DOCSLIB.ORG

Comparison of Immunohistochemistry Assay Results with Gene

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Comparison of Immunohistochemistry Assay Results with Gene r Biomar ula ke c rs Schaffer et al., J Mol Biomark Diagn 2018, 9:2 le o & M D f i DOI: 10.4172/2155-9929.1000386 a o g l Journal of Molecular Biomarkers n a o n r s i u s o J ISSN: 2155-9929 & Diagnosis Research Article Open Access Comparison of Immunohistochemistry Assay Results with Gene Expression Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification in Matched Patient Samples Michael Schaffer1, Shalini Chaturvedi1, JD Alvarez1, Sandy Frans2, Regina Aquino1, Brett Hall1, Mark Wildgust3 and Sriram Balasubramanian1* 1Janssen Oncology Translational Research, Spring House, PA, USA 2Janssen Research and Development, Beerse, Belgium 3Janssen Research and Development, Raritan, NJ, USA Abstract Background: The cell of origin (COO) in diffuse large B-cell lymphoma (DLBCL) has prognostic importance. While the COO was originally classified into germinal center B-cell (GCB) or activated B-cell (ABC) subtypes by microarray analysis, routine use was not practical. Immunohistochemistry (IHC)–based methods are widely used with varying results due to lack of standardization. Several classification methods have been developed recently; understanding the concordance between these and existing methods is essential to their practical application. Therefore, we evaluated concordance between 3 commercial assays: a standardized Hans-based IHC method and 2 gene expression profiling (GEP) methods and compared these to the accepted microarray classification. Methods: 137 DLBCL-confirmed tumor samples were evaluated using a standardized Hans-based IHC method for GCB or non-GCB subtype, by a published microarray-based assay, a digital gene expression-based Lymphoma Subtyping Test (LST) and a next-generation sequencing-based assay (EdgeSeq COO). Subtype calls from the 3 GEP methods were harmonized to “GCB” or “non-GCB” and assessed for concordance. Results: Concordance between the Hans-based IHC assay and the microarray-based assay, LST assay and EdgeSeq assay was 79.6% (N=137), 80.0% (N=125) and 78.2% (N=64), respectively; the positive percent agreement (PPA) in non-GCB was 88.9%, 87.0% and 78.1%, respectively. Concordance for the Hans-based IHC assay versus GEP methods was especially high for direct GCB calls (91.0%, 88.3% and 78.1% for microarray, LST and EdgeSeq COO methods, respectively). The newly developed GEP assays performed well against the microarray GEP method, against which they were calibrated (concordance 93.7% and 87.5% and PPA 94.3% and 92.9%, respectively, for LST and EdgeSeq COO). Conclusion: These results demonstrated good consistency between various platforms for stratification of DLBCL into COO subtype classifications. Application of a standardized Hans-based IHC assay offers a robust, rapid and easily accessible platform to classify DLBCL into prognostically important subtypes. Keywords: Diffuse large B-cell lymphoma; Hans-based have shown favorable responses to the Bruton’s tyrosine kinase (BTK) immunohistochemistry; Gene expression profiling inhibitor ibrutinib [11] or the immunomodulatory drug lenalidomide [12], alone or in combination with chemotherapy. This provides a Introduction rationale for the selective development of treatments for subtypes of Diffuse large B-cell lymphoma (DLBCL) is the most common DLBCL classified by COO, which further emphasizes the need for rapid type of lymphoma, accounting for 30% to 35% of non-Hodgkin and accurate identification of these subtypes. lymphoma [1,2]. DLBCL is a heterogeneous disease and has distinct Most patients with PMBL have a favorable outcome with a 3-year molecular subtypes based on the cell of origin (COO). Methods such overall survival rate of 97% [2]. In clinical practice today, PMBL is as immunohistochemistry (IHC) and gene expression profiling (GEP) easily diagnosed by other methods; thus, DLBCL COO classification are among the more common approaches used to classify subtypes efforts have focused on distinguishing the remaining subtypes. of DLBCL. GEP using DNA microarrays has been the standard The accepted standard for classification of DLBCL subtypes has experimental approach to molecularly classify DLBCL into major COO subtypes: germinal center B-cell–like (GCB), activated B-cell–like (ABC) and primary mediastinal B-cell lymphoma (PMBL) [3-6]. The small number of patients who cannot be classified clearly into these *Corresponding author: Sriram Balasubramanian, Janssen Oncology Translational Research, 1400 McKean Road, Spring House, PA 19087, USA, Tel: molecular subtypes are referred to as “unclassified” (UNC). +1 2157937757; E-mail: [email protected] Depending on the molecular subtype, the pathobiology of the Received March 03, 2018; Accepted March 21, 2018; Published March 24, 2018 disease is diverse; other than PBML, the initial clinical presentation Citation: Schaffer M, Chaturvedi S, Alvarez JD, Frans S, Aquino R, et al. (2018) of each subtype is similar but associated with very different clinical Comparison of Immunohistochemistry Assay Results with Gene Expression outcomes and large underlying molecular diversity. Of these biological Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification factors, the COO has proved to be the most important, and it is now in Matched Patient Samples. J Mol Biomark Diagn 9: 386. doi: 10.4172/2155- accepted that COO is a prognostic factor in DLBCL [7-9]. 9929.1000386 DLBCL subtypes respond to therapy in very different ways [10]. For Copyright: © 2018 Schaffer M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits example, patients with ABC DLBCL who failed R-CHOP (rituximab, unrestricted use, distribution, and reproduction in any medium, provided the cyclophosphamide, doxorubicin, vincristine and prednisone) therapy original author and source are credited. J Mol Biomark Diagn, an open access journal Volume 9 • Issue 2 • 1000386 ISSN:2155-9929 Citation: Schaffer M, Chaturvedi S, Alvarez JD, Frans S, Aquino R, et al. (2018) Comparison of Immunohistochemistry Assay Results with Gene Expression Profiling Methods for Diffuse Large B-Cell Lymphoma Subtype Identification in Matched Patient Samples. J Mol Biomark Diagn 9: 386. doi: 10.4172/2155-9929.1000386 Page 2 of 8 been using microarray GEP [13]. However, a reproducible, easy-to-use samples and allows more subtle evaluation of the sample and staining and validated microarray-based GEP assay for DLBCL subtyping has characteristics by a pathologist. not been accessible to all medical communities and any microarray Each methodology described has its own limitations; however, GEP-based approach generally has a turnaround time of 2 to 3 weeks, optimized assays using some of these methodologies are becoming which makes it difficult to implement as a prospective selection marker. commercially available, and thus multiple assays may be used to identify Therefore, routine clinical practice in DLBCL has used standard IHC- DLBCL patients and select the optimum therapy in the clinic. Therefore, based methods, which are widely available in most clinical settings, it becomes important to compare multiple platforms and evaluate to classify patients for therapy. Much effort has been dedicated to the concordance between these IHC and GEP assays, as well as the developing robust IHC assays using various combinations of CD10, concordance across the different GEP assays. To this end, we compared BCL6, BCL2, MUM1/IRF4, FOXP1, GCET1 and LMO2 proteins, all the Hans-based IHC assay with the traditional microarray GEP-based of which have been identified as having differential expression in the method and the novel gene expression-based assays described above DLBCL subtypes. These markers have been used to develop at least 9 under standard conditions, using the same set of clinical specimens. different IHC tests [14]. The Hans method was developed in 2004, based on 3 markers: CD10, BCL6 and MUM1/IRF4 [15], and has remained Materials and Methods the most popular due to its simplicity with only 3 widely used markers, Tissue samples a consistent 30% cutoff for all 3 markers and demonstrated prognostic value. This binary algorithm segregates patients into 2 groups – GCB All clinical samples used in this study were from FFPE blocks (in 42% of patients) and non-GCB (in 58% of patients) – instead of the purchased from Molecular Response (San Diego, CA, USA) or continuum from which discrete classes are derived from population- Conversant Bio (Huntsville, AL, USA), and obtained with informed based numeric cutoffs [15]. The non-GCB group incorporates the ABC consent. All samples were reviewed by a certified pathologist to subtype along with the unclassified