Screening Reagents Research Articles

Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter.

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.

How to Achieve Standardization? Diluted Russell Viper Venom Test for Lupus Anticoagulant Detection in a Chinese Female Population.

On an international scale, guidelines and proposals for lupus anticoagulant detection have been published over the last 20 years, but until now, standardization has not been completely realized. The aim of this study was to evaluate the different ways of interpreting the results of lupus anticoagulant detection for standardization. A retrospective review of 15 447 instances of lupus anticoagulant detection by the diluted Russell Viper Venom test for female patients presenting with problems relating to the areas of reproduction, gynecology and obstetrics was performed. Lupus anticoagulant data were compared between different departments, months, reagent lots and cutoffs. Significant differences were found in patient data between different reagent lots, especially between lots of screening reagents (monthly average: highest 37.96 s vs lowest 33.88 s) and in the positive rates of lupus anticoagulant by different detection cutoffs (47.58% by using LA1/LA2 > 1.20 without normalization as a cutoff in Lot 1 vs 1.52% by using LA1 > 44 s as a cutoff in Lot 3). Compared with the cutoff using the value above the 99th percentile of LA1 for the healthy donors per lot, the cutoff using integrated tests with normalization had the smaller deviation of positive rate between different reagent lots. Pregnant women had higher LA1/LA2 levels than nonpregnant women. Based on the results, normalization is needed because there are significant lot-to-lot variations. Integrated tests with normalization might be a better standard by which to confirm lupus anticoagulant. Pregnant women should have population-specific cutoffs because they have higher LA1/LA2 levels.

A Study on the Methods to Determine the Re-Entry of NAT-Reactive Blood Donors in China.

The false reactivity results of nucleic acid screening reagents have been reported in China and abroad. To identify false reactivity, effectively protect the rights of blood donors, and cherish limited blood resources, many countries study the methods of re-entry of NAT reactive blood donors. ELISA non-reactive and NAT-reactive donors who donated blood in 2012 - 2017 were selected, and informed consent was obtained. The collected blood samples were tested by ELISA and NAT. Then, the samples were tested by ECLA for HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe, anti-HCV, and anti-HIV. Forty-two donors were called back and tested: 15 of them were ELISA non-reactive/NAT-reactive, and 27 of them were ELISA non-reactive/NAT non-reactive. The ECLA results indicated that 90.5% ELISA non-reactive/NAT non-reactive donors were anti-HBc-reactive and/or anti-HBe-reactive (21 cases anti-HBc/anti-HBe-reactive, 17 cases anti-HBe-reactive). After 6 mouths, anti-HBc-reactive or anti-HBe-reactive donors were also anti-HBc-reactive and/or anti-HBe-reactive, and these donors were deferred permanently. Four cases that were non-reactive to all tested results may be eligible for re-entry. In view of blood safety, it is important to establish a set of safety criteria to allow re-entry of ELISA non-reactive/NAT-reactive blood donors.

