DOCSLIB.ORG

304: Light-Scattering Assays

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
304: Light-Scattering Assays TechNote 304 Light-Scattering Assays 9025 Technology Dr. • Fishers, IN 46038-2886 800.387.0672 • 317.570.7020 • Fax 317.570.7034 [email protected] • www.bangslabs.com B E A D S A B O V E T H E R E S T ™ Nephelometric Assay Measure Scattered Light Measure Transmitted Light Turbidimetric Assay Pass through Optical Counter Particle Counting Sample Assay CONTENTS I. BACKGROUND I. Background A. Principles of Light Scattering Immunoassays A. Principles of Light-Scattering Immunoassays Immunoassays based on the measurement of scattered or absorbed light 1. Turbidimetry are an extension of the basic principles underlying latex agglutination tests. 2. Nephelometry The change in the light scattered or blocked by Ab (or Ag) solutions is used 3. Particle Counting to measure the amount of Ag (or Ab) which causes the immunological Ab-Ag B. Kinetics of Immunoprecipitation precipitation reaction or agglutination reaction (if latex is used). II. Particle-Enhanced Immunoassay A. Assay Formats In a basic light-scattering immunoassay, polyvalent antigens react with B. Assay Instrumentation divalent antibodies to form large complexes, the antibody effectively forming 1. Spectrophotometer/Fast Centrifugal Analyzer a bridge between antigen molecules. A protein antigen, which can be 2. Nephelometer considered multivalent, with possibly multiple copies of the same epitope as 3. Optical Cell Counter well as different epitopes, can produce a large immune complex made up of C. Assay Parameters several molecules. 1. Particle Size 2. Optimum Wavelength 1. Turbidimetry 3. Kinetic vs. Endpoint Monitoring Turbidimetry is the measurement of light-scattering species in solution by 4. Antibody/Antigen Considerations means of a decrease in intensity of the incident beam after it has passed 5. Antigen/Antibody Loading through solution.1 For turbidimetric assays, the change in the amount of 6. Buffers and Ionic Species/Optimal pH light absorbed (inverse of amount transmitted) can be related to the amount 7. Influence of Polymers of agglutination which occurs. Hence, the amount of analyte (the species 8. Effect of Temperature causing agglutination) in the sample can be easily determined. D. Roles of Different Types of Microspheres 1. Size/Size Distribution 2. Nephelometry 2. Density Nephelometry is the technique for measuring the light-scattering species in 3. Refractive Index solution by means of the light intensity at an angle away from the incident 4. Colloidal Stability light passing through the sample.1 Nephelometric assays present an indirect 5. Streptavidin-Coated Microspheres method of measurement of the amount of analyte in a sample by measuring 6. Polystyrene/Magnetic Polystyrene the amount of light scattered or reflected at a given angle (typically 90˚) from E. Options for Ligand Attachment the origin. In the presence of the protein antigen, the antibody reacts with 1. Adsorption the antigen, and a precipitation reaction begins. The measurement is taken 2. Covalent Attachment early in this precipitation reaction time sequence. A quantitative value is 3. Streptavidin/Biotin Coupling obtained by comparison with a standard curve, which has been established III. New Developments previously. In order to increase the sensitivity of the detection, you can IV. References adsorb or covalently attach the protein to polymeric microspheres. In this way, a greater signal is produced with less reagent. (The main practical difference between these two approaches is the Bangs Laboratories, Inc. TechNote 304 Rev. #003, Active: 20/March/2013 << COPY >> Page 1 of 7 TECH NOTE 304 concentration of the solutions used in the assay. Nephelometry is best Figure 1 shows the general relationship between antigen concentration performed with dilute solutions, as at higher concentrations, destructive light and antibody precipitated.2 There are three distinct elements to the curve scattering might cause a loss of sensitivity. Conversely, turbidimetry requires in Figure 1. The first region, in which the antigen concentration increases, a higher density of particles to achieve a measurable and precise signal.) is one of antibody excess when the immune complexes are small, with some bridging. The second region, ‘equivalence,’ represents an optimum 3. Particle Counting ratio of antibody bridging in relation to antibody concentration, i.e., enough Particle counting is unique in that it requires a particle-enhanced format. polyvalent antigen molecules bind both ‘arms’ of the divalent antibodies in It is an immunoassay technique that recognizes microspheres