group, and as with ABC DLBCL, confirm the disease diagnosis and verify tumor content before IHC or these non-GCB patients have a less favorable outcome with standard GEP analysis. A total of 194 samples were first evaluated for disease immunochemotherapy [16]. Algorithms for IHC have 80% to 87% diagnosis confirmation and then tested. concordance with GEP and have gained the most clinical acceptance Sample preparation, RNA extraction and GEP [17-19]. However, there can be limitations to using IHC algorithms, including reproducibility concerns and variability between laboratories; 6 sections of 10 μm each were cut from the tissue block per sample also, the various IHC methods have not been uniformly reported to have and de-paraffinized using xylene as described in the AllPrep DNA/ the same prognostic utility as GEP [20,21]. A standardized Hans-based
Load more

Recommended publications
  • Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development
    Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development Gerlie Gieser, Ph.D. Office of Clinical Pharmacology, Div. IV Objectives • Outline the Phase 1 studies conducted to characterize the Clinical Pharmacology of a drug; describe important design elements of and the information gained from these studies. • List the Clinical Pharmacology characteristics of an Ideal Drug • Describe how the Clinical Pharmacology information from Phase 1 can help design Phase 2/3 trials • Discuss the timing of Clinical Pharmacology studies during drug development, and provide examples of how the information generated could impact the overall clinical development plan and product labeling. Phase 1 of Drug Development CLINICAL DEVELOPMENT RESEARCH PRE POST AND CLINICAL APPROVAL 1 DISCOVERY DEVELOPMENT 2 3 PHASE e e e s s s a a a h h h P P P Clinical Pharmacology Studies Initial IND (first in human) NDA/BLA SUBMISSION Phase 1 – studies designed mainly to investigate the safety/tolerability (if possible, identify MTD), pharmacokinetics and pharmacodynamics of an investigational drug in humans Clinical Pharmacology • Study of the Pharmacokinetics (PK) and Pharmacodynamics (PD) of the drug in humans – PK: what the body does to the drug (Absorption, Distribution, Metabolism, Excretion) – PD: what the drug does to the body • PK and PD profiles of the drug are influenced by physicochemical properties of the drug, product/formulation, administration route, patient’s intrinsic and extrinsic factors (e.g., organ dysfunction, diseases, concomitant medications,
    [Show full text]
  • Challenges Associated with the Development and Validation of Flow Cytometry Assays
    Challenges associated with the development and validation of flow cytometry assays Carolyne Dumont, Eliane Moisan, Martin Poirier, Marie-Hélène Côté, and Marie-Soleil Piché 1 INTRODUCTION 2 Case Study 1 3 Case Study 2 Development of a PD marker by flow cytometry to assess the efficacy of Development of a flow cytometry assay for the measurement of basophil Flow cytometry is a technology allowing multi-parametric analysis of thousands of particles per second and helps to a chemokine neutralizing antibody activation in the context of a Phase III clinical study adequately identify or functionally characterize complex cell populations of interest. It is often used in basic research, Assay Design: The assay was required for the evaluation of pharmacodynamics (inhibition an agonist’s granulocytes activation activity) in Assay Design: Human whole blood samples were spiked with the different controls (or compounds in the clinical study) and further discovery, preclinical and clinical trials. With the increasing proportion of biologics in the pipeline, flow cytometry has proven preclinical studies. Non-Human Primate (NHP) or rat blood from treated animals was incubated at 3 conditions and granulocyte activation stained with an anti-CCR3 and anti-CD63 antibody. The validations stimulation conditions tested included a negative control (PBS) and itself to be an indispensable tool to assess safety, receptor occupancy (RO) or pharmacodynamics (PD). was measured as CD11b expression by flow cytometry (MFI). The conditions tested included a negative control (PBS), a positive control two positive controls (anti-FcεRI and fMLP). Basophils were identified as CCR3+ and upon activation, CD63 became externalized and (fMLP) and the test condition (agonist).
