Udvikling af en Spyt Antibody Multiplex Immunoassay at måle menneskelig eksponering for miljømæssige patogener
Summary
In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.
Abstract
Ætiologien og virkningerne af menneskers eksponering for miljømæssige patogener er af stor bekymring i hele verden og dermed vil evnen til at vurdere eksponering og infektioner ved hjælp omkostningseffektive, high-throughput tilgange være uundværlig. Dette håndskrift beskriver udviklingen og analyse af en perle-baserede multiplex immunoassay stand til at måle tilstedeværelsen af antistoffer i humant spyt til flere patogener samtidigt. Spyt er særligt attraktivt i denne ansøgning, fordi det er noninvasiv, billigere og lettere at samle end serum. Antigener fra miljømæssige patogener blev koblet til carboxylerede mikrosfærer (perler) og anvendt til at måle antistoffer i meget små volumener af humane spytprøver bruger bead-baseret, opløsning-fase-assay. Perler blev koblet med antigener fra Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, norovira (G I.1 og G II.4) og hepatitis A-virus. At sikre, at antigenerne tilstrækkeligt koblet for atperlerne, kobling blev bekræftet ved anvendelse artsspecifikke, animalsk afledte primære fangantistoffer, efterfulgt af inkubation med biotinylerede anti-species sekundære afsløring antistoffer og streptavidin-R-phycoerythrin reporter (SAPE). Som en kontrol for at måle ikke-specifik binding, blev en kanttråd sæt behandlet identisk til de andre undtagen det ikke blev koblet til nogen antigen. De antigen-koblet og kontrol Perlerne blev derefter inkuberet med prospektivt-indsamlede humane spytprøver, målt på et højt gennemløb analysator baseret på principperne om flowcytometri, og tilstedeværelsen af antistoffer mod hvert antigen blev målt i gennemsnitlig fluorescensintensitet enheder (MFI) . Denne multiplex immunoassay har en række fordele, herunder flere data med mindre prøve; reducerede omkostninger og arbejdskraft; og evnen til at tilpasse assayet til mange mål af interesse. Resultaterne viser, at spyt multiplex immunoassay kan være i stand til at identificere tidligere eksponeringer og infektioner, som kan være especially nyttigt i overvågning undersøgelser med store befolkningsgrupper.
Introduction
Firs-otte procent af diarré-sygdom på verdensplan er forbundet med menneskers eksponering for forurenet vand, farlige fødevarer og dårlig sanitet / hygiejne, der forårsager ca. 1,5 millioner dødsfald, hvoraf de fleste er børn 1. Dette er en væsentlig årsag til bekymring for den offentlige sundhed embedsmænd og politikere. I et forsøg på at undersøge eksponeringer og sygdomme forbundet med vandbaseret og andre miljømæssige patogener, udviklede vi en multiplex immunoassay til måling af antistoffer i humane prøver 2-4. Denne metode kan anvendes på epidemiologiske undersøgelser for at bestemme menneskers udsættelse for disse patogener og for bedre at definere immunoprevalence og hændelser infektioner.
Spyt besidder betydelig lovende som et alternativ til serum til human biomarkør forskning. Blandt fordelene ved at anvende spyt er de ikke-invasivitet og let prøveindsamling, lave omkostninger, og prøver kan let indsamles fra børn 5-7 </ Sup>. Serum- og spytprøver er blevet omfattende undersøgt for antistoffer mod H. pylori 2,3,8, Plasmodium falciparum 9, Entamoeba histolytica 10, Cryptosporidium parvum 3,11, Streptococcus pneumonia 12, hepatitisvira A og C 13-14, norovira 2-4,15, T. gondii 2-4, dengue virus 16, humant immundefektvirus (HIV) 17, og Escherichia coli O157: H7 18.