that are solution; this is the point of maximum lattice, and thus precipitate, formation. not agglutinated (i.e., it determines the difference between the amount of In the third region, antigen excess, there is, in effect, a reduced supply of signal generated when antigen is present or absent). The reagent antigen or ‘bridging’ antibody molecules in relation to the amount of antigen now in antibody is coupled to microspheres and then forms immunocomplexes with solution. the sample antibody or antigen, respectively; the number of unagglutinated microspheres is thus inversely proportional to the analyte concentration. Region 1 + + (Antibody Excess) Table 1 shows a comparison of the detection limits for the measurement Region 2 + ( (Equivalence, ( Maximum of proteins in biological samples1 by turbidimetry, nephelometry, or particle n Aggregation) counting detection methods. Region 3 + + (Antigen Excess) Table 1: Comparison of Detection Limits Figure 2: Diagrammatical Representation of the Heidelberger-Kendall Curve Figure 2. Diagrammatical representation of the Heidelberger-Kendall Monitoring curve. System Analyte Sample [Conc.] Molar Turbidimetry: Human placental Serum 1.6mg/L 5.5x10-8 Figures 1 and 2 illustrate two important considerations when developing Nonenhanced Iactogen these types of assays. First, that there are two potential antigen concentrations that will generate the same apparent light-scattering -9 Turbidimetry: Retinol-binding Urine 25µg/L 12x10 signal; one when there is antibody excess and one when there is antigen Latex particle- protein enhanced excess. For this reason, the time at which measurements are made is an important factor. Second, assuming that the antigen used is divalent, Turbidimetry: Chloriogonadotropin Serum 50µg/L 1.6x10-10 the largest complex formed outside of the region of equivalence will be a Gold sol triplet (Antibody-Antigen-Antibody). Depending on the sophistication of the particle-enhanced instrumentation and the size of the antibodies and antigens used, this is normally not large enough to be measured. As is shown in Figure 1, the Nephelometry: Immunoglobulin M Cerebral 6.1mg/L 6.3x10-9 Nonenhanced Spinal Fluid region of equivalence represents a relatively high concentration of sample (antigen). For this reason, the direct turbidimetric immunoassay lacks Nephelometry: Myoglobin Serum 6.1µg/L 3.4x10-10 sensitivity for analytes for which small concentrations may hold clinical Latex particle- significance. The solution to these limitations is the particle-enhanced enhanced immunoassay, for the following reasons: Rate Immunoglobulin M Cerebral 11.1mg/L 1.1x10-9 1. Sensitivity is increased by increasing the relative light-scattering Nephelometry Spinal Fluid signal; 2. It provides the opportunity to use a different assay format. Particle C-reactive protein Serum 1.0µg/L 0.9x10-11 Specifically, the ability to test for haptens, whose single epitopic Counting: sites make them unsuitable for a direct turbidimetric assay; and 3. The problems that can result from antigen excess, as discussed B. Kinetics of Immunoprecipitation previously, can be avoided with assay optimization. II. PARTICLE-ENHANCED IMMUNOASSAY Antibody Antigen II. Particle-Enhanced Immunoassay analyzers and other discrete analyzers, in which either the reaction Excess Equivalence Excess A. Assay Formats cuvettes or the optics are rotated while the other remains stationary creating a regular scanning mode with respect to time, have also A. Assay formats proved to be very precise turbidimeters. Sample (Analyte Present) 2. Nephelometer- + ) d e r e t t a c S t h g i L ( Agglutination (Positive) strongly influenced by the angle from the incident beam at which d e t a t i p i c e r P y d o b i t n A detection occurs. sensitivity for larger scattering species (such as those found in par- Free No Free Antibody Coated Sample Antibody ticle-enhanced immunoassays). Antibody Microspheres (No Analyte Present) in Supernatant in Supernatant ment can be dif its use will require that the forward scattered light can be dif No Agglutination (Negative) Antigen Concentration ated from the incident beam. Secondly clinical applications that both scattered and transmitted light can be Figure 1. The HiedelbFigureerger -1:Ke Thenda lHeidelberger-Kendalll curve, showing the Curve general relationship Figure 3a:Figu Directre 3a. DAgglutinationirect Agglutination measured with the same analyzer between antigen concentration and antibody precipitated.2 ter is typically used for nephelometric monitoring. One advantage Bangs Laboratories, Inc. TechNote 304 Rev. #003, Active: 20/March/2013 << COPY >> Page 2 of 7 of nephelometric monitoring is that the sensitivity can be adjusted to specific assay requirements by setting the detector sensitivity ap- propriately Sample creased noise that will result. For these instruments, the intensity of + (Analyte Present) No Agglutination (Positive) the light source also plays a large role in the sensitivity of the assay Free Antibody