    [Show full text]
  • Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes
    Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes Paula Denney Eason, Renée C. Benson, Jenny T. Ly, Rachel A. Saxer, James L. Wilbur, George B. Sigal, Eli N. Glezer, Hans A. Biebuyck, Jacob N. Wohlstadter, and Robert M. Umek TM TM A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes Abstract This poster presents a robust, receptor-ligand binding assay based upon a novel assay platform developed by Meso Scale DiscoveryTM (MSDTM). MSD’s platform combines array technologies and electrochemiluminescence detection to achieve ultra-fast, highly sensitive assays in a homogeneous format. Cellular membranes containing the EGF receptor were passively adsorbed to MSD proprietary coated electrodes embedded in multi-well plates. Binding of EGF to the EGF receptor was detected by inducing and measuring electrochemi- luminescence from a labeled EGF ligand. Approximately 1000 cell equivalents per well yielded a signal to background ratio of 20. The observed KD agrees with that reported in the literature and demonstrates that immobilization of the membranes and modification of the ligand do not alter the binding affinity. Binding specificity was confirmed with two inhibitors. The assay can be readily adapted to facilitate analysis of a broad array of receptor-ligand interactions. TM TM A division of Meso Scale Diagnostics,TM LLC. Simple, Rapid Ligand-Binding Assay Using Immobilized Cellular Membranes TM Multi-Array TechnologyTM Multi-Array Technology Unified technology platform with instruments, plates and reagents for drug discovery for drug discovery. Combines the power of microarrays with the sensitivity of electrochemiluminescence Combines the power of microarrays with the sensitivity of electrochemiluminescence.96-, 384- and 1536 microplate formats 96-,Multi-Spot 384- TMand plates 1536 with microplate high density formats.
    [Show full text]
  • UBI® SARS-Cov-2 ELISA INSTRUCTIONS for USE
    UBI® SARS-CoV-2 ELISA INSTRUCTIONS FOR USE FOR IN VITRO DIAGNOSTIC USE ONLY FOR EMERGENCY USE AUTHORIZATION ONLY FOR PRESCRIPTION USE ONLY INTENDED USE The UBI® SARS-CoV-2 ELISA is an Enzyme-Linked Immunosorbent Assay (ELISA) intended for qualitative detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (sodium heparin or dipotassium (K2) EDTA). The UBI® SARS-CoV-2 ELISA is intended for use as an aid in identifying individuals with an adaptive immune response to SARS-CoV-2, indicating recent or prior infection. At this time, it is unknown for how long antibodies persist following infection and if the presence of antibodies confers protective immunity. The UBI® SARS-CoV-2 ELISA should not be used to diagnose or exclude acute SARS-CoV-2 infection. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C 263a, that meet requirements to perform high complexity testing. Results are for the detection of IgG SARS CoV-2 antibodies. IgG antibodies to SARS-CoV-2 are generally detectable in blood several days after initial infection, although the duration of time antibodies are present post-infection is not well characterized. Individuals may have detectable virus present for several weeks following seroconversion. Laboratories within the United States and its territories are required to report all results to the appropriate public health authorities. The sensitivity of the UBI® SARS-CoV-2 ELISA early after infection is unknown. Negative results do not preclude acute SARS-CoV-2 infection. If acute infection is suspected, direct testing for SARS-CoV-2 is necessary.
    [Show full text]
  • Molecular Diagnostic Assay Validation Update to the 2009 AMP Molecular Diagnostic Assay Validation White Paper
    Molecular Diagnostic Assay Validation Update to the 2009 AMP Molecular Diagnostic Assay Validation White Paper Members of the 2013 and 2014 Clinical Practice Committees of the Association for Molecular Pathology The 2013 AMP Clinical Practice Committee consisted of Matthew J. Bankowski, Milena Cankovic, Jennifer Dunlap, Larissa V. Furtado*, Jerald Gong, Thomas Huard, Linda Jeng*, Loren Joseph (Chair), Annette Kim, Bryan Krock, Marilyn M. Li, Mary Lowery-Nordberg, Melissa Miller, Caroline Sue Richards, and Paul Rothberg. *Project leads Standard of practice is not defined by this article, and there may be alternatives. See Disclaimer for further details. INTRODUCTION The goal of method validation in the molecular diagnostics laboratory is to ensure that a given test is ready for implementation in the clinical laboratory. To reach that goal, each step of the testing process must be carefully evaluated and documented. Such validation is relatively standardized for high volume automated assays in fields such as clinical chemistry. It is challenging to apply those standardized practices to molecular diagnostics laboratories, which rely heavily on low-volume labor-intensive tests, both FDA approved and laboratory- developed. Excellent and comprehensive documents have recently been published on this topic 1-10. This overview is intended to provide a brief practical reference that can be used to guide the validation process in molecular diagnostics laboratories. In addition, a sample summary checklist is provided in the appendix as a template to facilitate documentation of laboratory director review and sign off on the validation process when new assays are developed or adopted. SUMMARY CLIA (42 CFR 493.1253) and CAP (MOL 30785-31705) require that laboratories validate the performance of tests before reporting patient results.
