Utvikling av en spytt antistoff Multiplex Immunoassay å måle menneskelig eksponering for miljø 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
Etiologi og konsekvensene av menneskelig eksponering for miljø patogener er av stor bekymring over hele verden, og dermed vil evnen til å vurdere eksponering og infeksjoner ved bruk av kostnadseffektive, high-throughput tilnærminger være uunnværlig. Dette manuskriptet beskriver utvikling og analyse av en kule-baserte multipleks immunoassay stand til å måle nærværet av antistoffer i humant spytt til flere patogener samtidig. Spytt er spesielt attraktivt i denne anvendelse fordi den er ikke-invasiv, billigere og enklere å samle enn serum. Antigener fra miljøet patogener ble koblet til karboksylerte mikrosfærer (perler) og brukes til å måle antistoffer i meget små volumer av human spyttprøver ved hjelp av en vulst basert, løsning-fase-analysen. Perler ble kombinert med antigener fra Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 og G II.4) og hepatitt A virus. For å sikre at antigenene var tilstrekkelig koblet tilkulene, kobling ble bekreftet ved bruk av artsspesifikke, animalske primære fangst-antistoffer, etterfulgt av inkubasjon med biotinylerte anti-arter sekundære deteksjon-antistoffer og streptavidin-R-fykoerytrin reporter (SAPE). Som en kontroll for å måle ikke-spesifikk binding, ble en perle sett behandlet identisk med de andre, bortsett fra at det ikke ble koblet til en hvilken som helst antigen. De antigen-koblet og kontroll perler ble deretter inkubert med fremadrettet oppsamlede humane spyttprøver, målt på en høy gjennomstrømning analysator basert på prinsippene om flowcytometri, og nærværet av antistoffer mot hvert antigen ble målt i Median fluorescensintensitet enheter (MFI) . Denne multiplex immunoassay har en rekke fordeler, blant annet mer data med mindre prøven; reduserte kostnader og arbeid; og evne til å tilpasse analysen til mange mål av interesse. Resultater indikerer at spytt multipleks immunoassay kan være i stand til å identifisere tidligere eksponeringer og infeksjoner, som kan være especially nyttig i overvåking studier som involverer store befolkningsgrupper.
Introduction
Åtti-åtte prosent av diaré-relaterte sykdom på verdensbasis er forbundet med menneskelig eksponering for forurenset vann, utrygg mat, og dårlig hygiene / hygiene, forårsaker om lag 1,5 millioner dødsfall, de fleste av dem er barn 1. Dette er en viktig årsak til bekymring for offentlige helsemyndigheter og politikere. I et forsøk på å undersøke eksponeringer og sykdommer forbundet med vannbåren og andre miljø patogener, har vi utviklet en multiplex immunologisk å måle antistoffer i humane prøver 2-4. Denne fremgangsmåten kan anvendes på epidemiologiske studier for å bestemme human eksponering for disse patogener og for å bedre definere immunoprevalence og innfallende infeksjoner.
Spytt holder betydelige løftet som et alternativ til serum for human biomarkør forskning. Blant fordelene med å bruke spytt er den ikke-invasivitet og enkel prøvetaking, lav pris, og prøvene kan lett hentes fra barn 5-7 </ Sup>. Serum og spyttprøver har blitt studert for antistoffer mot H. pylori 2,3,8, Plasmodium falciparum 9, Entamoeba histolytica 10, Cryptosporidium parvum 3,11, Streptococcus pneumonia 12, hepatittvirus A og C 13-14, noroviruses 2-4,15, T. gondii 2-4, dengue virus 16, humant immunsviktvirus (HIV) 17, og Escherichia coli O157: H7 18.
En multipleks immunoassay tillater analyse av multiple analytter samtidig innenfor en enkelt prøvevolum og i løpet av en enkelt syklus eller løp. Multipleksede antigener fra C. jejuni, T. gondii, H. pylori, hepatitt A virus, og to noroviruses ble brukt til å måle menneskelig spytt IgG 2-4 og IgA 3,4 og plasma IgG 2,3 antistoffresponser mot disse patogener ved hjelp av enperle-baserte multipleksing immunoassay. Når den brukes sammen med epidemiologiske studier av eksponering for mikrober i vann, jord og mat, kan den typen av analysen beskrevet i denne studien gi verdifull informasjon for å forbedre forståelsen av infeksjoner forårsaket av miljø patogener. Videre kan spytt antistoff-data oppnådd fra slike undersøkelser anvendes for å forbedre risikovurderinger modeller 19-22.
Protocol
Representative Results
Discussion
Disse resultatene indikerer at multipleks-immunoassay-metoden er nyttig for å skille mellom spyttprøver, som er immunopositive eller immunonegative. For å bestemme immunopositivity, ble en eneste cut-off point utviklet ved å beregne middelverdien pluss tre standardavvik logg forvandlet MFI reaksjoner fra kontroll koblede perler testet med alle de spyttprøver. Cut-off point gis muligheten til å vurdere eksponering og immunoprevalence til enten en enkelt eller flere patogener. Dette diskriminerende makt kan være ny…
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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