Kinds Of Assays Research Articles

Abstract 7505: Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays

Abstract Introduction: Androgen-receptor splice variant 7 (AR-V7) is constitutively activated isoform of AR and has been associated with resistance towards AR targeting therapies and development into mCRPC. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the drug, but the detection of AR-V7 with tissues has limitations such as clinical challenge to poorly accessible tumor tissues before and after treatment. Here, we developed the method of AR-V7 detection in circulating tumor cells (CTC) after isolation with CytoGen’s Smart BiopsyTM CTC isolator by in vitro assays of qPCR and immunofluorescence (IF) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Method: qPCR and IF methods were developed for high sensitivity and specificity of AR-V7 detection. Sensitivity and specificity were tested using AR-V7 positive cells (VCaP and 22Rv1) and negative cells (PC-3 and Raji). Limit of detection (LOD) test of AR-V7 expression was performed with AR-V7 positive cells (200 cells to 50 cells, 3 points) mixed with Raji cells (1 × 10^5cells). Then, AR-V7 positive cells (200 cells to 50 cells, 3 points) were spiked into peripheral blood mononuclear cells (PBMC) of blood sample (5mL, healthy donor) to mimic CTC after isolation by CytoGen’s Smart BiopsyTM CTC isolator, subsequently AR-V7 expression was tested with qPCR and IF assay. Results: AR-V7 expression was clearly detected by qPCR and IF in the LOD test of AR-V7 positive cells mixed with Raji cells and was not detected at all in the negative controls. The similar clear results were also observed in the spiking test of AR-V7 positive cells into PBMC isolated by CytoGen’s Smart BiopsyTM CTC isolator, suggesting that clinical samples of prostate cancer could be applied with excellent sensitivity and specificity for AR-V7. Conclusions: AR-V7 expresses only a part of cancer tissue, so it is difficult to accurately diagnose with traditional histological assays. Detection of AR-V7 in intact live CTC isolated by CytoGen’s Smart BiopsyTM CTC isolator might overcome limitations of tissue biopsy. In this study, we developed the method to detect AR-V7 in CTC with high sensitivity and specificity with qPCR and IF assay, and presented the results of verifying AR-V7 expression by double check using two kinds of assays. We plan to conduct clinical validation of the developed method through additional study using patient samples. Citation Format: Hoin Kang, Jihyun Lee, Min Hyung Kim, Soee Kim, Hyoyong Kim, Jubi Lee, Hae Ung Lee, Hai-Chon Lee, Jung Won Kim. Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7505.

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Current Trends in Molecular Imprinting: Strategies, Applications and Determination of Target Molecules in Spain.

Over the last decades, an increasing demand for new specific molecular recognition elements has emerged in order to improve analytical methods that have already been developed in order to reach the detection/quantification limits of target molecules. Molecularly imprinted polymers (MIPs) have molecular recognition abilities provided by the presence of a template molecule during their synthesis, and they are excellent materials with high selectivity for sample preparation. These synthetic polymers are relatively easy to prepare, and they can also be an excellent choice in the substitution of antibodies or enzymes in different kinds of assays. They have been properly applied to the development of chromatographic or solid-phase extraction methods and have also been successfully applied as electrochemical, piezoelectrical, and optical sensors, as well as in the catalysis process. Nevertheless, new formats of polymerization can also provide new applications for these materials. This paper provides a comprehensive comparison of the new challenges in molecular imprinting as materials of the future in Spain.

External quality assessment program for biochemical assays of human seminal plasma: a French 6-years experience