    [Show full text]
  • Assay Qualification Recall…
    Assay Qualification Recall… Assay Qualification: • ACCURACY: Orthogonal method • PRECISION: Reproducibility: same day replicates, day to day, different operators • ROBUSTNESS: sensitivity to assay parameters • SPECIFICITY: sensitivity to matrix effects • DYNAMIC RANGE AND RESPONSE FUNCTION: Instrument benchmarking. +/- controls. Calibration curve. Limit of detection Goal Establish confidence in genomic assays used to identify on-target and off- target genome editing genomic locations and sequence variants. Simplified Genome Editing Process Design & Execute Source sample Screen for genome disruption Target Editing molecule Targeted Confirm sequence change Editing molecule sequence (e.g. T7 Endonuclease, Surveyor, HRMA, TIDE) Targeted (sequencing, qPCR) Editing molecule formulation Genome Wide Whole exome sequencing Delivery (e.g. GUIDE-Seq, Digenome-Seq, Whole genome sequencing CIRCLE-Seq, SITE-Seq, BLESS/BLISS) Assay Reference qualification Materials Activities and outputs: 1. Qualify genomic assays used to identify on-target and off-target genome editing genomic locations and sequence variants. 2. Develop & qualify reference materials – designed to mimic genome edited DNA complexity for use in benchmarking sequence verification assay limits of detection and sensitivity. Assay Qualification Process Assess assay precision (initially within NIST laboratories) Assess sources of variability and improve assay protocols and robustness (DoE) (NIST) Identify assay(s) of primary importance (community consensus) Refined assay protocols will be evaluated
    [Show full text]
  • A Polyacrylamide Gel Electrophoresis (PAGE) Method for Assay of Phytic Acid Species
    INTERNATIONAL ZINC NUTRITION CONSULTATIVE GROUP WWW.IZiNCG.ORG IZiNCG practical tips A Polyacrylamide Gel Electrophoresis (PAGE) Method for Assay of Phytic Acid Species The PAGE method illustrated here was first was described in Losito et al. (2009). The basic principle of chromatography is that different compounds will migrate at different rates down the gel and thus be separated. A video demonstration can be found at http://www.jove.com/details.php?id=3027, as described in Loss et al. (2011). As described in the video, this method was initially developed for the isolation and identification of compounds related to phytic acid called “inositol pyrophosphates”. These compounds have seven or eight phosphate esters per molecule, as compared with phytic acid (inositol hexaphosphate or IP6), which has six phosphate esters per molecule. PAGE is typically used to chromatographically separate and detect molecules of much higher molecular weight than inositol phosphates, such as proteins and nucleic acid fragments. To provide a gel of sufficient density to retain these lower molecular weight inositol phosphates, this method uses a much higher concentration of 33.3% acrylamide. 1. Required Reagents The following chemicals are widely available from many commercial sources. 1. 40% Acrylamide/Bisacrylamide (19:1) 2. Ammonium Persulfate (APS) 3. Tetramethylethylenediamine (TEMED) 4. Tris(hydroxymethyl)aminomethane (TRIS) Base 5. Boric Acid 6. Ethylenediaminetetraacetic acid (EDTA) 7. Toluidine Blue 8. Sodium Hydroxide 9. Orange G 2. Experimental Standards Standards are readily available from many commercial sources. 1. 1.0 mM IP6 prepared using phytic acid dodecasodium salt hydrate prepared from rice (Sigma, St. Louis MO, USA).
