Abstract 16877: Correlation of Antibody Binding Strength to Complement Fixation

Background: In patients (pts) who are highly sensitized, desensitization therapy is commonly applied to reduce antibody (Abs) load and to reduce potential morbidity and mortality following heart transplant (HTx). It is not known as to what level of antibody binding is needed to initiate desensitization therapy. It is believed that high binding Abs may have the ability to fix complement and lead to cytotoxicity. Methods: Between 2011 and 2012 we assessed 13 highly sensitized pts who were noted to have standardized fluorescence intensity (SFI) >100,000 pretransplant. A complement binding assay (C1q binding assay) was then applied to these Abs to assess for a correlation of binding to cytotoxicity. Correlation statistics were used to derive significance between higher antibody binding and capacity to bind complement. Results: There seems to be no correlation between the different binding strength and C1q positivity. However, when evaluating Luminex 1:8 test, there seems to be correlation between class I binding strength with C1q positivity. There was C1q positive rate of 10.8% in SFI 200,000 range. Conclusions: Undiluted antibodies do not correlate to C1q binding ability. However the 1:8 dilution test seems to provide an better prediction of complement binding. 1:8 dilutions should be performed to accurately assess the ability for antibodies to bind complement.

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Stard 3 (Steroidogenic Acute Regulatory-Related Lipid Transfer Domain-Containing Protein 3) Play Important Role in Preadipocyte Differentiation

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2960 ◽

2022 ◽

Vol 12 (4) ◽

pp. 827-833

Author(s):

Zhonge Chen ◽

Yanhua Tang ◽

Wenyong Jiang ◽

Xiaoqian Zhou

Keyword(s):

Fluorescence Intensity ◽

Lipid Transfer ◽

Gene Expressions ◽

Preadipocyte Differentiation ◽

Protein Expressions ◽

Positive Rate ◽

Steroidogenic Acute Regulatory ◽

Oil Red O Staining ◽

Oil Red O

Aim: To evaluate Stard 3’s effects and relative mechanisms in preadipocyto differentiation by vitro study. Materials and Methods: The 3T3-L1 cell were divided into 5 groups as NC, si-Stard 3, ROS agonist, ROS inhibitor and si-Stard 3+ROS agonist groups. The cell of different groups were evaluated by Oil red O staining and Triglyceride. Evaluating ROS production by DHE and NBT assay. Using RT-qPCR and WB methods to evaluate gene and protein expressions. Results: Compared with NC group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly down-regulation in si-Stard 3 and ROS inhibitor groups (P < 0.001, respectively), and were significantly up-regulation in ROS agonist group (P < 0.001, respectively); However, with si-Stard 3 transfection and ROS agonist treatment, compared with si-Stard 3 group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly increased in si-Stard 3+ROS agonist group (P < 0.001, respectively). With RT-qPCR and WB assay, Compared with NC group, Stard 3 gene and protein expressions of si-Stard 3 and si-Stard 3+ROS agonist group were significantly depressed (P < 0.001, respectively), AMPK, PPARγ, CEBPα and FABP4 gene expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively) and p-AMPK, PPARγ, CEBPα and FABP4 protein expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively), with si-Stard 3 transfection and ROS agonist the relative gene and protein expressions were significantly resumed compared with si-Stard 3 group (P < 0.001, respectively). Conclusion: Stard 3 knockdown had effects to suppress 3T3-L1 cells transformation into adipocytes in vitro study.

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A Radioimmunometric Antibody-Binding Assay for Evaluation of Xenoantisera to Melanoma-Associated Antigens23

JNCI Journal of the National Cancer Institute ◽

10.1093/jnci/62.3.455 ◽

1979 ◽

Vol 62 (3) ◽

pp. 455-463 ◽

Cited By ~ 26

Author(s):

Richard P. McCabe ◽

Vito Quaranta ◽

Luciano Frugis ◽

Soldano Ferrone ◽

Ralph A. Reisfeld

Keyword(s):

Binding Assay ◽

Antibody Binding

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Dibenzopyridoimidazocinnolinium cations: a new family of light-up fluorescent DNA probes

Organic Chemistry Frontiers ◽

10.1039/c8qo00236c ◽

2018 ◽

Vol 5 (12) ◽

pp. 1916-1927 ◽

Cited By ~ 6

Author(s):

Pedro Bosch ◽

David Sucunza ◽

Francisco Mendicuti ◽

Alberto Domingo ◽

Juan J. Vaquero

Keyword(s):

Dna Binding ◽

Fluorescence Intensity ◽

Living Cells ◽

Dna Probes ◽

Live Cell ◽

Binding Ability ◽

Active Uptake ◽

New Family ◽

Cell Staining ◽

New Compounds

A new family of weakly fluorescent azonia cations with DNA-binding ability by intercalation whose fluorescence intensity increases significantly upon DNA addition is reported. A live-cell staining cells analysis showed the capacity of these new compounds for active uptake and accumulation by living cells.

