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Context: Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer., Aims: The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS., Settings and Design: In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported., Subjects and Methods: 111 In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals., Statistical Analysis Used: Statistical analyses were performed using SPSS 10.0., Results: 111 In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111 In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site., Conclusions: 111 In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors., Competing Interests: None

In this study, [ 111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [ 111 In]In-DOTA-hIgG and [ 111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [ 111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [ 111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [ 111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology., (© 2020 John Wiley & Sons, Ltd.)

Background: Osteosarcoma (OS) is the basic bone neoplasm with lower survival and poor prognosis. It is distinguished by its offensive nature and metastatic potential. The fundamental death source in OS patients is lung metastasis. In addition, the proliferation and cell migration are thus essential for cancer progression, especially for intrusion and transformation. Several studies have illustrated that 1,25-Dihydroxyvitamin D (1,25(OH)2D) has a critical role in the growth and differentiation of bone. However, knowledge of the outcome of 1,25(OH)2D on the progression and incursion of osteosarcoma cells is minimal., Objective: The present study aimed to analyze the effect of different concentrations of 1,25(OH)2D on the multiplication, progression, and intrusion of OS cells and verify the effective doses of 1,25(OH)2D that can decrease the intensity of the disease and improving the prognosis in OS patients., Methods: Saos-2 cells were treated with 1,25(OH)2D (0, 50, 100, and 200 nM) for 48, 72, and 96 hours. Proliferation, invasion, and migration were determined by MTT assay, Transwell assay, and Scratch test, respectively. The levels of c-Myc and FOXO1 proteins were determined by Western blotting., Results: The proliferation, invasiveness, and migration of Saos-2 cells that were treated with 1,25(OH)2D were significantly decreased compared with untreated cells. Although 1,25(OH)2D notably decreased c-Myc protein levels (after 48 and 72 hours), FOXO1 protein levels have been significantly increased after 48 and 72 hours. 1,25(OH)2D and the vitamin D receptor (VDR) suppress c-Myc function through regulating the c-Myc/MXD1 network and thus, providing a molecular basis of 1,25(OH)2D related to the cancer-preventive actions., Conclusion: Based on the present results, 1,25(OH)2D by targeting c-Myc and FOXO1 expression displays anti-invasive, anti-migration and anti-proliferative effects on OS cells in vitro. Our findings suggest that effective doses of the 1,25(OH)2D may reduce the aggressive potential of the OS cell line. However, further investigation and clinical trials are needed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objective: Breast cancer radioimmunoscintigraphy targeting MUC1 expression is a growing field of work in nuclear medicine research. PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast tumors. In this study, we report production, quality control and preclinical qualifications of a copper-64 labeled PR81 for PET imaging of breast cancer., Methods: PR81 was conjugated with DOTA-NHS-ester and purified by molecular filtration followed by chelate:mAb ratio determination by spectrophotometric method. DOTA-PR81 was labeled with (64)Cu followed by radiochemical purity, in vitro stability, in vitro internalization and immunoreactivity determination. The tissue biodistribution of the (64)Cu-DOTA-PR81 and (64)Cu-DOTA-hIgG was evaluated in BALB/c mice with breast carcinoma tumors using tissue counting and imaging., Results: The radiochemical purity of radioimmunoconjugate was >95±1.9% (ITLC) (specific activity; 4.6 μCi/μg). The average number of chelators per antibody was 3.4±0.3:1. The (64)Cu-DOTA-PR81 showed immunoreactivity towards MUC1 antigen and MCF7 cell line with significant in vitro stability (>89% in PBS and 78±0.5% in human serum) over 48 h. Maximum internalized activity of radiolabeled PR81 in 4-8 h was 81.5%. The biodistribution and scintigraphy studies showed the accumulation of the complex at the site of tumors with high sensitivity and specificity compared to control probes., Conclusion: The results showed that (64)Cu-DOTA-PR81 may be considered as a potential PET tracer for diagnosis and follow-up of MUC1 expression in oncology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In the present work, we report characterization studies of antigen(Ag)-antibody (Ab) interaction based on the multi-walled carbon nanotube array-modified gold electrode for label-free electrochemical immunosensor. -COOH functionalization of MWCNT was suggested by FTIR spectroscopy. Images from atomic force microscopy (AFM) and RAMAN spectroscopy confirmed the interaction of Ab on the gold interface based on the MWCNT array. A cyclic voltammetric study exhibited a linear response in phosphate buffered saline (PBS) solution including 1mM K3Fe (CN) 6 at pH 7.4 with PSA concentrations over a range of 2 to 40 ngmL(-1), and a calculated detection limit of 0.56 ngmL(-1).

With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.