Manipulation of Tropomysin 1 in iPSCs to Enhance in Vitro Blood Cell Production

Donor-derived blood transfusions are critical to our healthcare system, but do not fully meet the needs of patients with multiple alloantibodies, rare blood types, or HLA-sensitization. These needs have fueled the concept of ex vivo blood cell production, which might address issues related to demographic aging, infectious outbreaks transmitted by transfusions, and rare blood types. Blood cells produced in vitro could be used for transfusions, and could also be used as blood bank testing reagents (Coleman et al, Transfusion 2019). One major challenge in deploying this system is efficiently scaling up cell production. We used machine learning and genome-edited induced pluripotent stem cell (iPSC) models to determine that Tropomyosin 1 (TPM1) normally inhibits in vitro hematopoiesis. TPM1 knockout (TPM1KO) iPSCs produced 2-fold more hematopoietic progenitor cells (HPCs) than controls, thereby increasing production of mature blood cells that were functionally normal (Thom et al, BMC Biol 2020). During human hematopoiesis, HPCs arise from specialized vascular 'hemogenic endothelial' cells (HE) with distinct surface markers that can be used for identification and isolation. To define molecular mechanisms by which TPM1 regulates in vitro primitive hematopoiesis, we performed RNA sequencing analysis on sorted KDR+CD31+ endothelial cells and CD43+ HPCs from TPM1KO and control cultures. TPM1KO endothelial cells and HPCs had altered expression of genes and pathways known to regulate HE biology, including cell adhesion, integrin expression, and integrin-mediated signaling (p<0.05). 'Anoikis' is an apoptosis-like programmed cell death that occurs after extracellular matrix detachment. This process may limit nascent non-adherent HPC production in vitro, but has not been previously studied. TPM1KO cells showed increased expression of N-cadherin and RAP1-activating genes; increased N-cadherin and activated RAP1 limit anoikis in other biological contexts. In sum, these results suggested that TPM1KO cultures increased HE production and/or survival. To analyze HE production at the single cell level, we sorted KDR+CD31+CD43- endothelial cells and plated them in limiting dilution. We cultured sorted cells in hematopoietic cytokines for 7 days, and analyzed the number of wells in which CD43+ HPCs arose from sorted TPM1KO and control cells. Using limiting dilution analysis (Hu & Smyth,J. Immunol. Methods 2009), we found that TPM1KO cultures produced 2-fold more HE than controls. These results show that TPM1KO enhances in vitro hematopoiesis by increasing HE and subsequent HPC production, perhaps by limiting anoikis in nascent HPCs. TPM1-mediated regulation at the HE stage represents a novel mechanism that may be genetically or pharmacologically exploited to augment in vitro hematopoiesis. These findings will help boost in vitro HPC and blood cell production to clinically relevant scales, supporting efforts to produce blood cells for direct transfusion and/or to be used as clinical screening reagents. Disclosures No relevant conflicts of interest to declare.

Evaluation of immunoassays for the determination of MDMA and cannabinoids in urine samples

Methylenedioxymethamphetamine (MDMA) is structurally related to methamphetamine (MA). There are many different commercially available immunoassay (IA) reagents for the initial screening of amphetamine and/or methamphetamine. These reagents may be employed to detect MDA/MDMA in urine samples. In order to select a suitable reagent for the initial screening of MDMA in urine samples, we evaluated 7 different amphetamine immunoassay reagents: Emit d.a.u. Monoclonal Amphetamine/Methamphetamine; Emit II Plus Monoclonal Amphetamine/Methamphetamine; Emit d.a.u. Amphetamine Class; DRI Amphetamine; AxSYM Amphetamine/Methamphetamine II; Abuscreen Online Amphetamine and Cedia Amphetamine/Ecstasy. We also determined the cross reactivity of these reagents with MDA, MDMA, MBDB, MDEA and other phenethylamines. These IA reagents were employed to screen a group of 146 urine samples collected from pub patrons. Results of the initial screening were compared with results obtained with gas chromatography/mass spectrometry (GC/MS). Five of the IA assays were acceptable for the initial screening of MDMA, except the Emit II Plus Monoclonal Amphetamine/Methamphetamine reagent and Emit d.a.u. Class Amphetamine reagent. Results obtained with Emit II reagent showed high false negatives, while results obtained with Emit d.a.u. Class reagent showed high false positives. We evaluated 5 different IA for cannabinoids. Results of the initial screening of 74 urine samples collected from pub patrons were compared with results obtained by GC/MS. There are 12 confirmed positives with GC/MS. Results obtained with DRI reagent showed no false negatives, while results obtained with Emit, Abuscreen Online, AxSYM and Cedia reagents have 4, 2, 3 and 4 false negatives, respectively.

