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The EliSpot Method
ELISPOT分析方法
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- EliSpot- a short introduction to the method
- AID and the emerging EliSpot field
- Publications with regard to the EliSpot method
- Typical images of wells
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EliSpot- a short introduction to the method
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EliSpot (or ElisaSpot, short for Enzyme-linked Immunosorbent Spot Assay)originally was developed as a method todetectantibody-secretingB-cells. Later the method was adaptedtodetermine T-cell reactionto a specific antigen, usuallyrepresentedas number of activatedcells per million. Mostresearchers useInterferon gamma (IFN-g)production as a read-out foractivation ofsingle cells.
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Elispot serves to determine cytotoxic T-cell activityinviral(HIV/Aids, Hepatitis) or tumor related studies ortodetermineTh1-Th2 balance. EliSpot Assays are routinely performedin96-wellmicotiter plates with a membrane bottom to each well.Themethod isnot new but has only in recent years been made usablewiththedevelopment of EliSpot analyzers (analysers), imagingdevicesthattake a picture of a single well of the EliSpot plateandenumeratethe spots.
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AID and the emerging EliSpot field
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AID was one of the first companies to offerprofessionallydesignedEliSpot readers as well as ready-to-useEliSpot kits. Since1989AID develops and manufactures immunoassaysand imaging devicestoread them. Since 1997, AID has been makingEliSpot assay kitsandthe AID EliSpot Reader System. Assays,hardware and softwareareoriginal AID developments. The AID EliSpotAssay kits containaprecoated EliSpot plate, a secondary (detection)antibody,anenzyme conjugate and the substrate. |
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Kits are available for most human cytokines as well asforsomeanimal parameters. Like all other AID products, ElispotAssayKitsare manufactured under strict ISO-certified qualitycontroltoinsure standardized response. Kits are also customizedondemand.The classic AID EliSpot Reader System has been the choiceofmanydistinguished research labs on all continents. Compact ,robustanduser-friendly, it is one of the most successfuldevelopmentsinthis area. We are particularly proud to equip theIAVIclinicaltrials for HIV vaccine delopment with several of ourAIDEliSpotReader HR.
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Publications with regard to the EliSpot method
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A selection of papers we found of technical interst totheEliSpotmethod (if you have published or find a paper weshouldinclude inthis section, please email!).
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J Immunol Methods. 2002 Feb 1;260(1-2):157-72. A panelofMHCclass I restricted viral peptides for use as a qualitycontrolforvaccine trial ELISPOT assays.
Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L,JanetzkiS,Ferrari G, Birx DL, Cox JH. The US Military HIVResearchProgram,Suite 200, 13 Taft Court, Rockville, MD 20851,USA.
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Vaccines in general and HIV vaccines in particulararefocusingever more on the induction of cellularimmunityspecifically thegeneration of cytotoxic T cells (CTL). Asprogressis made towardsdeveloping a safe and effective HIV vaccine,thereis a need for arobust, sensitive and reproducible assaytoevaluatevaccine-induced cellular immunogenicity in PhaseII/IIItrials. Theenzyme-linked immunosorbent spot (ELISPOT) assay fitsthesecriteria and isa technology that is readily transferableandamenable tohigh-through-put screening. There is a need forreagentsthat canbe used as positive controls and for optimizingandstandardizingthe assay. We selected a panel of 23 8-11merInfluenza virus(Flu), Cytomegalovirus (CMV) and Epstein Barrvirus(EBV) epitopesrecognized by CD8 positive T cells and presentedby11 class IHLA-A and HLA-B alleles whose cumulativefrequenciesrepresent>100% of Caucasian individuals.
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We examined interferon-gamma (IFN-gamma) secretioninperipheralblood mononuclear cells (PBMC) incubated withthepeptides using amodified ELISPOT assay. IFN-gamma secretionwasdetected in 15/17(88%) HIV-1 seronegative and 14/20 (70%)HIV-1seropositiveindividuals. Release of IFN-gamma in response tothepool ofpeptides was CD8+ T cell mediated and HLA restricted.Invitrostimulation of PBMC with individual peptides or thepoolofpeptides led to the expansion of T cells capable ofkillingtargetcells expressing the appropriate viral antigen in aCTLassay. Thesize, shape and appearance of the spots producedusingthis peptidepanel provided a standard for the establishmentofacceptancecriteria of spots for the evaluation of ELISPOTplatesusing anautomated reader system. |
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J Immunol Methods. 2004 Feb;285(1):89-92. Poorlysolublepeptidescan mimic authentic ELISPOT responses. Karlsson RK,JennesW,Page-Shafer K, Nixon DF, Shacklett BL.