BackgroundIn 1999, despite a longstanding use, the WHO manual for the examination of human semen finally proposed to assay several biochemical components of the seminal plasma for a functional exploration of the male accessory glands. At the same time, an international effort was made to standardize laboratory tests and to increase their performance through ISO 15189 accreditation. In this setting, participation to relevant external quality assessment (EQA) schemes is an essential requirement for laboratories. To fulfil this injunction, we have organized an EQA program for seminal biochemistry using presumed commutable samples. In this study, we aimed to report an overview of the French laboratory offer, the kinds of assays used, their performance as well as their likelihood of satisfying ISO15189 requirements for EQA.ResultsBetween 2014 and 2019, we performed seven surveys. A median of six laboratories participated to each survey giving a ratio of one laboratory per 11.2 million inhabitants. Seven biomarkers are routinely assayed but the core set shared by all laboratories comprised citrate and zinc (prostate), fructose (seminal vesicles) and α-1, 4 glucosidase (epididymis). The use of CE-IVD marked methods concerned between 0 to 75% of overall assays. According to analytical specifications, 100% of laboratories results were compliant for zinc, 75% for citrate and α-1,4 glucosidase and 67% for fructose. By combining overall data in an empirical scoring system, we identified several types of seminal biomarkers: citrate, fructose and zinc appear as good candidates for a full accreditation, α-1,4 glucosidase still presents an analytical weakness, but prostatic acid phosphatase, free L-carnitine and glycerophosphocholine cannot be accredited in the current state.ConclusionsWe organized the first French EQA program for seminal biochemistry to help local laboratories to face their legal requirement to be fully accredited by 2020. It could be improved still further but it gave us an oversight on the analytic landscape. Effective methods are available for a confident biochemical exploration of prostate and seminal vesicles. However, that of epididymis appeared unexpectedly fragile. This andrological issue should be addressed by dedicated recommendations from health authorities and scientific societies.

Composition of bound polyphenols from carrot dietary fiber and its in vivo and in vitro antioxidant activity

Qualitative analysis of bound polyphenols from carrot dietary fiber (CDF-PP) was performed by ultra-performance liquid chromatography equipped with an electrospray ionization and quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS). Eleven organic acids, nine hydroxybenzoic acids and derivatives, six hydroxycinnamic acids and derivatives, four phenolic alcohols and derivatives, three flavonoids and derivatives, seven esters and derivatives, two other compounds, were detected by matching their retention times, secondary mass spectrometry fragment information with authentic standards or literature data. Furthermore, in vitro antioxidant activity was determined by different kinds of assays, including DPPH, ORAC, PSC, demonstrated that CDF-PP could scavenge radicals in a dose dependent manner. Moreover, CDF-PP exhibited significantly reactive oxygen species (ROS) scavenging activity in living Caenorhabditis elegans. To our knowledge, this is the first comprehensive research to investigate composition and in vitro/in vivo antioxidant activity of bound polyphenols in CDF, which implied that CDF-PP could be a promising source of antioxidants.

Visual detection of the prostate specific antigen via a sandwich immunoassay and by using a superwettable chip coated with pH-responsive silica nanoparticles.

A pH-responsive superwettable chip is described whose surface can switch between superhydrophobic and superhydrophilic. It can be used for the visual detection of the prostate specific antigen (PSA) based on contact angle readout. Magnetic beads were modified with primary antibody against PSA. After immunobinding, gold nanoparticles loaded with secondary antibody labeled with glucose oxidase is added. On addition of glucose, gluconic acid is formed which causes a drop in the local pH value. This results in a wettability switch of the pH-responsive superwettable chip from hydrophobic to hydrophilic. Under the optimized conditions, PSA can be quantified with a 3.2pgmL-1 limit of detection by analyzing the contact angle and the related color that changes from blue via orange to red. The method is applicable to PSA detection in serum samples and for visual classification by cancer patients and healthy persons. It is also suitable for color-blind and color-weak individuals. Conceivably, this kind of assay can be transferred to the determination of various kinds of other bioanalytes including nucleotide, proteins, and even of ions and small organic molecules, and thus is has a wide scope. Graphical abstract Schematic presentation of a pH-responsive superwettable chip coated with silica nanoparticles for the visual detection of prostate specific antigen (PSA) by reading the contact angle. The superwettable chip achieves reliable clinical detection of serum PSA from prostate cancer patients.

Proposal of open field test as a model of varenicline pharmacokinetic study in rats

Varenicline is a medication used for smoking treatment that acts as a partial agonist for nicotinic cholinergic receptors α4β2 and α3β4 and as a total agonist of receptor α7 in the central nervous system. Pharmacokinetic is important information for medications that acts in the central nervous system. This kind of assay is commonly done by expensive and complex analytical techniques. Therefore, the aim of this study was to evaluate the possibility of using the open field test as a pharmacokinetic model for varenicline in male rats exposed to a single dose of varenicline. Male rats received a single dose orally (gavage) of three different concentrations of varenicline: 0.03 (therapeutic dose for humans), 0.1 and 0.3 mg/kg or water (control group). The open field observations were recorded 30 min, 1, 2, 4, 6, 24, 48, 72 h and 7 days after the administration of varenicline or water. The results showed alterations in locomotion and rearing frequencies, as well as in immobility time observed in open field, which is consistent with this drug’s plasma peak. Consequently, this behavioral test apparently can be considerate as a model for pharmacokinetic evaluation of varenicline.