Rapid establishment of a COVID-19 biobank in NHRI by National Biobank Consortium of Taiwan

By the request of the Minister of Health and Welfare, NHRI Biobank was assigned to establish a COVID-19 biobank in early Feb, 2020 to collect COVID-19 patients’ blood samples for Taiwan researchers and industries in an emergent way. It was set up in less than 3 weeks and quickly opened for application. By August 5, 2020, this COVID-19 biobank has collected 165 blood samples of 110 patients from more than 10 hospitals across north, middle and south part of Taiwan, including both COVID-19 (+) and (-) pneumonia patients. This biobank can provide applicants with biosamples, such as serum, DNA and RNA, and also the clinical and genomic data, so as to accelerate the COVID-19 treatment and prevention research in Taiwan. This COID-19 biobank already received 15 applications. It has become the most important research resource for the COVID-19 pandemic in Taiwan, including new screening reagents, disease mechanism, the variable human responses and epidemic preventions. Since it is publicly available for both academic and industrial applicants.

Low prevalence red blood cell antigens: transfusions, babies, and changing demographics

See article on page 870–874, in this issue

De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

BackgroundThe promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.ResultsFluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L.ConclusionsFACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Designer covalent heterobivalent inhibitors prevent IgE-dependent responses to peanut allergen

Allergies are a result of allergen proteins cross-linking allergen-specific IgE (sIgE) on the surface of mast cells and basophils. The diversity and complexity of allergen epitopes, and high-affinity of the sIgE-allergen interaction have impaired the development of allergen-specific inhibitors of allergic responses. This study presents a design of food allergen-specific sIgE inhibitors named covalent heterobivalent inhibitors (cHBIs) that selectively form covalent bonds to only sIgEs, thereby permanently inhibiting them. Using screening reagents termed nanoallergens, we identified two immunodominant epitopes in peanuts that were common in a population of 16 allergic patients. Two cHBIs designed to inhibit only these two epitopes completely abrogated the allergic response in 14 of the 16 patients in an in vitro assay and inhibited basophil activation in an allergic patient ex vivo analysis. The efficacy of the cHBI design has valuable clinical implications for many allergen-specific responses and more broadly for any antibody-based disease.

HUMAN INDUCED PLURIPOTENT STEM CELL REGION DETECTION IN BRIGHT-FIELD MICROSCOPY IMAGES USING CONVOLUTIONAL NEURAL NETWORKS

Human induced pluripotent stem (iPS) cells represent an ideal source for patient specific cell-based regenerative medicine. For practical uses of iPS cells, large-scale, cost- and time-effective production of fully reprogrammed iPS cells from a number of patients should be achieved. To achieve this goal, culture protocols for inducing iPS cells as well as methods for selecting fully reprogrammed iPS cells in a mixture of cells which are still in reprogramming and non-iPS differentiated cells, should be improved. This paper proposes a convolutional neural network (CNN) structure to classify a bright-field microscopy image as respective probability images. Each probability image represents regions of differentiated cells, fully reprogrammed iPS cells or cells still in reprogramming, respectively. The CNN classifier was trained by multiple types of image patches which represent differentiated, reprogramming and reprogrammed iPS cells, etc. Classification of an image containing the confirmed iPS cells by the trained CNN classifier shows that high classification accuracy can be achieved. Classifications of sets of time-lapse microscopy images show that growth and transition from CD34[Formula: see text] human cord blood cells through reprogramming to reprogrammed iPS cells can be visualized and quantitatively analyzed by the output time-series probability images. These experiment results show our CNN structure yields a potential tool to detect the differentiated cells that possibly undergo reprogramming to iPS cells for screening reagents or culture conditions in human iPS induction, and ultimately further understand the ideal culturing conditions for practical use in regenerative medicine.

不規則抗体スクリーニング試薬0.8%セルスクリーンJ -Alba-の検討

不規則抗体スクリーニング試薬0.8%セルスクリーンJ -Alba-の検討

How to Optimize Activated Partial Thromboplastin Time (APTT) Testing: Solutions to Establishing and Verifying Normal Reference Intervals and Assessing APTT Reagents for Sensitivity to Heparin, Lupus Anticoagulant, and Clotting Factors.