Load more

Recommended publications
  • "Epitope Mapping: B-Cell Epitopes". In: Encyclopedia of Life Sciences
    Epitope Mapping: B-cell Advanced article Epitopes Article Contents . Introduction GE Morris, Wolfson Centre for Inherited Neuromuscular Disease RJAH Orthopaedic Hospital, . What Is a B-cell Epitope? . Epitope Mapping Methods Oswestry, UK and Keele University, Keele, Staffordshire, UK . Applications Immunoglobulin molecules are folded to present a surface structure complementary to doi: 10.1002/9780470015902.a0002624.pub2 a surface feature on the antigen – the epitope is this feature of the antigen. Epitope mapping is the process of locating the antibody-binding site on the antigen, although the term is also applied more broadly to receptor–ligand interactions unrelated to the immune system. Introduction formed of highly convoluted peptide chains, so that resi- dues that lie close together on the protein surface are often Immunoglobulin molecules are folded in a way that as- far apart in the amino acid sequence (Barlow et al., 1986). sembles sequences from the variable regions of both the Consequently, most epitopes on native, globular proteins heavy and light chains into a surface feature (comprised of are conformation-dependent and they disappear if the up to six complementarity-determining regions (CDRs)) protein is denatured or fragmented. Sometimes, by acci- that is complementary in shape to a surface structure on the dent or design, antibodies are produced against linear antigen. These two surface features, the ‘paratope’ on the (sequential) epitopes that survive denaturation, though antibody and the ‘epitope’ on the antigen, may have a cer- such antibodies usually fail to recognize the native protein. tain amount of flexibility to allow an ‘induced fit’ between The simplest way to find out whether an epitope is confor- them.
    [Show full text]
  • Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives
    sensors Review Ten Years of Lateral Flow Immunoassay Technique Applications: Trends, Challenges and Future Perspectives Fabio Di Nardo * , Matteo Chiarello , Simone Cavalera , Claudio Baggiani and Laura Anfossi Department of Chemistry, University of Torino, 10125 Torino, Italy; [email protected] (M.C.); [email protected] (S.C.); [email protected] (C.B.); [email protected] (L.A.) * Correspondence: [email protected] Abstract: The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed. Citation: Di Nardo, F.; Chiarello, M.; Cavalera, S.; Baggiani, C.; Anfossi, L.
    [Show full text]
  • Comparison of Immunohistochemistry with Immunoassay (ELISA
    British Journal of Cancer (1999) 79(9/10), 1534–1541 © 1999 Cancer Research Campaign Article no. bjoc.1998.0245 Comparison of immunohistochemistry with immunoassay (ELISA) for the detection of components of the plasminogen activation system in human tumour tissue CM Ferrier1, HH de Witte2, H Straatman3, DH van Tienoven2, WL van Geloof1, FJR Rietveld1, CGJ Sweep2, DJ Ruiter1 and GNP van Muijen1 Departments of 1Pathology, 2Chemical Endocrinology and 3Epidemiology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands Summary Enzyme-linked immunosorbent assay (ELISA) methods and immunohistochemistry (IHC) are techniques that provide information on protein expression in tissue samples. Both methods have been used to investigate the impact of the plasminogen activation (PA) system in cancer. In the present paper we first compared the expression levels of uPA, tPA, PAI-1 and uPAR in a compound group consisting of 33 cancer lesions of various origin (breast, lung, colon, cervix and melanoma) as quantitated by ELISA and semi-quantitated by IHC. Secondly, the same kind of comparison was performed on a group of 23 melanoma lesions and a group of 28 breast carcinoma lesions. The two techniques were applied to adjacent parts of the same frozen tissue sample, enabling the comparison of results obtained on material of almost identical composition. Spearman correlation coefficients between IHC results and ELISA results for uPA, tPA, PAI-1 and uPAR varied between 0.41 and 0.78, and were higher for the compound group and the breast cancer group than for the melanoma group. Although a higher IHC score category was always associated with an increased median ELISA value, there was an overlap of ELISA values from different scoring classes.