    [Show full text]
  • Sample and Assay Technologies
    QIAGEN – Sample and Assay Technologies Cheuvreux 7th German Corporate Conference Frankfurt, January 21, 2008 Dr. Joachim Schorr Senior Vice President Global Research & Development -1- Sample & Assay Technologies Forward Looking Statements Safe Harbor Statement: Certain of the statements contained in this presentation may be considered forward-looking statements within the meaning of Section 27A of the U.S. Securities Act of 1933, as amended, and Section 21E of the U.S. Securities Exchange Act of 1934, as amended. To the extent that any of the statements contained herein relating to QIAGEN's products and markets and operating results are forward-looking, such statements are based on current expectations that involve a number of uncertainties and risks. Such uncertainties and risks include, but are not limited to, risks associated with management of growth and international operations (including the effects of currency fluctuations), variability of operating results, the commercial development of the DNA sequencing, genomics and synthetic nucleic acid-related markets, as well as the nucleic acid-based molecular diagnostics, applied testing markets and genetic vaccination and gene therapy markets, competition, rapid or unexpected changes in technologies, fluctuations in demand for QIAGEN's products (including fluctuations for certain events including funding, budgets, and others), difficulties in successfully adapting QIAGEN’s products to integrated solutions and producing such products, the ability of QIAGEN to identify and develop new products and to differentiate its products from competitors, the management of intellectual property, and the integration of acquisitions of technologies and businesses. For further information, refer to the discussion in reports that QIAGEN has filed with or furnished to the U.S.
    [Show full text]
  • Analytic Validation of Immunohistochemical Assays: a Comparison of Laboratory Practices Before and After Introduction of an Evidence-Based Guideline
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Arch Pathol Manuscript Author Lab Med. Author Manuscript Author manuscript; available in PMC 2019 December 31. Published in final edited form as: Arch Pathol Lab Med. 2017 September ; 141(9): 1247–1254. doi:10.5858/arpa.2016-0558-CP. Analytic Validation of Immunohistochemical Assays: A Comparison of Laboratory Practices Before and After Introduction of an Evidence-Based Guideline Patrick L. Fitzgibbons, MD, Jeffrey D. Goldsmith, MD, Rhona J. Souers, MS, Lisa A. Fatheree, BS, SCT(ASCP), Keith E. Volmar, MD, Lauren N. Stuart, MD, MBA, Jan A. Nowak, MD, PhD, J. Rex Astles, PhD, Raouf E. Nakhleh, MD Department of Pathology, St Jude Medical Center, Fullerton, California (Dr Fitzgibbons); the Department of Pathology, Boston Children’s Hospital, Boston, Massachusetts (Dr Goldsmith); the Biostatistics Department (Ms Souers) and the Pathology and Laboratory Quality Center (Ms Fatheree), College of American Pathologists, Northfield, Illinois; the Department of Pathology, UNC REX Healthcare, Raleigh, North Carolina (Dr Volmar); the Department of Pathology, Treasure Coast Pathology, East Ocean, Florida (Dr Stuart); the Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York (Dr Nowak); the Office of Laboratory Systems Development, Division of Laboratory Systems, US Centers for Disease Control & Prevention, Atlanta, Georgia (Dr Astles); and the Department of Pathology, Mayo Clinic Jacksonville, Jacksonville, Florida (Dr Nakhleh). Abstract Context.——Laboratories must demonstrate analytic validity before any test can be used clinically, but studies have shown inconsistent practices in immunohistochemical assay validation. Objective.——To assess changes in immunohistochemistry analytic validation practices after publication of an evidence-based laboratory practice guideline.
    [Show full text]
  • ELISA) for Detection of Renibacterium Salmoninarum Antigen in Fish Tissue
    CHAPTER 6 Enzyme Linked Immunosorbent Assay (ELISA) for Detection of Renibacterium salmoninarum Antigen in Fish Tissue Kimberly True USFWS – California/Nevada Fish Health Center Anderson, California NWFHS Laboratory Procedures Manual - Second Edition, June 2004 Chapter 6 - Page 1 Chapter 6 - Table of Contents I. INTRODUCTION ........................................................................................................ 6-4 A. Standardization of Reagents 1. Standardization of Antibody Reagents 2. Optimization of Antibody Dilutions B. Tissue Collection and Processing C. Performing the Assay D. Interpretation of Data E. Quality Control II. ELISA - Day 1 Preparation ........................................................................................ 6-6 A. Coating Immunoplates with Antibody B. Preparing Wash Solution and Washer Reservoirs C. Thaw and Centrifuge Test Samples III. ELISA - Day 2 Running the Assay ............................................................................ 6-9 A. Washing Plates 1. Initial Priming of Washer 2. Wash Protocol B. Loading Tissue Samples and Controls C. Washing unbound antigen D. Adding Secondary HRP-labeled Conjugate E. Washing unbound antibody F. ABTS Substrate - Color Development G. Stop Solution and Plate Reading H. Equipment Maintenance upon completion of assay IV. INTERPRETATION OF DATA ............................................................................... 6-16 A. Quality Control B. Negative-Positive Determination V. BIBLIOGRAPHY ........................................................................................................