Selective capture and sensitive fluorometric determination of Pseudomonas aeruginosa by using aptamer modified magnetic nanoparticles.

A fluorometric assay is described for the detection of the food pathogen Pseudomonas aeruginosa (P. aeruginosa). It is based on the hybridization of aptamer and fluorescein-labeled complementary DNA (FAM-cDNA) in combination with magnetic separation. In the absence of P. aeruginosa, FAM-cDNA is assembled on the surface of aptamer modified magnetic particles (MNPs) via hybridization between aptamer and cDNA. Upon addition of P. aeruginosa, FAM-cDNA is replaced by the bacteria and released from the MNPs since the aptamer preferentially binds to bacteria. After magnetic separation, the amount of bacteria can be quantified by determination of the fluorescence intensity (λexc/em = 494/525nm) of the supernatant containing the released FAM-cDNA. This kind of assay allows for both selective enrichment and sensitive fluorometric determination of bacteria in a single step. The assay has a response to the logarithm of P. aeruginosa concentration that is linearin the range between 10 and 108cfu·mL-1, with a detection limit as low as 1cfu·mL-1. The detection process can be finished within <1.5h. The feasibility of the assay was verified by detecting P. aeruginosa in spiked food samples. Graphical abstract Hybridization of aptamer and carboxyfluorescein labeled complementary DNA is combined with magnetic separation for detection of as low as 1cfu·mL-1 Pseudomonas aeruginosa. This kind of assay allows for both selective enrichment and sensitive fluorometric determination of bacteria in a single step.

Electronic Detection of DNA Hybridization by Coupling Organic Field-Effect Transistor-Based Sensors and Hairpin-Shaped Probes.

In this paper, the electronic transduction of DNA hybridization is presented by coupling organic charge-modulated field-effect transistors (OCMFETs) and hairpin-shaped probes. These probes have shown interesting properties in terms of sensitivity and selectivity in other kinds of assays, in the form of molecular beacons (MBs). Their integration with organic-transistor based sensors, never explored before, paves the way to a new class of low-cost, easy-to-use, and portable genetic sensors with enhanced performances. Thanks to the peculiar characteristics of the employed sensor, measurements can be performed at relatively high ionic strengths, thus optimizing the probes’ functionality without affecting the detection ability of the device. A complete electrical characterization of the sensor is reported, including calibration with different target concentrations in the measurement environment and selectivity evaluation. In particular, DNA hybridization detection for target concentration as low as 100 pM is demonstrated.

Identification of the membrane-spanning domain of glycoprotein 45 in bovine immunodeficiency virus.

The membrane-spanning domain (MSD) of the transmembrane subunit (TM) anchors the envelope glycoprotein (Env) on the lipid bilayer of the host cell membrane and virions. Its functions include membrane fusion efficiency and intracellular trafficking of the lentivirus envelope protein. Our study aimed to determine the MSD of bovine immunodeficiency virus (BIV) glycoprotein 45 (gp45) and reveal structural characteristics of the BIV Env protein. We have predicted the region of the BIV MSD and obtained the sequence using bioinformatics software. Various kinds of assays, including analogy analysis, fluorescence microscopy, and dye-transfer-based assays, were carried out to validate the prediction. The results, for the first time, show that the BIV MSD is located at the D170 to M191 amino acids of gp45, and the identified MSD divides gp45 into the extracellular domain (ED), MSD and cytoplasmic domain (CT). We further found that the BIV MSD had a similar structure and function as the HIV MSD using amino acid sequence alignment and fluorescence microscopy. Additionally, the dye-transfer-based assay demonstrates that deletion of the BIV MSD efficiently decreases cell-cell fusion. Based on the identification of the MSD, a "snorkeling" model, in which the flanking charged amino acid residues are buried in the lipid bilayer while their side chains interact with polar head groups, was proposed for the BIV MSD. Ultimately, we further improved the primary structure of the BIV envelope glycoprotein.