En multiplex immunoassay tillader analyse af flere analytter samtidigt i en enkelt prøve volumen og inden for en enkelt cyklus eller løbe. Multipleksede antigener fra C. jejuni, T. gondii, H. pylori, blev hepatitis A virus og to norovira anvendes til måling humant spyt IgG 2-4 og IgA 3,4 og plasma IgG 2,3 antistofreaktioner til disse patogener under anvendelse af enperle-baserede multiplexing immunoassay. Når det bruges sammen med epidemiologiske undersøgelser af eksponering for mikrober i vand, jord og fødevarer, kan type assay er beskrevet i denne undersøgelse give værdifuld information til at øge forståelsen af infektioner forårsaget af miljømæssige patogener. Desuden kan spyt antistof data fra sådanne undersøgelser anvendes til at forbedre risikovurderingsmodeller 19-22.
Protocol
Representative Results
Discussion
Disse resultater indikerer, at multiplex immunoassay metode er nyttig til at skelne mellem spytprøver, der er immunpositive eller immunonegative. For at bestemme immunopositivitet, blev en enkelt skæringspunkt udviklet ved at beregne gennemsnittet plus tre standardafvigelser af de log transformeret MFI svarene fra de testede med alle de spytprøver kontrol koblede perler. Den skæringspunkt gav mulighed for at vurdere eksponeringen og immunoprevalence til enten en enkelt eller flere patogener. Denne diskriminerende ma…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Clarissa Curioso was supported through an appointment to the Research Participation Program at the U.S. Environmental Protection Agency administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. EPA.
Materials
| Equipment and Software | |||
| Microcentrifuge | Thermo Electron Corporation | 75002446 | Used to centrifuge samples |
| Vortex Mixer | VWR | G560 | Used to mix samples |
| Sonicator (mini) | Fisher Scientific | 15-337-22 | Used to separate beads |
| Pipettors P10, P20, P100, P1000, 8 ch. | Capp | Various | |
| Hemacytometer (Bright Line) | Housser Scientific | 3200 | Used to count coupled beads |
| Multiscreen Vacuum Manifold | Millipore | MSVMHTS00 | Used in washing steps to remove supernatant |
| MicroShaker | VWR | 12620-926 | Used to agitate beads during incubations |
| Tube rack (1.5mL and 0.5mL) (assorted) | VWR | 30128-346 | |
| Weighing Scale | Mettler or other | Used to measure wash reagents for making buffers | |
| Dynabead Sample Mixer | Invitrogen | 947-01 | Used during coupling incubation step |
| MatLab (R2014b) | The MathWorks, Inc. | Used to analyze antibody response data | |
| Microsoft Excel 2014 | Microsoft Corporation | Used to analyze antibody response data | |
| Luminex Analyzer with xPonent 3.1 software | Luminex Corporation | LX200-XPON3.1 | Instrument and software used to run assay |
| Antigens | |||
| GI.1 Norwalk Virus : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| GII.4 Norovirus VA387 : p-particle | Xi Jiang (CCHMC)* | NA | *Cincinnati Childrens' Hospital. Final conc. 5 µg. |
| Hepatitis A Virus : grade II concentrate from cell culture | Meridian Life Sciences | 8505 | Antigen coupled at 100 µg |
| Helicobacter pylori : lysate | Meridian Life Sciences | R14101 | Antigen coupled at 25 µg |
| Toxoplasma gondii : recombinant p30 (SAG1) | Meridian Life Sciences | R18426 | Antigen coupled at 25 µg |
| Campylobacter jejuni : heat killed whole cells | KPL | 50-92-93 | Antigen coupled at 50 µg |
| Primary Antibodies | |||
