Dissociation-enhanced Lanthanide Fluorescent Immunoassay Research Articles

Installation of AutoDELFIA: A new addition in Bangladesh for the Screening of Congenital Hypothyroidism in Newborn Babies

Newborn Screening for the diagnosis of Congenital Hypothyroidism (CH) was first started in Bangladesh in 1999 by the Immunoradiometric assay (IRMA). Dissociation enhanced lanthanide fluorescent immunoassay (DELFIA) was started in 1986 due to its superior sensitivity, speed, and non-radioactive methodology. Auto DELFIA 1235 automatic immunoassay system was introduced in the In vitro Division of National Institute of Nuclear Medicine & Allied Sciences (NINMAS) under the ADP Project “Screening of Congenital Hypothyroidism in Newborn Babies (Phase 2)”. It is an automatic system that can minimize the pipetting error. In this experiment, dried blood spots (DBS) were punched into an antibody-coated microtitration well with an automated Panthera Puncher and analyzed by AutoDELFIA. Six different standards of 0.8, 10.3, 24.5, 49.3, 93.2, 239μU/mL were used and the calibration curve was drawn by using these standards. Standard error and Standard deviation were found 12.4 and 30.5. Limit of detection (LOD) and Limit of Quantification (LOQ) of X variables were 0.16 and 0.47. The average of the percent recovery was 96.7. The R2 value of the calibration curve was 0.9985. The accuracy of the standard samples was 96.7 ±10.6. For this assay two Quality Control (QC) of 16.39 μU/mL and 63.09, μU/mL were used. All of the measured values from the calibration curve were close to ±1 standard deviation. All these findings indicate appropriateness of Auto DELFIA System for the CH screening from dried blood spots in the newborn screening laboratory of the In vitro division of NINMAS. Bangladesh J. Nuclear Med. 23(1&2): 15-20, 2020

Development of an automated assay for accelerated in vitro detection of DNA adduct-inducing and crosslinking agents

Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and 32P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time–resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate.

Diagnostic evaluation of heel prick newborn screening of thyroid stimulating hormone on dissociation-enhanced lanthanide fluorescence immunoassay with the establishment of reference value in Pakistani neonates.

To develop and validate a method on Dissociation Enhanced Lanthanide Fluorescence Immunoassay for neonatal heel prick blood human thyroid stimulating hormone and the establishment of its reference value in the local population. The multi-centre cross-sectional validation study was conducted from September 2016 to September 2018 at Zahra Beau Naqvi Foundation Welfare Trust laboratory, Islamabad, Pakistan, and comprised samples related to newborns aged 1 month or less taken from neonatal units of 39 hospitals based in Punjab, Khyber Pakhtunkhuwa, Gilgit-Baltistan and Azad Jammu and Kashmir. Samples were collected after 24 hours of birth using the heel prick test. The samples were dried and sent to the laboratory for assessment where Dissociation Enhanced Lanthanide Fluorescent Immunoassay was used to estimate thyroid stimulating hormone levels. Data recorded included age, gender, and birth detail, like gestational age, mode of delivery etc. Data was analysed using SPSS 21. Method validation and reference value were manually calculated. Of the 14,147 samples received, 8,207(58%) related to boys and 5,940(42%) to girls. Most samples 4903(34.6%) came from Peshawar. The overall mean age of the newborns was 5.6±4.8 days. Thyroid stimulating hormone data was divided into three groups; positive with median value 27.8±36.6 uIU/ml, negative with median 1.42±1.60 uIU/ml, and borderline with median 11.4±4.12uIU/ml. Prevalence of congenital hypothyroidism in high-risk population in the positive group was 39(0.3%), negative 14,012(99.0%) and borderline 96(0.7%). Reference cut-off was calculated as 7.06uIU/ml for screening of healthy and positive cases of congenital hypothyroidism. Method Validation results showed limit of detection -0.5uU/ml, limit of quantitation LOQ 0.8uU/ml, accuracy 100±5%, precision coefficient of variation at each level of calibrators -4, 8.8, 1.2, 11.3, 7.2 and 4.3% respectively, and linearity from to 0.8uU/ml to 254.1uU/ml. Neonatal human thyroid stimulating hormone by heel prick blood was found to be an affordable and highly sensitive method of screening for congenital hypothyroidism.