    [Show full text]
  • CELL CULTURE BASICS Handbook
    CELL CULTURE BASICS Cell Culture Basics Culture Cell www.invitrogen.com/cellculturebasics Handbook B-087243 0110 Contents Introduction . .1 Purpose of the Handbook . 1. Introduction to Cell Culture . 2. What is Cell Culture? . 2 Finite vs Continuous Cell Line . .2 Culture Conditions . .2 Cryopreservation . .2 Morphology of Cells in Culture . 3 Applications of Cell Culture . .3 Cell Culture Laboratory . .4 Safety . 4. Biosafety Levels . 4 SDS . 5 Safety Equipment . 5 Personal Protective Equipment (PPE) . 5 Safe Laboratory Practices . .5 Cell Culture Equipment . 6. Basic Equipment . 6 Expanded Equipment . .6 Additional Supplies . .6 Cell Culture Laboratory . 7. Aseptic Work Area . 7 Cell Culture Hood . .7 Cell Culture Hood Layout . .8 Incubator . 9 Storage . 9 Cryogenic Storage . .10 Cell Counter . .10 Cell Culture Basics | i Contents Aseptic Technique . .11 Introduction . .11 Sterile Work Area . .11 Good Personal Hygiene . .11 Sterile Reagents and Media . .12 Sterile Handling . .12 Aseptic Technique Checklist . .13 Biological Contamination . .14 Introduction . .14 Bacteria . .14 Yeasts . .15 Molds . .15 Viruses . .16 Mycoplasma . 16 Cross-Contamination . .17 Using Antibiotics . .17 Cell Culture Basics . .18 Cell Lines . .18 Selecting the Appropriate Cell Line . .18 Acquiring Cell Lines . 18 Culture Environment . .19 Adherent vs Suspension Culture . .19 Media . .20 pH . .21 CO2 . .21 Temperature . .21 Cell Morphology . .22 Mammalian Cells . .22 Variations in Mammalian Cell Morphology . .22 Morphology of 293 Cells . .23 Insect Cells . .24 Morphology of Sf21 Cells . .24 Morphology of Sf9 Cells . .25 ii | Cell Culture Basics Contents Methods . .26 Guidelines for Maintaining Cultured Cells . .26 What is Subculture? . .26 When to Subculture? . .27 Media Recommendations for Common Cell Lines .
    [Show full text]
  • Characterization of Critical Reagents for Ligand Binding Assays
    Characterization of Critical Reagents for Ligand Binding Assays 03_app_fly_Application Text Placeholder Ligand binding assays are utilized throughout the large molecule drug development process. However their robustness, accuracy and reproducibility depend on the quality of critical reagents. The unique characteristics of these reagents are crucial to assay performance, and require careful characterization. To ensure their consistent characterization in bioanalytical laboratories, a team of pharmaceutical scientists have outlined recommendations and best practices for this process, particularly during the early stages of drug development. Drug development relies on ligand binding assays (LBAs), This article summarizes the guidelines for characterizing which are bioanalytical procedures widely used to measure critical reagents, particularly during the early stages of drug the immunogenicity of biotherapeutic molecules, and development. In this context, characterization refers to the determine drug concentrations for pharmacokinetic analyses. process of identifying the physicochemical attributes of The accuracy and performance of LBAs, however, depends on these reagents (O’Hara et al. 2012). In addition, examples are the quality of key reagents, called critical reagents. The Global shown to provide insight into the analytic methods applied Bioanalysis Consortium (GBC) and the European Medicines at Bio-Rad for quality control and analytical antibody batch Agency classify critical reagents as analyte specific or binding characterization as
    [Show full text]