| Guinea pig anti-Norovirus | (CCHMC)* | NA | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| Mouse anti-Hepatitis A IgG | Meridian Life Sciences | C65885M | Used for coupling confirmation |
| BacTraceAffinity Purified Antibody to Helicobacter pylori | KPL | 01-93-94 | Used for coupling confirmation |
| Goat pAb to Toxoplasma gondii | Abcam | Ab23507 | Used for coupling confirmation |
| BacTrace Goat anti-Campylobacter species | KPL | 01-92-93 | Used for coupling confirmation |
| Secondary Antibodies | |||
| Biotin-SP-Conjugated AffiniPure Donkey anti-Goat IgG (H+L) | Jackson | 705-065-149 | Used for coupling confirmation |
| Biotinylated Rabbit anti-Goat IgG (H+L) | KPL | 16-13-06 | Used for coupling confirmation |
| Biotinylated Goat anti-Mouse IgG (H+L) | KPL | 16-18-06 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Rabbit IgG(H+L) | KPL | 176-1506 | Used for coupling confirmation |
| Affinity Purified Antibody Biotin Labeled Goat anti-Human IgG(ᵞ) | KPL | 16-10-02 | Used for Salivary Immunoassay |
| Consumables | |||
| 1.5 mL copolymer microcentrifuge tubes | USA Scientific | 1415-2500 | Used as low binding microcentrifuge tubes |
| 10 µL pipette tip refills | BioVentures | 5030050C | |
| 200 µL pipette tip refills | BioVentures | 5030080C | |
| 1000 µL pipette tip refills | BioVentures | 5130140C | |
| Aluminum foil | Various Vendors | Used keep beads in the dark during incubations | |
| Deep Well plates | VWR | 40002-009 | Used for diluting saliva samples |
| Multiscreen Filter Plates | Millipore | MABVN1250 | Used to run assays |
| Oracol saliva collection system | Malvern Medical Developments Limited | Used for saliva collection | |
| Reagents | |||
| Carboxylated microspheres (beads) | Luminex Corporation | Dependent on bead set | Antigens are coupled to the microspheres |
| EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) | Pierce | 77149 or 22980 | Used in bead activation |
| Sulfo-NHS (N-hydroxysulfosuccinimide) | Pierce | 24510 | Used in bead activation |
| Steptavidin-R-phycoerythrin (1mg/mL) | Molecular Probes | S-866 | Used as reporter |
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | M-2933 | Used for coupling |
| Tween-20 (Polyoxyethylenesorbitan monolaurate) | Sigma | P-9416 | Used in wash buffer to remove non-specific binding |
| Protein Buffers | |||
| PBS-TBN Blocking/ Storage Buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Azide, pH 7.4)** | Filter Sterilize and store at 4°C | ||
| PBS, pH 7.4 | Sigma | P-3813 | 138 mM NaCl, 2.7 mM KCl |
| BSA | Sigma | A-7888 | 0.1% (w/v) |
| Tween-20 | Sigma | P-9416 | 0.2% (v/v) |
| Sodium Azide (0.05% azide)** | Sigma | S-8032 | **Caution: Sodium azide is acutely toxic. Avoid contact with skin and eyes. Wear appropriate PPE's. Dispose of according to applicable laws. |
| MES/ Coupling Buffer (0.05 M MES, pH 5.0) | |||
| MES (2-[N-Morpholino]ethanesulfonic acid) | Sigma | S-3139 | |
| 5 N NaOH | Fisher | SS256-500 | |
| Assay Buffer (PBS, 1% BSA, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 1% BSA, pH 7.4 | Sigma | P-3688 | 138 mM NaCl, 2.7 mM KCl, 1% BSA |
| Activation Buffer (0.1 M NaH2PO4, pH 6.2) | Filter Sterilize and store at 4°C | ||
| NaH2PO4 (Sodium phosphate, monobasic anhydrous) | Sigma | S-3139 | 0.1M NaH2PO4 |
| 5 N NaOH | Fisher | SS256-500 | |
| Wash Buffer (PBS, 0.05% Tween-20, pH 7.4) | Filter Sterilize and store at 4°C | ||
| PBS, 0.05% Tween-20, pH 7.4 | Sigma | P-3563 | 138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN |
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