    [Show full text]
  • Immunoassay - Elisa
    IMMUNOASSAY - ELISA PHUBETH YA-UMPHAN National Institute of Health, Department of Medical Sciences 0bjective After this presentation, participants will be able to Explain how an ELISA test determines if a person has certain antigens or antibodies . Explain the process of conducting an ELISA test. Explain interactions that take place at the molecular level (inside the microtiter well) during an ELISA test. Outline - Principal of immunoassay - Classification of immunoassay Type of ELISA - ELISA ELISA Reagents General - Applications Principal of ELISA ELISA workflow What is immunoassay? Immunoassays are bioanalytical methods that use the specificity of an antigen-antibody reaction to detect and quantify target molecules in biological samples. Specific antigen-antibody recognition Principal of immunoassay • Immunoassays rely on the inherent ability of an antibody to bind to the specific structure of a molecule. • In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. Classification of Immunoassays Immunoassays can be classified in various ways. Unlabeled Labeled Competitive Homogeneous Noncompetitive Competitive Heterogeneous Noncompetitive https://www.sciencedirect.com/science/article/pii/S0075753508705618 Classification of Immunoassays • Unlabeled - Immunoprecipitation • Labeled Precipitation of large cross-linked Ag-Ab complexes can be visible to the naked eyes. - Fluoroimmnoassay (FIA) - Radioimmunoassay (RIA) - Enzyme Immunoassays (EIA) - Chemiluminescenceimmunoassay(CLIA) - Colloidal Gold Immunochromatographic Assay (ICA) https://www.creative-diagnostics.com/Immunoassay.htm Classification of Immunoassays • Homogeneous immunoassays Immunoassays that do not require separation of the bound Ag-Ab complex. (Does not require wash steps to separate reactants.) Example: Home pregnancy test. • Heterogeneous immunoassays Immunoassays that require separation of the bound Ag-Ab complex.
    [Show full text]
  • Analysis of Proteins by Immunoprecipitation
    Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate).
    [Show full text]
  • Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein a in Positive Blood Culture Samples
    diagnostics Article Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples Arpasiri Srisrattakarn 1 , Patcharaporn Tippayawat 1, Aroonwadee Chanawong 1, Ratree Tavichakorntrakool 1, Jureerut Daduang 1, Lumyai Wonglakorn 2 and Aroonlug Lulitanond 1,* 1 Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; [email protected] (A.S.); [email protected] (P.T.); [email protected] (A.C.); [email protected] (R.T.); [email protected] (J.D.) 2 Clinical Microbiology Unit, Srinagarind Hospital, Khon Kaen University, Khon Kaen 40002, Thailand; [email protected] * Correspondence: [email protected]; Tel.: +66-(0)-4320-2086 Received: 11 August 2020; Accepted: 21 September 2020; Published: 7 October 2020 Abstract: Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively).
    [Show full text]
  • Ige – the Main Player of Food Allergy
    DDMOD-431; No of Pages 8 Vol. xxx, No. xx 2016 Drug Discovery Today: Disease Models Editors-in-Chief Jan Tornell – AstraZeneca, Sweden DRUG DISCOVERY Andrew McCulloch – University of California, SanDiego, USA TODAY DISEASE MODELS IgE – the main player of food allergy 1 2,3 2 Henrike C.H. Broekman , Thomas Eiwegger , Julia Upton , 4, Katrine L. Bøgh * 1 Department of Dermatology/Allergology, University Medical Centre Utrecht (UMCU), Utrecht, The Netherlands 2 Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program, The Department of Paediatrics, Hospital for Sick Children, Toronto, Canada 3 Research Institute, Physiology and Experimental Medicine, The University of Toronto, Toronto, Canada 4 National Food Institute, Technical University of Denmark, Søborg, Denmark Food allergy is a growing problem worldwide, presently Section editor: affecting 2–4% of adults and 5–8% of young children. IgE Michelle Epstein – Medical University of Vienna, is a key player in food allergy. Consequently huge Department of Dermatology, DIAID, Experimental Allergy, Waehringer Guertel 18-20, Room 4P9.02, A1090, efforts have been made to develop tests to detect Vienna, Austria. either the presence of IgE molecules, their allergen binding sites or their functionality, in order to provide allergen ingestion [1], and involve one or more of the follow- information regarding the patient’s food allergy. The ing systems; the skin (pruritus, urticaria, or angioedema), the ultimate goal is to develop tools that are capable of gastro-intestinal tract (diarrhea, vomiting, contractions, in- creased bowel movement), the respiratory tract (asthma at- discriminating between asymptomatic sensitization tack, hoarseness, stridor/laryngeal angioedema) or the and a clinically relevant food allergy, and between cardiovascular system (dizziness, drop in blood pressure, loss different allergic phenotypes in an accurate and trust- of consciousness) [2,3].