Chemiluminescence Immunoassay: Basic Mechanism and Applications

Chemiluminescence immunoassay (CLIA) has been widely applied in different fields including environmental monitoring, liquid chromatography, clinical diagnosis; food safety, pharmaceutical analysis, immuno- and gene probe assays, as a promising approach for selective, sensitive, rapid and simple analysis. It is often necessary to detect a large number of complicated or low-abundance samples. However, traditional methods need great consumption of time, reagents and labor, which limit their clinical applications. As a result, rapid, high-throughput, sensitive and low-cost detection methods have become the development trend of CLIA. A new chemiluminescence immunoassay analyzer with advanced acridinium ester (AE) technology will be installed at the radioimmunoassy laboratory of National Institute of Nuclear Medicine and Allied Sciences (NINMAS) shortly. In conjunction with the existing Radioimmunoassay (RIA), Immunoradiometric assay (IRMA) and DissociationEnhanced Lanthanide Fluorescent Immunoassay (DELFIA), introduction of chemiluminescence immunoassay (CLIA) technology will expand the activities of in vitro immunoassay. It will also improve the research and development activities of in vitro division of NINMAS. In this review article, we have summarized the basic principle of chemiluminescence immunoassay and its applications. A comparison of RIA, CLIA and enzyme-linked immunosorbent assay (ELISA) was also briefly described in the article.Bangladesh J. Nuclear Med. 18(2): 171-178, July 2015

Newborn screening for congenital hypothyroidism in a tertiary care centre

Background: Congenital hypothyroidism (CH) is one of the most common preventable causes of mental retardation in paediatric population. Universal screening though possible is still not available for neonates even in tertiary care centers because of low priority in spite of proven community benefits. The aim and objective was to determine the prevalence of CH in neonates in a tertiary care center. Methods: The study was conducted in the neonatal unit of RSRM Lying in hospital- Stanley medical College as a prospective cross sectional study from March 2012 to May 2012. All newborns with gestational age of 34 weeks or more delivered in the hospital were included in the study. Sample was collected after getting informed consent after 48 hours of age till 7 days of age . 1695 babies who met the inclusion criteria were enrolled, from whom sample was collected. The blood sample was collected by heel prick on to a filter paper and TSH levels estimated by dissociation enhanced lanthanide fluorescent immunoassay (DELFIA). Exclusion criteria were preterm infants with gestational age less than 34 weeks, those who have received blood transfusion prior to sampling, neonates more than 7 days of age and refusal of informed consent were excluded from the study. Results: The study population consists of 1695 newborns. Among 1695 newborns 3 babies had elevated TSH representing a prevalence of 1.7 per 1000. Conclusions: Our Study results revealed the prevalence of hypothyroidism among infants is 1.7 per thousand.

New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10(-6)). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

Functionalized Metal Chelates Based on Diethylenetriaminetetraacetic Acids for Chemical Modification of Proteins and Small Biomolecules

Bifunctional reagents based on diethylenetriaminetetraacetic acid containing a bound metal ion and a reactive functional group for the interaction with proteins and low-molecular-weight substances have been synthesized. An Amino-derivative of a complexonate was obtained by acylation of monosubstituted diamine with diethylenetriaminepentaacetic acid dianhydride followed by deprotection ofthe amino group, purification by anion exchange chromatography and chelation of Eu3+. This metal chelate derivative was used for labeling 17α-hydroxyprogesterone 3-(O-carboxymethyl)oxime and horseradish peroxidase. The enzyme modified with the Eu3+ complexonate at the carbohydrate component and with a cortisol derivative at the polypeptide chain was used in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) as well as in an enzyme immunoassay of the steroid hormone. DELFIA showed that labeled 17α-hydroxyprogesterone retained the affinity for corresponding antibodies. A Eu(3+)-complexonate carboxy-derivative N-succinimide ester was obtained by acylation of the aminochelate with p-phthalic acid di-N-succinimide ester. It was used for modification of amino groups of lysine residues in polypeptide chains of human serum albumin and some immunoglobulins G. Purification of Eu3+ complexonate-protein conjugates by gel-chromatography on a Superose- 12 column allowed to separate the modified proteins from unreacted low molecular weight Eu(3+)-derivatives and to determine a degree of lanthanide inclusion into a protein. The amount of Eu3+ covalently attached to a protein was determined by measuring the fluorescence of a conjugate in the dissociative-enhancement solution. The obtained values correlated well with the results of ICP-MS determination of Eu3+ concentration in a conjugate solution. It was shown that conjugates of monoclonal antibodies obtained by the proposed method possessed the required characteristics of fluorescence intensity, signal-to-noise ratio, sensitivity and specificity in DELFIA medical diagnostic systems.