The activated partial thromboplastin time (APTT) assay is a very common coagulation test, used for several reasons. The test is conventionally used for assessing the contact factor (intrinsic) pathway of blood coagulation, and thus for screening deficiencies in this pathway, most typically factors VIII, IX, and XI. The APTT is also sensitive to contact factor deficiencies, including factor XII, prekallikrein, and high-molecular-weight kininogen. The APTT may also be elevated in a variety of conditions, including liver disease, vitamin K deficiency, and disseminated intravascular coagulation. The APTT can also be used for monitoring unfractionated heparin (UFH) therapy, as well as for screening lupus anticoagulant (LA) or for assessing thrombosis risk. Which of these separate uses is important to a given laboratory or clinician depends on the laboratory and the clinical context. For example, UFH sensitivity is important in hospital-based laboratories, where UFH therapy is used, but not in hospital-based laboratories where low-molecular-weight heparin (LMWH) is largely employed or where UFH may be assessed by anti-factor Xa testing, or in private/community laboratories not associated with a hospital system. High sensitivity to (low levels of) factors VIII, IX, and XI is generally preferred, as their deficiencies are clinically significant. Also preferred, but not usually achieved, is low sensitivity to factor XII and other contact factors, as these deficiencies are usually asymptomatic. Nevertheless, a good knowledge of factor sensitivity is usually needed, if only to help explain the reasons for a prolonged APTT in a given patient, or whether factor testing or other investigation is required. A good working knowledge of reagents sensitivity to LA is also advisable, especially when the reagent is used as part of a LA test panel, or else as a "general-purpose screening reagent." The current report is aimed at providing some guidance around these questions, and is intended as a kind of "how to" guide, that will enable laboratories to assess APTT reagents in regard to their sensitivity to heparin, LA, and clotting factors. The report also provides some advice on generation of normal reference ranges, as well as solutions for troubleshooting prolonged APTTs, when performing factor testing or searching for inhibitors.

Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment.

Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.

Novel approach for the rapid screening of banned aromatic amines in dyed textiles using a chromogenic method.

A novel and simple method utilizing a chromogenic reaction on filter paper is introduced for the rapid screening of banned aromatic amines released from azo dyes. The proposed method involves the sample preparation protocols outlined by the current standard method and the chromogenic reaction of extracted aromatic amines on filter paper. Based on the principle of the reaction between primary amines and aldehydes, p-dimethylaminobenzaldehyde (DMAB) was used as the chromogenic reagent for the rapid screening of 24 carcinogenic aromatic amines and aniline without any chromatographic instruments under optimized experimental conditions. The detection limit for all the aromatic amines in this study was less than 15mg/kg. A total of 727 dyed textile samples were analyzed using both the present standard method and the proposed method simultaneously. Using the proposed method, a total of 471 samples did not require further instrumental analysis, which can dramatically save instrumental detection time (61.2%), can decrease instrumental detection costs, and can avoid the use of large amounts of toxic reagents. The proposed method has been applied to detect banned aromatic amines in some inspection institutions and dye factories and has large social and economic benefits. Graphical abstract Chromogenic reaction methods.

A compartmentalized culture device for studying the axons of CNS neurons

We report here the development of a compartmentalized culture device that allows the spatial separation of the somatodendrites and axons of central nervous system (CNS) neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, axons are the only processes that can migrate through the septum to the bottom compartment. The axons in the bottom compartment can be studied directly in real-time or through immunofluorescence staining after fixation. Axons containing ∼3 μg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed.

Purification, identification and molecular mechanism of two dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from Antarctic krill (Euphausia superba) protein hydrolysate.

Dipeptidyl peptidase IV (DPP-IV) played an important role in blood glucose regulation. Inhibition of DPP-IV may improve glycemic control in diabetics by preventing the rapid breakdown of incretin hormones and prolonging their physiological action. In this study, Antarctic krill (Euphausia superba) protein was hydrolyzed using animal proteolytic enzymes. The hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and reversed phase high-performance liquid chromatography (RP-HPLC). DPP-IV inhibitory activity of the fractions achieved from Antarctic krill protein was determined by DPP-IV screening reagent kit. Two purified peptides were identified by Xevo G2-XS QTof mass spectrometer (QTOF-MS). One peptide purified was Ala-Pro (AP) with IC50 values of 0.0530mg/mL, the other Ile-Pro-Ala (IPA) with IC50 values of 0.0370mg/mL. They both exhibited strong DPP-IV inhibitory activity. The molecular docking analysis revealed that DPP-IV inhibition by AP and IPA was mainly due to formation of a strong interaction surface force with the 91-96 and 101-105 amino acids of the DPP-IV. Our results suggested that the protein hydrolysate from Antarctic krill can be considered as a promising natural source of DPP-IV inhibitory peptides in the management of diabetes.