A new kind of highly sensitive competitive lateral flow immunoassay displaying direct analyte-signal dependence. Application to the determination of the mycotoxin deoxynivalenol.

A new kind of competitive immunochromatographic assay is presented. It is based on the use of a test strip loaded with (a) labeled specific antibodies, (b) a hapten-protein conjugate at the control zone, and (c) antibodies interacting with the specific antibodies in the analytical zone. In the case where a sample does not contain the target antigen (hapten), all labeled antibodies remain in the control zone because of the selected ratio of reactants. The analytical zone remains colorless because the labeled antibodies do not reach it. If an antigen is present in the sample, it interferes with the binding of the specific antibodies in the control zone and knocks them out. Some of these antibodies pass the control zone to form a colored line in the analytical zone. The intensity of the color is directly proportional to the amount of the target antigen in the sample. The assay has an attractive feature in that an appearance in coloration is more easily detected visually than a decoloration. Moreover, the onset of coloration is detectable at a lower concentration than a decoloration. The new detection scheme was applied to the determination of the mycotoxin deoxynivalenol. The visual limit of detection is 2ng·mL-1 in corn extracts (35ng per gram of sample). With the same reagents, this is lower by a factor of 60 than the established test strip. The assay takes only 15min. This new kind of assay has wide potential applications for numerous low molecular weight analytes. Graphical abstract Competitive immunochromatography with direct analyte-signal dependence is proposed. It provides a 60-fold decrease of the detection limit for mycotoxin deoxynivalenol. The analyte-antibody-label complexes move along the immobilized antigen (control zone) and bind with anti-species antibodies (test zone).

DNA aptamer selection and aptamer-based fluorometric displacement assay for the hepatotoxin microcystin-RR

Microcystin-RR (MC-RR) is a highly acute hepatotoxin produced by cyanobacteria. It is harmful to both humans and the environment. A novel aptamer was identified by the systemic evolution of ligands by exponential enrichment (SELEX) method as a recognition element for determination of MC-RR in aquatic products. The graphene oxide (GO) SELEX strategy was adopted to generate aptamers with high affinity and specificity. Of the 50 aptamer candidates tested, sequence RR-33 was found to display high affinity and selectivity, with a dissociation constant of 45.7 ± 6.8 nM. Aptamer RR-33 therefore was used as the recognition element in a fluorometric assay that proceeds as follows: (1) Biotinylated aptamer RR-33 is immobilized on the streptavidinylated wells of a microtiterplate, and carboxyfluorescein (FAM) labelled complementary DNA is then allowed to hybridize. (2) After removal of excess (unbound) cDNA, sample containing MC-RR is added and incubated at 37 °C for 2 h. (3) Displaced free cDNA is washed away and fluorescence intensity measured at excitation/emission wavelengths of 490/515 nm. The calibration plot is linear in the 0.20 to 2.5 ng·mL−1 concentration range, and the limit of detection is 80 pg·mL−1. The results indicate that the GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins. The detection scheme was applied to the determination of MC-RR in (spiked) water, mussel and fish and gave recoveries between 91 and 98 %. The method compares favorably to a known ELISA. Conceivably, this kind of assay is applicable to other toxins for which appropriate aptamers are available.

Determination of clinically significant tests for antiphospholipid antibodies and cutoff levels for obstetric antiphospholipid syndrome.

The objective of this paper is to determine which kinds of assays for antiphospholipid antibodies (aPL) should be tested for clinical practice for patients with recurrent pregnancy loss (RPL). We studied 560 patients with a history of RPL prospectively. We determined the obstetric significance of 11 commercially available tested assays for lupus anticoagulant (LA)-aPTT StaClot, phosphatidylserine-dependent antiprothrombin (aPS/PT) IgG, IgM, classical cardiolipin (CL) IgG, IgM, CL IgG, IgM, IgA, and β2glycoprotein I (β2GPI) IgG, IgM, IgA Phadia. Obstetric significance was defined as the potential for anticoagulant therapy to improve the subsequent live birth rate, or a difference in the live birth rate between positive and negative untreated cases. The LA-aPTT StaClot assay and aPS/PT IgG assay, but not CL IgG, were found to have obstetric significance. Our conventional tests covered positive cases with the aPS/PT IgM and classical CL IgG assays. The results of the LA-aPTT StaClot, LA-aPTT and LA-RVVT assays showed different distributions, although strong or moderate correlation was observed. LA-aPTT StaClot and aPS/PT IgG might be suitable for use in routine practice for patients with RPL. Each test for aPL should be ascertained for obstetric significance, because similar assays may have different outcomes.

Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes.

BackgroundSLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na+/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein.ResultsWe detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK293 cells. But this chimera also did not show any transport activity, even when the N-terminal domain of SLC10A4 was deleted by mutagenesis.ConclusionsAlthough different kinds of assays were used to screen for transport function, SLC10A4 failed to show transport activity for a series of neurotransmitters and neuromodulators, indicating that SLC10A4 does not seem to represent a typical neurotransmitter transporter such as DAT, SERT, CHT1 or VMAT2.

Electrochemiluminescence immunosensor for ketamine detection based on polyamidoamine-coated carbon dot film

In this work, we reported the synthesis of water-soluble carbon dots (CDs) by a one-step hydrothermal synthetic route using polyamidoamine (PAMAM) as platform and passivant, where the formation of CDs capped with PAMAM and the surface passivation were realized simultaneously. As a result, the prepared PAMAM-CDs were uniform, monodispersed sphere particles with an approximate diameter of about 2 nm, and the fluorescence and electrochemiluminescence (ECL) response of the nanocomposite film were enhanced. By combining the high sensitivity of ECL and the selectivity of immunoreactions, an ECL immunosensor for facile, rapid, and ultrasensitive detecting of ketamine was constructed, and the further application of the immunoassay was investigated. Under optimal experimental conditions, the obtained immunosensor showed a wide linear detection range from 0.2 to 200 ng mL−1 with a detection limit at 0.067 ng mL−1 in 0.1 M phosphate buffer solutions (pH 7.4). The immunosensor was successfully applied to the determination of ketamine in blood plasma. This kind of assay is expected to pave new avenues in label-free drug assays.

Real-Time Quantitative Polymerase Chain Reaction for Apolipoprotein E genotyping

Background : Apolipoprotein E (ApoE) is an important substance responsible for the lipoprotein metabolism and thetransportation of lipid and known to play an important role in the body's physiological and pathological processes. Recently,ApoE genotyping test has been applied to the field of diagnostic methods for predicting high risk of Alzheimer's disease,cardiovascular diseases, as well as new diseases such as immune regulation disorders. Therefore, a faster and more accurateApoE genotyping test is required for the prevention of related disease and the general public health.Methods : We evaluated 220 serum specimens using two kinds of assays: one-stop Real-Q ApoE genotyping (BioSewoom,Seoul, Korea) and Seeplex ApoE ACE genotyping (Seegene, Seoul, Korea). The results were finally confirmed using sequencingas the reference. Statistical analysis was performed for the concordance rates between each assay and sequencing and thesensitivity and specificity were calculated with their 95% confidence intervals.Results : Sequencing results of 220 DNA samples showed 1 case of E2/E2, 139 of E3/E3, 9 of E4/E4, 17 of E2/E3, 4 ofE2/E4, 50 of E3/E4. The concordance rate between One-step Real-Q ApoE genotyping and sequencing was 100% (220/220),and that between Seeplex ApoE ACE genotyping and sequencing was 99.5% (219/220). Both sensitivity and specificity of OnestepReal-Q ApoE genotyping for every genotypes was 100%. The sensitivities and specificities of Seeplex ApoE ACEgenotyping were almost 100%, with two exception: 99.3% sensitivity in E3/E3 and 99.5% specificity in E2/E2.Conclusions : The performances of One-step Real-Q ApoE genotyping and Seeplex ApoE ACE genotyping were comparableoverall. However, the former showed better sensitivity and specificity comparable with those of sequencing. Hence, One-stepApoE genotyping could be a faster and more accurate option for ApoE genotyping.