Gladstone Institute of Virology and Immunology,UniversityofCalifornia, San Francisco, CA 94141, USA
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The ELISPOT assay is a specific, sensitive, quantitativeassayforassessing cell-mediated immune responses to a varietyofantigensincluding HIV-1 peptides. In aninterferon(IFN)-gamma-ELISPOTassay, peripheral blood mononuclearcells (PBMC)from two HIV-1exposed seronegative (ESN) individualsappeared torespond stronglyto an HIV Gag peptide. |
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Analysis of this peptide revealed that itwasincompletelydissolved and induced non-specific spot formation,evenin theabsence of cells. In subsequent experiments, the peptidewasfoundto interact with avidin and the ELISPOT membrane.Filteringthepeptide prevented non-specific spot formation.Thesefindingsunderscore the need for appropriate controls andproperpeptidepreparation in order to reduce the risk offalse-positiveELISPOTresponses. J Immunol Methods. 2002 Dec1;270(1):99-108.
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Enhanced ELISPOT detection of antigen-specific Tcellresponsesfrom cryopreserved specimens with addition of bothIL-7andIL-15--the Amplispot assay. Jennes W, Kestens L,NixonDF,Shacklett BL. Gladstone Institute of VirologyandImmunology,University of California-San Francisco, SanFrancisco,CA, USA.
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The importance of the enzyme-linked immunosorbentspot(ELISPOT)assay as a tool for studying immune responses in vitroisbecomingincreasingly apparent. However, there remains a needforenhancedsensitivity for the detection of lowfrequencyantigen-specific Tcell responses. We reasoned that theaddition ofa combination ofthe cytokines interleukin (IL)-7 andIL-15 wouldselectivelyincrease interferon-gamma (IFN-gamma)productionfromantigen-stimulated CD4+ and CD8+ effector memory Tcells.Freshlyisolated or cryopreserved peripheral blood mononuclearcells(PBMC)from four healthy donors were analysed by ELISPOT forthefrequencyof purified protein derivative (PPD)-specific CD4+ Tcellsorcytomegalovirus (CMV) peptide-specific CD8+ T cells. |
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Addition of IL-7 and IL-15 increased the number ofPPD-specificCD4+T cells up to 2.4-fold in fresh PBMC and up to18-foldincryopreserved PBMC. The cytokines also increased thenumber ofCMVpeptide-specific CD8+ T cells in fresh PBMC up to7.5-fold.Noadditional increases were seen when antibodiestoco-stimulatorymolecules CD28 and CD49d were applied togetherwiththe cytokinecombination. These data demonstrate thatthesensitivity of theELISPOT assay may be significantly augmentedbyaddition of thecytokines IL-7 and IL-15 toantigen-stimulatedcells. This methodwill be particularly useful forthe assessmentofantigen-stimulated cytokine production by T cellsincryopreservedbiological specimens. |
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Typical images of wells
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Example 1:
IFN-gamma, human. Although the wells need not be as darkasthis,this well represents a good assay. The spots areclearlydefinedbut reflect their origin of diffusion, the rim of thewelles cleanand the background is even. There are no patterns tothespots. |
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Example 2:
IFN-gamma, human. The PVDF membrane has been rinsed withethanoltoimprove coating with the capture antibody. |
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Example 3:
IFN-gamma, human. The PVDF membrane has beencoatedwithoutpretreatment. Poorly defined spots and a tendency togive amessyrim. |
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Example 4:
B-cell EliSpot often gives huge, imprecisely outlined spotsbutthisis a very nice IgG assay. |
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Example 5
IFN-gamma, mouse. Poor coating and prolonged cellculturegivepatches of confluent spots, messy rim andoverallquestionablecount results. |
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