Human induced pluripotent stem cell region recognition in microscopy images using Convolutional Neural Networks.

We present a deep learning architecture Convolutional Neural Networks (CNNs) for automatic classification and recognition of reprogramming and reprogrammed human Induced Pluripotent Stem (iPS) cell regions in microscopy images. The differentiated cells that possibly undergo reprogramming to iPS cells can be detected by this method for screening reagents or culture conditions in iPS induction. The learning results demonstrate that our CNNs can achieve the Top-1 and Top-2 error rates of 9.2% and 0.84%, respectively, to produce probability maps for the automatic analysis. The implementation results show that this automatic method can successfully detect and localize the human iPS cell formation, thereby yield a potential tool for helping iPS cell culture.

Breeding of peanut variety Yuhua 4 by in vitro mutagenesis

The embryonic leaflets of peanut (Arachis hypogaea) variety Huayu 20 were used as explants and pingyangmycin as a mutagen to induce somatic embryos. Four weeks after the inoculation, the survived explants were transferred to somatic embryo germination medium containing screening reagent hydroxyproline, and finally 15 regenerated plants were obtained. Pedigree breeding method was used during the following selection breeding, and three lines with significantly increased yield and 23 lines with high oil content were obtained from these mutant offsprings. The line with both high yield and high oil content has passed peanut variety multi-location in Anhui province and was named "Yuhua 4". Its yield was 16.63% higher than that of the control variety Baisha 1016, ranking the first in all the testing varieties. Yuhua 4 showed the characteristics of early maturity, small pod and high oil content. The oil content of kernels was 56.10%, higher than that of original parent Huayu 20 with 49.50% oil content, tested by the Ministry of Agriculture of Oil and Products Quality Supervision, Inspection and Test Center (Wuhan), and the yield was 15% higher than that of Huayu 20. It was concluded that in vitro mutagenesis and target screening was an effective way on creating new germplasm and breeding new variety in peanut.

Frequency of Red Cell Alloantibodies in Pregnant Females of Navsari District: An Experience that Favours Inclusion of Screening for Irregular Erythrocyte Antibody in Routine Antenatal Testing Profile.

Alloimmunisation due to irregular erythrocyte antibodies is a recognised cause of hemolytic disease of the fetus and newborn (HDFN). Prior knowledge of red cell alloimmunisation in pregnant females guides the obstetrician to monitor the foetus for HDFN and if required for appropriated intervention. As limited data are available on prevalence of red cell alloimmunisation in pregnant females in India, the current study was carried out to know the prevalence of red cell alloimmunisation in pregnant females coming at our laboratory. Screening for irregular erythrocyte antibodies was performed in 1960 pregnant females after obtaining informed consent between June 2015 and June 2016. MatrixTM screening and identification reagent red cells from Tulip Diagnostics (P) Ltd were used, and column agglutination technique was employed as a method for the test. Twenty antibodies (all of single specificity) were detected in 1960 samples giving a prevalence rate of alloimmunisation of 1.02%. Out of the 20 antibodies, 18 were identified to be anti-D, 1 was anti-c and 1 antibody was anti-H. The results obtained were then compared with those reported in the literature. Red cell alloimmunisation is not uncommonly observed in pregnant females; the information gained can help the obstetrician to identify high-risk cases to timely start antenatal and post-natal treatment. Obstetricians should request screening for irregular red cell antibody desirably in all pregnant females; however, if limiting factors are there, it should be done at least in select group of pregnant females having bad obstetric history.

Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M−1 s−1 and 4300 M−1 s−1, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (kcat/Km) of 5200 M−1 s−1. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.