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

BackgroundThe ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery.MethodsPlasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors.ResultsIn both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition.ConclusionsThe two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

Newborn Screening in Slovenia.

IntroductionNewborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH.MethodsBlood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA).ResultsFrom 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively.ConclusionsSuccessful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1–21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

Seroepidemiology of Norovirus‐Associated Travelers' Diarrhea

Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India. Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay. NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50 > 200, a level that has been described previously as a possible correlate of protection. We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

Newborn screening program for congenital hypothyroidism in Montenegro

Objective – The aim of this study was to assess the prevalence of congenital hypothyroidism (CH) over the last 5 years and analyze and summarize the status of newborn screening in Montenegro. Materials and methods – This is a population-based retrospective study. Blood samples were collected from the heels of newborns 48 – 96 hours after birth and thyroid-stimulating hormone (TSH) was determined. The cut off value in our laboratory was 10 mIU/l in whole blood. Dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) was used for detection. Results – Over the period January 2008 – December 2012, a total of 40,758 newborns were screened and 17 cases were confirmed as CH - both transient and permanent. The program covered all live born infants (100%). Recall rate was 0.97 – 1.36%. Mean age of starting treatment was 21.4±6.4 days. Conclusion – During the period of analysis in our study, 17 cases of CH were detected. The overall prevalence of CH was 1 in 2397 live births. This is the first report on newborn screening for congenital hypothyroidism in Montenegro.

A SENSITIVE TIME-RESOLVED FLUOROIMMUNOASSAY FOR DETERMINATION OF MEDIAN LEVELS OF PREGNANCY-ASSOCIATED PLASMA PROTEIN A IN PREGNANT WOMEN IN CHINA

Pregnancy-associated plasma protein A (PAPP-A) is an important serum marker for first trimester screening. Its weekly median value varies with ethnicity. A one-step time-resolved fluoroimmunoassay (TRFIA) using two monoclonal antibodies against PAPP-A with Eu3+ chelates as labels has been developed. Using the assay described here, we evaluated 5,301 normal serum samples from Chinese women at 7–13 weeks of gestation. The detection limit using this assay was 1.2 mIU/L, and the maximum detection range was up to 10,000 mIU/L. The intra-assay and inter-assay coefficients of variation were <3.0% and <5.0%, respectively, and the mean recovery rate was 98.0%. PAPP-A concentrations measured in 516 maternal serum samples correlated well with those obtained by Dissociation-Enhanced Lanthanide Fluorescent Immunoassay (DELFIA) PAPP-A assay (r = 0.988, P < 0.001). The medians for 7–13 weeks of maternal serum PAPP-A were higher in the women from China compared to reports from other countries. The present assay possesses accuracy and high sensitivity and exhibits great potential for the clinical analysis of PAPP-A. Our investigation on the median concentrations of PAPP-A will help establish reference values that are specific for China and study the importance of ethnic factors in biochemical screening.

Development of a Pharmacodynamic Assay Based on PLCγ2 Phosphorylation for Quantifying Spleen Tyrosine Kinase (SYK)–Bruton’s Tyrosine Kinase (BTK) Signaling

Spleen tyrosine kinase (SYK) and Bruton’s tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVI-mediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.

Capillary electromigration based techniques in diagnostics of prion protein caused diseases

Transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases with long incubation time. This group includes Creutzfeld-Jakob disease, kuru, scrapie, chronic wasting disease, and bovine spongiform encephalopathy. Sensitive and specific detection of abnormal prion protein as "a source agent" of the above-mentioned diseases in blood could provide a diagnostic test or a screening assay for animal and human prion protein diseases diagnostics. Therefore, diagnostic tests for prion protein diseases represent unique challenge requiring development of novel assays exploiting properties of prion protein complex. Presently, diagnostic methods such as protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, fluorescence correlation spectroscopy, and/or flow microbead immunoassay are used for abnormal prion protein (PrP(Sc) ) detection. On the other hand, using of CE for PrP(Sc) detection in body fluids is an attractive alternative; it has been already applied for the blood samples of infected sheep, elk, chimpanzee, as well as humans. In this review, assays for prion protein detection are summarized with special attention to capillary electromigration based techniques, such as CE, CIEF, and/or CGE. The potential of the miniaturized and integrated lab-on-chip devices is highlighted, emphasizing recent advances of this field in the proteomic analysis.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus

ObjectivesTo evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA)...

Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

BackgroundCollagen Triple Helix Repeat Containing-1 (CTHRC1) and Nuclear factor (erythroid-derived 2)-like 3 (NFE2L3) may be useful biomarker candidates for the diagnosis of colorectal cancer (CRC) since they have shown an increase messenger RNA transcripts (mRNA) expression level in adenomas and colorectal tumours when compared to normal tissues.MethodsTo evaluate CTHRC1 and NFE2L3 as cancer biomarkers, it was generated and characterised several novel specific polyclonal antibodies (PAb), monoclonal antibodies (MAbs) and soluble Fab fragments (sFabs) against recombinant CTHRC1 and NFE2L3 proteins, which were obtained from different sources, including a human antibody library and immunised animals. The antibodies and Fab fragments were tested for recognition of native CTHRC1 and NFE2L3 proteins by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA) in colorectal cell lines derived from tumour and cancer tissues.ResultsBoth, antibodies and a Fab fragment showed high specificity since they recognised only their corresponding recombinant antigens, but not a panel of different unrelated- and related proteins.In Western blot analysis of CTHRC1, a monoclonal antibody designated CH21D7 was able to detect a band of the apparent molecular weight of a full-length CTHRC1 in the human colon adenocarcinoma cell line HT29. This result was confirmed by a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the monoclonal antibodies CH21D7 and CH24G2, detecting CTHRC1 in HT29 and in the colon adenocarcinoma cell line SW620.Similar experiments were performed with PAb, MAbs, and sFab against NFE2L3. The immunoblot analysis showed that the monoclonal antibody 41HF8 recognised NFE2L3 in HT29, and leukocytes. These results were verified by DAS-ELISA assay using the pairs PAb/sFab E5 and MAb 41HF8/sFab E5.Furthermore, an immunoassay for simultaneous detection of the two cancer biomarkers was developed using a Dissociation-Enhanced Lanthanide Fluorescent Immunoassay technology (DELFIA).ConclusionsIn conclusion, the antibodies obtained in this study are specific for CTHRC1 and NFE2L3 since they do not cross-react with unrelated- and related proteins and are useful for specific measurement of native CTHRC1 and NFE2L3 proteins. The antibodies and immunoassays may be useful for the analysis of CTHRC1 and NFE2L3 in clinical samples and for screening of therapeutic compounds in CRC.

A Cell-Based High-Throughput Screening Assay to Measure Cellular Histone H3 Lys27 Trimethylation with a Modified Dissociation-Enhanced Lanthanide Fluorescent Immunoassay

Histone proteins are subject to several modifications, including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These posttranslational modifications play critical roles in chromatin structure and gene transcription. Because of their involvement in the progression of a variety of diseases, histone modifications are attracting increased attention. We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of “freeze-thaw” and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for high-throughput screening. The findings presented here should be applicable to other assays involving binding and extraction of histone proteins.

Serological responses to experimental Norwalk virus infection measured using a quantitative duplex time-resolved fluorescence immunoassay.

A quantitative duplex time-resolved fluorescence assay, dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), was developed to measure Norwalk virus (NV)-specific IgA and IgG antibodies simultaneously. The duplex assay showed superior performance by detecting seroconversion following experimental NV infection at an earlier time point than a reference total immunoglobulin enzyme-linked immunosorbent assay (ELISA).

First trimester and second-trimester integrated screening for Down's syndrome

To evaluate the effect of first and second-trimester integrated screening so as to provide an efficient screening protocol for Down's syndrome. Using the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), the freeβhCG (beta human chorionic gonadotropin), PAPP-A (pregnancy associated plasma protein-A) and NT (nuchal translucency) value of type B ultrasound were assayed in the pregnancy serum during the first trimester (11-13W(+6) d) and free βhCG and AFP (alpha fetoprotein) during the second trimester(15-20W(+6) d). By the risk calculation software, the risks during both trimesters and their integrated risk were calculated for each patient respectively. Amniocentesis and venepuncture were employed for diagnosing the high-risk patients (> 1/270). Electronic network follow-up was carried out after delivery. In a total of 4237 pregnant women, 98 were found to carry a high risk during the first trimester, 241 during the second trimester and 101 during the integrated screening respectively. And 2, 3 and 4 cases were diagnosed with Down's symptom at a detection rate of 50%, 75% and 100% and a detection efficiency of 1:50, 1:80 and 1:25 respectively. Integrated screening is superior to either the first or second-trimester screening. With a lower false positive rate and a higher detection rate, it reduces the chance of invasive puncture. Advanced type B ultrasonic technology is needed to improve the first-trimester diagnostic efficiency and to develop a better integrated screening protocol.