Transformation of macrophages to foam cells is determined by the rates of cholesterol uptake and efflux. This study uses a real time RT-PCR technique to investigate the role of conjugated linoleic acid (CLA), α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) in the regulation of the ATP-binding cassette A1 (ABCA1) and liver X receptor α (LXR) genes, which are involved in cholesterol homeostasis. Accordingly, these fatty acids significantly reduced the total, free and esterified cholesterols within the foam cells. While the expression of the ABCA1 and LXRα genes was increased in the presence of the pharmacological LXRα ligand, T0901317, their mRNA expression was not significantly affected by CLA, ALA and EPA. These results suggest that although polyunsaturated fatty acids have an effect on cholesterol homeostasis, they cannot change the expression of the ABCA1 and LXRα genes. Alternatively, several other genes and proteins may be involved.

Digoxin is widely used as a cardiac glycoside drug in the treatment of various heart conditions. Because it is a toxic drug, it should be regularly monitored in the serum of patients under treatment. In this study, colloidal nanogold is synthesized and the preparation of nanogold-labeled monoclonal antibody probe to digoxin is described under optimal conditions. In addition, an immunochromatographic (IC) method for digoxin analysis employing nanogold-labeled probe is developed. With this technique, it requires only 5 min to complete the quantitative detection of digoxin. The detection time is decreased 20-30 times in comparison to radioimmunoassay (RIA). The sensitivity to digoxin was about 2 ng/ml by naked eye, which is within the therapeutic and toxic ranges of digoxin. The results of serum samples obtained by IC strip were in agreement with those obtained by RIA. The IC strip was sufficiently sensitive and accurate to be used for the rapid detection of digoxin in serum samples.

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to compare the two labeling methods (direct and indirect radioiodination) for application of this antibody against MUC1 as a radioimmunotherapeutical agent.Monoclonal antibody (PR81) against the tandem repeat of the core protein (MUC1) was prepared, characterized, purified, and labeled with 131I using the direct (chloramin-T) and indirect (Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH (YYK) attached to N-hydroxysuccinimide as a linker between PR81 and 131I) methods. The immunoreactivity of 131I-PR81 and 131I-TP-PR81 complexes with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of 131I-PR81 and 131I-YYK-peptide-PR81 complexes in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the 131I-PR81 and 131I- YYK-peptide -PR81 complexes was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The labeling efficiency was determined by measuring the percentage recovery of radioactivity in the final product relative to the initial activity in the shipment vial, was found to be 59.9% +/- 7.9% for direct and 50% +/- 3.2% for indirect methods. 131I-PR81 and 131I- YYK- peptide -PR81 complexes showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled products in human serum which was measured by thin layer chromatography (TLC) was found to be more than 50% over 24 hr for 131I-PR81 and 70% for 131I- YYK-peptide -PR81 complexes. Cell toxicity and in vitro internalization studies showed that the 131I-PR81 and 131I- YYK-peptide -PR81 complexes inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to 40% of the 131I-PR81 and 60% of the 131I- YYK-peptide -PR81 complexes internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection. Thyroid and stomach levels from PR81 labeled with 131I- YYK-peptide were two- to three- fold less than those with directly labeled 131I-PR81, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinase. These results show that the indirect labeling was better than the indirect labeling and 131I- YYK-peptide -PR81 may be considered as a promising candidate for therapy of breast cancer.

A simple, sensitive, rapid and specific enzyme linked immunosorbent assay (ELISA) for quantitative measurement of aflatoxin B(1) (AFB(1)) in urine samples was developed in this study. Polyclonal antibodies were raised against a C(1)-carboxymethyl oxime (CMO) derivative of AFB(1) conjugated bovine serum albumin (BSA). AFB(1)-C(8)-penicillinase (AFB(1)-C(8)-P) and AFB(1)-C(1)-carboxymethyl oxime-penicillinase (AFB(1)-CMO-P) were prepared and used as tracer molecule. A heterologous combination of antibody and enzyme conjugates (AFB(1)-C(1)-CMO-BSA and AFB(1)-C(8)-P) proved to work better with respect to specificity and sensitivity. Ig purified antibody (4 microg/well) was coated onto the pre-coated (BSA) wells of microtiter plate. The assay procedure was completed within 3 hr and the sensitivity was calculated to be from 200 pg/ml. The standard curve was linear up to 10 ng/ml so was able to detect high concentration of AFB(1) in sample. Affinities were calculated for homologous and heterologous system in which the heterologous system showed better affinities (1.9 x 10(8) M(-1)). The antibody prepared in this study showed minimal cross-reaction with structurally related molecules being affected by homology and heterology of the assay system that is the site of conjugation of carrier protein for antibody production using the hapten BSA conjugate and the site of enzyme conjugated on the hapten molecule used as tracer as well as direct and indirect coating of antibody on the surface of microtiter plat. The results reported here indicated that the heterologous combination of antibody and enzyme conjugate performs better in assay qualities in general. More than 90% recovery of AFB(1) added to stripped urine samples were observed in this type of assay. Inter and intra-assay percent of coefficient of variations for ten successive assays were found to be 10.2 and 6.9% respectively. Logit -log transformation of standard curve and sample dilution with urine sample containing no AFB(1) in a serial manner exhibited parallel line with the slope of -1.03 and -1.03 respectively. A correlation of 0.90 was found between the ELISA reported in this study and radioimmunoassay (RIA) of AFB(1) in urine samples.

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.