223. Consideration for Quality Control of NKG Cell Line Used for Adoptive Cellular Therapy

NK cells play the important role in the adoptive cell therapy. As we know, NK-92 cell, a NK cell line, has been used for the preparation of cell therapy product for clinical trials in some laboratory and had been showed the potent effect in cancer therapy. Except NK-92, another NK cell line named NKG was established by Dr Tian Zhigang in University of Science and Technology of China. NKG cell line was derived from a male patient with non-Hodgkin's lymphoma. It was isolated from patient peripheral blood samples and cultivated through long-lasting period in the medium containing horse and fatal bovine serum and IL-2. Finally it was gradually adopted to grow in serum-free medium suitable for clinical use. The surface market of NKG showed CD56brightCD16- phenotype by FACS and showed the cytotoxicity to several kinds of caner cells by in vitro and in vivo assays. Then it will be potential for applying for clinical trial in the near future. So, it is the first step to qualify the master cell bank of NKG thoroughly before it is into clinical trial.The strategy for qualification of NKG is based on the principle of risk assessment. The parameters which should be considered include the original donor eligibility, culture history and raw materials used during its isolation and cultivation, cell banking, cell characters and its intended use. So the qualification scheme for NKG includes the tests for identity and purity, the adventitious agents, the cell character and its potency.Several methods is used for test of identity and purity of NKG. Except the morphology, isoenzyme analysis is used to confirm its human origin. The STR profile analysis is used to identify it is derived from one human body and has no cross-contamination with other human cell lines. A group of cell surface makers is selected and detected by FACS. These makers will show several feactures of NKG including its NKbright celltype, its high purity with no other immune cell contamination, its low adhesion activity for serum-free adaption and its cytotoxic potency.For adventitious agents test, NKG is tested to confirm there is no presence of bacterial, mycoplasma and viruses contamination. Except for the traditional assays, the viruses is extend detected by induction assay by chemicals. The deep sequencing is also used for virus detection of NKG.Other characters of NKG is also analyzed for qualification, including the growth curve, the cytogenetic analysis, tumorigenicity and oncogenticity using NKG cell protein and DNA.In order to qualify the NKG, three kinds of assays are used for its potency test. The cell surface makers of NKG2D and NKp30 which is related with cytotoxiciey is detected by FACS. Then the cytokine secretion is detected by ELISA for IFN-gand by FACS for Granzyme B and perforin. Finally the cytotoxicity is analyzed by cell-based assay.In conclusion, thorough qualification of NKG cell lines established in China will be important and helpful to support its applying for clinical trial in the future.

SP0127 Basic Concepts: Analysis of Anti-Drug Antibodies and Serum Concentrations of Biologicals

BackgroundTreatment with biological drugs in the last fifteen years has changed the therapy of most inflammatory diseases, mainly in the field of rheumatology. The use of these drugs has supposed...

LEGO Plot for Simultaneous Application of Multiple Quality Requirements During Trueness Verification of Quantitative Laboratory Tests

We developed a two-dimensional plot for viewing trueness that takes into account potential shift and variable quality requirements to verify trueness using certified reference material (CRM). Glucose, total cholesterol (TC), and creatinine levels were determined by two kinds of assay in two levels of a CRM. Available quality requirements were collected, codified, and sorted in an ascending order in the plot's header row. Centering on the mean of measured values from CRM, the "mean ± US CLIA '88 allowable total error" was located in the header of the leftmost and rightmost columns. Twenty points were created in intervening columns as potential shifts. Uncertainties were calculated according to regression between certified values and uncertainties of CRM, and positioned in the corresponding columns. Cells were assigned different colors where column and row intersected based on comparison of the 95% confidence interval of the percentage bias with each quality requirement. A glucose assay failed to meet the highest quality criteria, for which shift of +0.13-0.14 mmol/l was required. A TC assay met the quality requirement and a shift of ±0.03 mmol/l was tolerable. A creatinine assay also met the quality requirement but any shift was not tolerable. The plot provides a systematic view of the trueness of quantitative laboratory tests.

Wound healing assay in a low-cost microfluidic platform

Cell migration is a very important stage in different pathological processes such as wound healing, vascularisation, immune response, tissue regeneration and cancer. Traditional wound healing assays are done by scratching a monolayer on standard culture surfaces. However this method can lead to data misinterpretation and poor comparability in results from different experiments. The microfluidic wound healing assay is a mature alternative to the traditional method, but it is expensive and bulky due to the need of syringe pumps and can be difficult to implement by the end user. Here we show an alternative low-cost microfluidic platform based on the MainSTREAM platform. We also demonstrate the feasibility of a microperistaltic pump to carry out these kind of assays. A laminar flow of PBS-Trypsin-PBS confined in a microfluidic channel with a previously cultured monolayer of IIB-Mel LES melanoma cells was used to generate the wound. Cell migration rate could be quantified successfully.