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Development of a Fluorescence Intensity Assay for the Mitotic Serine/Threonine Protein Kinase Aurora-A

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112459888 ◽

2012 ◽

Vol 18 (2) ◽

pp. 219-225 ◽

Cited By ~ 2

Author(s):

Andrew F. Slatter ◽

Spencer Campbell ◽

Richard M. Angell

Keyword(s):

Fluorescence Intensity ◽

Kinase Inhibitors ◽

Binding Assay ◽

Aurora A Kinase ◽

Aurora A ◽

Aurora Kinases ◽

Intramolecular Quenching ◽

Labeled Probe ◽

Key Steps ◽

Tumor Types

The Aurora kinases are a group of serine/threonine protein kinases that regulate key steps during mitosis, and deregulation of these proteins (e.g., by gene amplification or overexpression) has been linked to a wide variety of tumor types. Thus, Aurora-A and Aurora-B have been intensely studied as targets for anticancer therapy and are now clinically validated targets. Here we report on the development of a novel fluorescence intensity binding assay for Aurora-A kinase inhibitors using a fluorescently labeled probe compound that shows intramolecular quenching when unbound but exhibits a dramatic increase in fluorescence when bound to Aurora-A.

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The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

Cellular & Molecular Biology Letters ◽

10.2478/s11658-013-0092-1 ◽

2013 ◽

Vol 18 (3) ◽

Cited By ~ 31

Author(s):

Arezou Ghahghaei ◽

S. Bathaie ◽

Hoda Kheirkhah ◽

Elmira Bahraminejad

Keyword(s):

Electron Microscopy ◽

Fluorescence Intensity ◽

Amyloid Fibril ◽

Binding Assay ◽

Cd Spectroscopy ◽

Thioflavin T ◽

Drug Candidate ◽

Amyloid Formation ◽

Ans Binding ◽

Aβ Amyloid

AbstractAβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ40, and the less common but more amyloidogenic form, Aβ42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ42 amyloids. To further investigate the relationship between crocin and Aβ42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

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Improvements to immunoassays by use of covalent binding assay plates

Clinical Chemistry ◽

10.1093/clinchem/40.9.1833 ◽

1994 ◽

Vol 40 (9) ◽

pp. 1833-1837 ◽

Cited By ~ 8

Author(s):

A S Douglas ◽

C A Monteith

Keyword(s):

Alkaline Phosphatase ◽

Edge Effect ◽

Binding Assay ◽

Covalent Binding ◽

Blocking Agent ◽

Antibody Binding ◽

Plate Surface ◽

High Background ◽

Color Development ◽

Capture Antibody

Abstract The uniformity of Xenobind covalent binding microwell plates was compared with that of passive binding plates by binding streptavidin to the plate surface, reacting the streptavidin with biotinylated alkaline phosphatase, and developing color with phenolphthalein monophosphate. Absorbance of the covalent binding plates at 570 nm was 0.95 A (CV 1.9%) compared with 0.35-0.5 A (CV 6.7-16.9%), for passive plates. No edge effect was observed with Xenobind covalent plates. Use of both plates in a model immunoassay gave a CV of 4% for the covalent binding plate and 12-15% for the passive binding plates. Moreover, the background absorbance (color development when no antigen was present) for the covalent binding plates was < 0.05 A in all cases, whereas that for the passive binding plates ranged up to 0.45 A. The high background on passive plates results from displacement of capture antibody and (or) blocking agent from the plate, thereby providing a place for detector antibody binding.