    [Show full text]
  • The Immunoassay Guide to Successful Mass Spectrometry
    The Immunoassay Guide to Successful Mass Spectrometry Orr Sharpe Robinson Lab SUMS User Meeting October 29, 2013 What is it ? Hey! Look at that! Something is reacting in here! I just wish I knew what it is! anti-phospho-Tyrosine Maybe we should mass spec it! Coffey GP et.al. 2009 JCS 22(3137-44) True or false 1. A big western blot band means I have a LOT of protein 2. One band = 1 protein Big band on Western blot Bands are affected mainly by: Antibody affinity to the antigen Number of available epitopes Remember: After the Ag-Ab interaction, you are amplifying the signal by using an enzyme linked to a secondary antibody. How many proteins are in a band? Human genome: 20,000 genes=100,000 proteins There are about 5000 different proteins, not including PTMs, in a given cell at a single time point. Huge dynamic range 2D-PAGE: about 1000 spots are visible. 1D-PAGE: about 60 -100 bands are visible - So, how many proteins are in my band? Separation is the key! Can you IP your protein of interest? Can you find other way to help with the separation? -Organelle enrichment -PTMs enrichment -Size enrichment Have you optimized your running conditions? Choose the right gel and the right running conditions! Immunoprecipitation, in theory Step 1: Create a complex between a desired protein (Antigen) and an Antibody Step 2: Pull down the complex and remove the unbound proteins Step 3: Elute your antigen and analyze Immunoprecipitation, in real life Flow through Wash Elution M 170kDa 130kDa 100kDa 70kDa 55kDa 40kDa 35kDa 25kDa Lung tissue lysate, IP with patient sera , Coomassie stain Rabinovitch and Robinson labs, unpublished data Optimizing immunoprecipitation You need: A good antibody that can IP The right beads: i.
    [Show full text]
  • Wo2015188839a2
    Downloaded from orbit.dtu.dk on: Oct 08, 2021 General detection and isolation of specific cells by binding of labeled molecules Pedersen, Henrik; Jakobsen, Søren; Hadrup, Sine Reker; Bentzen, Amalie Kai; Johansen, Kristoffer Haurum Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Pedersen, H., Jakobsen, S., Hadrup, S. R., Bentzen, A. K., & Johansen, K. H. (2015). General detection and isolation of specific cells by binding of labeled molecules. (Patent No. WO2015188839). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/188839
    [Show full text]
  • Serotyping of Bdellovibrios by Agglutination and Indirect Immunofluorescence
    INTERNATIONAL JOURNAL OF SYSTEMATICBACT~RIOLOGY. Oct. 1983, p. 816-821 Vol. 33, No. 4 0020-7713/83/040816-06$02.00/0 Copyright 0 1983, Internationnl Union of Microbiologicdl Societie\ Serotyping of Bdellovibrios by Agglutination and Indirect Immunofluorescence M. E. SCHELLING’* AND S. F. CONTI’ Department Biology, Texus A &M Unitwsit?, College Station, Texus 77843‘ und University of Mussa ch us et t s , A rn ii erst, Muss N c h iiset t J 0 1 002’ A comparative serological study was undertaken to clarify the uncertain interrelationships among various bdellovibrios. A total of 17 predacious (host- dependent) strains and 12 nonpredacious strains were serotyped. Strains were grouped into serotypes on the basis of positive agglutination and strongly positive fluorescence cross-reactions. The results of division of the strains into serogroups by these two methods were identical. Bdellovihrio starrii strain A3.12 and Bdellovibrio stolpii strain UKi2 were found to be antigenically distinct from each other and from the group of strains comprising Bdellovihrio hacteriovorus; the latter was found to be an antigenically heterogeneous group consisting of at least nine serogroups. All nonpredacious strains were found to be related antigenically to their obligately predacious counterparts. An antigen(s) common to all of the Bdellovibrio strains examined was exhibited as weakly positive fluorescence. Selected strains of Bdellovihrio were studied by Ouchterlony immunodiffusion, which confirmed the serogrouping results and provided a technically simple method of serotyping. Further immunochemical characterization of the strains of Bdellovihrio in conjunction with increased knowledge of other differences among strains will likely result in the formation of additional species of Bdellovihrio.
