusia. Oleh karena itu, cara deteksi dini kontaminasi okratoksin yang cepat, akurat dan mudah perlu dikembangkan. Sebagai bagian dari usaha untuk mengembangkan teknik deteksi dini okratoksin pada pada berbagai komoditas, khususnya produk pertanian, pene-litian ini bertujuan menghasilkan reagen imuno-kimia yang menggunakan antibodi immune-globulin Y (IgY) berdasarkan metode imunokro-matografik untuk deteksi okratoksin. Hasil penelitian menunjukkan bahwa OTA-OVA dapat disintesis dengan metode ester aktif dengan menambahkan N-hidroksisuksiimida dan disiklo-karboimida. Senyawa antara yang dihasilkan memiliki absorpsi pada frekuensi 1600 cm-1 yang menunjukkan adanya vibrasi ulur ikatan C=O dan adanya banyak absorpsi pada 1300-1000 cm-1 yang mengindikasikan adanya serapan ulur yang kuat ikatan C-O. Konjugat antibodi-nanopartikel emas dihasilkan optimum pada pH 9 dan dengan pengenceran antibodi 1:7,5 (v/v). Hasil pengujian spektrofotometer visible menunjukkan adanya pergeseran serapan sebesar 50 nm setelah anti-bodi dikonjugasikan pada nanopartikel emas. Meskipun secara visual tidak begitu jelas, tetapi hasil pengujian pada teststrip menunjukkan bahwa nilai cut off konsentrasi okratoksin yang terdeteksi adalah10 ppb. Hasil ini menunjukkan bahwa teknik yang dikembangkan dapat diguna-kan untuk deteksi kontaminasi okratoksin.

Sintesis reagen imunokimia untuk deteksi okratoksin dengan metode imunokromatografik nanopartikel emas Synthesis of immunochemical reagent for ochratoxin detection using gold nanoparticle immunochromatographic method

The quality of Indonesiam coffee andcocoa products has been declined due to the contamination of fungi producing ochratoxin, a serious human mycotoxin. Therefore, develop-ment of fast, accurate and simple method for early detection of ochratoxin contamination is required. As a part of research attempted to develop early detection technique of ochratoxin in severall commodities, especially agricultural products, the objective of this study was to produce immunochemical reagent for ochratoxin detectionusing immunoglobulin Y (IgY) based on immunochromatographic method. Results showed that OTA-OVA could be synthesized using active ester method with addition of N-hydroxy-succiimide and dicyclocarboimide. The inter-mediate compound produced showed C=O stretching vibrational band at 1600 cm-1 and C-O stretching vibrational band at 1300-1000 cm-1. Antibody-gold nanoparticle conjugate was optimally produced at pH 9 and with antibody dilution of 1:7.5 (v/v). There was 50 nm absorb-tion shift in visible absorbtion after the antibody was conjugated with gold nanoparticle. Even though the test strip did not show clear visual-ization, the cut off of ochratoxin concentrationis obviously determined at 10 ppb. This results suggest thatthe technique could be used to detect ochratoxin contamination. Abstrak Komoditas kopi dan kakao Indonesia ter-kendala masalah mutu produk yang rendah akibat kontaminasi jamur yang menghasilkan okra-toksin. Okratoksin merupakan mikotoksin yang membahayakan kesehatan manusia. Oleh karena itu, cara deteksi dini kontaminasi okratoksin yang cepat, akurat dan mudah perlu dikembangkan. Sebagai bagian dari usaha untuk mengembangkan teknik deteksi dini okratoksin pada pada berbagai komoditas, khususnya produk pertanian, pene-litian ini bertujuan menghasilkan reagen imuno-kimia yang menggunakan antibodi immune-globulin Y (IgY) berdasarkan metode imunokro-matografik untuk deteksi okratoksin. Hasil penelitian menunjukkan bahwa OTA-OVA dapat disintesis dengan metode ester aktif dengan menambahkan N-hidroksisuksiimida dan disiklo-karboimida. Senyawa antara yang dihasilkan memiliki absorpsi pada frekuensi 1600 cm-1 yang menunjukkan adanya vibrasi ulur ikatan C=O dan adanya banyak absorpsi pada 1300-1000 cm-1 yang mengindikasikan adanya serapan ulur yang kuat ikatan C-O. Konjugat antibodi-nanopartikel emas dihasilkan optimum pada pH 9 dan dengan pengenceran antibodi 1:7,5 (v/v). Hasil pengujian spektrofotometer visible menunjukkan adanya pergeseran serapan sebesar 50 nm setelah anti-bodi dikonjugasikan pada nanopartikel emas. Meskipun secara visual tidak begitu jelas, tetapi hasil pengujian pada teststrip menunjukkan bahwa nilai cut off konsentrasi okratoksin yang terdeteksi adalah10 ppb. Hasil ini menunjukkan bahwa teknik yang dikembangkan dapat diguna-kan untuk deteksi kontaminasi okratoksin.

The Synthesis and Immune Identification of Bisphenol A Artificial Antigen

Bisphenol A (BPA) belongs to estrogenic substances, which influences the development of genital system in human being. In this paper, 4, 4-bis (4-hydroxyphenyl) valeric acid (BVA), a derivative of BPA, was chosen as the hapten to prepare artificial antigen. An active ester method was used for coupling BPA hapten with keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). Both UV spectra and immunoassay indicated that the two kinds of artificial antigen BVA-KLH and BVA-OVA were synthesized successfully. The artificial antigen of BPA can be used to develop monoclonal antibody of anti-BPA.

Enzyme-Linked Immunosorbent Assay for Okadaic Acid: Investigation of Analytical Conditions and Sample Matrix on Assay Performance

Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28±0.38 ng/mL, corresponding to 12.8±3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y=1.0064x–10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.