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P-064 Application of an artificial intelligence model for morphologic prediction of fertilization-competent human spermatozoa

Human Reproduction ◽

10.1093/humrep/deab127.043 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

T Y Leung ◽

C L Lee ◽

P C N Chiu

Keyword(s):

Artificial Intelligence ◽

Deep Learning ◽

Sperm Morphology ◽

Binding Assay ◽

Morphological Characteristics ◽

Selection Method ◽

Human Spermatozoa ◽

Sperm Selection ◽

Binding Ability

Abstract Study question What is the role of artificial intelligence in selecting fertilization-competent human spermatozoa according to their morphological characteristics? Summary answer The established AI model in this study can be potentially used to select semen samples with superior fertilization potential in clinical settings. What is known already Defective spermatozoa-zona pellucida (ZP) interaction causes subfertility and is a major cause of low IVF fertilization rates. While ICSI benefits patients with defective spermatozoa-ZP binding, a standard method to identify such patients prior to conventional IVF is lacking. The application of artificial intelligence to sperm morphology analysis has become a topic of growing interest owing to the fact that the conventional assessment is highly subjective and time-consuming. Deep-learning, a core element of artificial intelligence (AI), incorporates the convolutional neural networks (CNN) to process all the data composing a digital image through successive layers to identify the underlying pattern. Study design, size, duration The fertilization-competent spermatozoa were isolated according to their binding ability to the ZP. The ZP-bound and -unbound spermatozoa were collected for functional assays and to establish an AI model for morphologic prediction of sperm fertilization potential. Human spermatozoa (n = 289) were isolated from normozoospermic samples. Human oocytes (n = 562) were collected from an assisted reproduction program in Hong Kong. Sample collection has been ongoing and will continue until the end of this study in November 2021. Participants/materials, setting, methods Sperm-ZP binding assay was employed to collect ZP-bound and -unbound spermatozoa. The fertilization potential and genetic quality of the collected spermatozoa were evaluated by our established protocols. Diff-Quik- stained images of ZP-bound and -unbound spermatozoa were collected respectively for the establishment of an AI model. A novel algorithm for sperm image transformation and segmentation was developed to pre-process the images. CNN architecture was then applied on these pre-processed images for feature extraction and model training. Main results and the role of chance Our result showed that the sperm-ZP binding assay had no detrimental effect on sperm viability when compared with the raw samples and unbound-sperm subpopulations. ZP-bound spermatozoa were found with statistically higher acrosome reaction rates, improved DNA integrity, better morphology, lower protamine deficiency and higher methylation level when compared with the unbound spermatozoa. A deep-learning model was trained and validated by analyzing a total of 1,334 and 885 of ZP-bound/unbound spermatozoa to evaluate the predictive power of sperm morphology for ZP binding ability. Our newly trained AI-based model showed initial success in classifying the ZP-bound/ unbound spermatozoa according to their morphological characteristics with high accuracy of 85% and low computational complexity. Limitations, reasons for caution This sperm selection method requires micromanipulation and relatively long processing time to recover ZP-bound spermatozoa. In addition to limited availability, the use of human materials may result in interassay variations affecting the reproducibility of this method among laboratories. Wider implications of the findings In light of current findings, AI-based sperm selection method may provide high predictive values of sperm fertilization potential for clinical purposes. This method is particularly applicable to patients who had poor fertilization outcomes after conventional IVF treatments or those with high degree of defective sperm-ZP binding ability. Trial registration number not applicable

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Serological and fluorescence studies of the heat stability of bovine lactoferrin

Journal of Dairy Research ◽

10.1017/s0022029900016885 ◽

1979 ◽

Vol 46 (1) ◽

pp. 83-93 ◽

Cited By ~ 9

Author(s):

Augustin Baer ◽

Marko Oroz ◽

Bernard Blanc

Keyword(s):

Metal Binding ◽

Binding Sites ◽

Complement Fixation ◽

Heat Stability ◽

Heat Denaturation ◽

Bovine Lactoferrin ◽

Iron Binding ◽

Binding Ability ◽

Fluorescence Studies ◽

Tryptophan Residues

SUMMARYThe heat denaturation of Fe-saturated lactoferrin (If) and Fe-free lactoferrin (apo-lf) was studied using the methods of micro-complement fixation and fluorescence. It was established that the change in conformation of apo-lf, induced by iron binding, conferred a higher heat stability to the molecule: the changes were observed at temperatures above 40 °C for apo-lf and above 60 °C for If. The Fe-binding ability of the protein was partially independent of the degree of denaturation. Fluorescence analyses indicated that tryptophan residues were probably not directly involved in the metal binding. There was no evidence of antibodies interfering with the binding sites.

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