    [Show full text]
  • Role of Serologic Testing in Rheumatic Diseases
    Role of Serologic Testing in Rheumatic Diseases Debendra Pattanaik MD FACP Associate professor of Medicine UTHSC, Memphis TN Disclosure None Objectives Discuss commonly available serologic testing useful in daily clinical practice Recognize the serologic associations of rheumatic diseases Recognize their diagnostic utilities and limitations Diagnostic Accuracy for Lupus and other autoimmune diseases in the community setting 476 patients were evaluated at Autoimmunity Center of University of Florida, Gainesville for 13 months which were by from primary care physicians SLE was over diagnosed on many patients on the basis of + ANA 39 patients are taking prednisone 60 mg/day who have no autoimmune disease but only have + ANA Inappropriate diagnosis leads to inappropriate therapy, emotional and financial consequences The authors suggested continuing education in screening for autoimmune disease and identify patients who may benefit from early referral. Arch Intern Med. 2004;164:2435-2441 Antinuclear Antibody (ANA) Testing for Connective Tissue Disease British Columbia Population: 4.631 million. More than 94,000 ANA tests were performed in B.C. in fiscal year 2011/12 at a cost of $2.24 million annually. Incidence and Estimated New Cases in B.C. for Selected CTDs Connective Tissue Disease Disease incidence per million population Estimated new BC cases/year * Systemic lupus erythematosus 56 259 Scleroderma 19 88 Dermatomyositis & polymyositis < 10 < 46 Eighteen percent of first-time tested outpatients underwent unnecessary repeat testing in 2010/2011. In 57.2% of the repeat testing, both the initial and the repeat ANA tests were ordered by a GP. In 24.8% the initial test was ordered by a GP and the repeat test was ordered by a specialist, and in 10.2% both the initial and the repeat test were ordered by the same specialist.
    [Show full text]
  • Anti-GFP (Green Fluorescent Protein) Mab-Agarose Code No
    D153-8 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures. MONOCLONAL ANTIBODY Anti-GFP (Green Fluorescent Protein) mAb-Agarose Code No. Clone Subclass Quantity D153-8 RQ2 Rat IgG2a Gel: 200 L BACKGROUND: Since the detection of intracellular 5) Dragone, L. L., et al., PNAS. 103, 18202-18207 (2006) [IP] Aequorea Victria Green Fluorescent Protein (GFP) requires 6) Darzacq, X., et al., J. Cell Biol. 173, 207-218 (2006) [ChIP] only irradiation by UV or blue light, it provides an excellent 7) Hayakawa, T., et al., Plant Cell Physiol. 47, 891-904 (2006) [IP] means for monitoring gene expression and protein 8) Obuse, C., et al., Nat. Cell Biol. 6, 1135-1141 (2004) [IP] localization in living cells. Agarose conjugated anti-GFP monoclonal antibody can detect GFP fusion protein on As this antibody is really famous all over the world, a lot of Immunoprecipitation. researches have been reported. These references are a part of such reports. SOURCE: This antibody was purified from hybridoma (clone RQ2) supernatant using protein G agarose. This INTENDED USE: hybridoma was established by fusion of mouse myeloma For Research Use Only. Not for use in diagnostic procedures. cell PAI with Wister rat lymphnode immunized with GFP purified from GFP expressed 293T cells by affinity 1 2 chromatographic technique using mouse anti-GFP. kDa 66 FORMULATION: 100 g of anti-GFP monoclonal antibody covalently coupled to 200 L of agarose gel and 45 provided as a 50% gel slurry suspended in PBS containing GFP fusion protein preservative (0.1% ProClin 150) for a total volume of 400 30 L.
    [Show full text]