commiss decis februari amend annex council direct eec regard diagnost test african hors sick notifi document number text eea relev ec commiss european commun regard treati establish european commun regard council direct eec june anim health condit govern movement import countri equida amend decis ec articl thereof annex direct eec describ complement fixat test carri diagnosi african hors sick novemb commun refer laboratori alget spain host annual meet nation refer laboratori african hors sick eu member state meet scientif evid present complement fixat test annex direct eec limit suitabl detect antibodi recent infect vaccin test practic replac modern elisa test laboratori commun major export countri internation accept laboratori test detect antibodi african hors sick viru manual standard diagnost test vaccin offic intern de epizooti oie current edit mention elisa test appear modifi annex direct eec account technic develop internation approv standard measur provid decis accord opinion stand veterinari committe adopt decis articl annex direct eec replac annex decis articl decis address member state brussel februari commiss david byrn member commiss oj oj chapter fourth edit annex quot annex african hors sick diagnosi reagent enzym link immunosorb assai elisa obtain european commun refer laboratori oie refer laboratori african hors sick competit elisa detect antibodi african hors sick viru ahsv prescrib test competit elisa detect specif ahsv antibodi sera speci equida broad spectrum polyclon immun anti ahsv guinea pig serum hereinaft quot guinea pig antiserum quot serogroup specif detect serotyp ah viru principl test interrupt reaction ahsv antigen guinea pig antiserum test serum sampl ahsv antibodi test serum sampl compet guinea pig antiserum result reduct expect colour addit enzym label anti guinea pig antibodi substrat sera test singl dilut spot test method titrat serum titrat method give dilut end point inhibit valu higher regard posit test protocol hereinaft region refer laboratori african hors sick pirbright unit kingdom test procedur prepar plate coat elisa plate ahsv antigen extract infect cell cultur dilut carbon bicarbon buffer ph incub elisa plate overnight wash plate time flood empti well phosphat buffer salin pb ph ph blot dry adsorb paper control well titrat posit control sera twofold dilut seri column block buffer pb tween skim milk powder cadburi marveltm adult bovin serum give final volum μl add μl neg control serum dilut μl serum μl block buffer well column add μl block buffer well column blank add μl block buffer well column guinea pig control spot test method add dilut test serum block buffer duplic well column μl sera μl block buffer serum titrat method prepar twofold dilut seri test sampl block buffer well singl column add μl guinea pig antisera pre dilut block buffer well blank well elisa plate well final volum μl incub hour orbit shaker wash plate time blot dry add μl rabbit anti guinea pig horseradish peroxidas hrp conjug pre dilut block buffer incub hour orbit shaker wash plate time blot dry chromogen prepar chromogen opd opd ortho phenyldiamin solut manufactur instruct mg ml steril distil water add substrat hydrogen peroxid give final concentr solut add μl opd solut leav plate bench minut ambient temperatur stop reaction addit μl sulphur acid read read spectrophotometr nm express result softwar packag print optic densiti od valu percentag inhibit pi test control sera base record guinea pig control well data express od pi valu determin test perform accept limit upper control limit ucl lower control limit lcl guinea pig control od valu end point titr posit control base pi rang plate fail conform criteria reject posit control serum titr greater test sampl neg neg test sampl accept duplic neg control serum well duplic blank well record pi valu failur limit invalid plate suggest background colour develop diagnost threshold cut test sera pi sampl record pi valu greater record posit sampl record pi valu lower record neg sampl record pi valu threshold duplic well consid doubt sampl test spot test titrat posit sampl titrat provid indic degre posit spot test layout gt tabl gt ve cont neg control ve cont posit control gp cont guinea pig control test sera gt tabl gt ve cont neg control ve cont posit control gp cont guinea pig control indirect elisa detect antibodi african hors sick viru ahsv prescrib test test hereinaft accord test descript chapter oie manual standard diagnost test vaccin fourth edit recombin vp protein antigen ah viru antibodi determin high index sensit specif advantag stabl infect test procedur solid phase elisa plate coat recombin ahsv vp dilut carbon bicarbon buffer ph incub plate overnight wash plate time distil water tween wash solut gentli tap plate absorb materi remov residu wash block plate phosphat buffer salin pb skim milk nestlé dry skim milktm μl hour remov block solut gentli tap plate absorb materi test sampl serum sampl test posit neg control sera dilut pb skim milk tween μl incub hour titrat make twofold dilut seri μl serum plate column posit neg control incub hour wash plate step conjug dispens μl horseradish peroxidas hrp conjug anti hors gamma globulin dilut pb milk tween ph incub hour wash plate step cromogen substrat add μl chromogen substrat solut ml mm dmab dimethyl aminobenzaldehyd ml mm mbth methyl benzo thiazolin hydrazon hydrochlorid μl colour develop stop ad μl approxim minut neg control begin colour chromogen abt azino bi ethylbenzothiazolin sulphon acid tmb tetramethyl benzidin opd ortho phenyldiamin read plate nm nm interpret result calcul cut ad neg control standard deviat deriv group neg sera test sampl give absorb valu lower cut regard neg test sampl give absorb valu greater cut regard posit test sampl give intermedi absorb valu doubt techniqu emploi confirm result block elisa detect antibodi african hors sick viru ahsv prescrib test block elisa design detect specif ahsv antibodi sera suscept speci vp major antigen viral protein ahsv conserv serotyp monoclon antibodi mab direct vp assai give high level sensit specif recombin vp antigen complet innocu guarante high degre safeti princip test interrupt reaction recombin vp antigen bound elisa plate conjug mab specif vp antibodi test sera block reaction antigen mab result reduct colour test hereinaft carri european commun refer laboratori african hors sick alget spain test procedur elisa plate coat elisa plate recombin ahsv vp dilut carbon bicarbon buffer ph incub overnight wash plate time phosphat buffer salin pb tween pbst stabilis plate treatment stabilis solut order long term storag loss activ blot dry adsorb materi test sampl control gt tabl gt gt tabl gt conjug add μl pre dilut horseradish peroxidas hrp conjug mab monoclon antibodi specif vp mix gentli ensur homogen incub minut wash plate time pbst blot dry chromogen substrat add μl chromogen substrat solut ml abt azino bi ethylbenzothiazolin sulphon acid mg ml ml substrat buffer phosphat citrat buffer ph incub minut room temperatur colour develop stop ad μl sd sodium dodecyl sulphat read read nm elisa reader interpret result assai valid test valid optic densiti od neg control nc higher od posit control pc lower cut calcul posit cut gt refer graphic gt neg cut gt refer graphic gt nc od neg control pc od posit control interpret result sampl od lower posit cut consid posit ahsv antibodi sampl od higher neg cut consid neg ahsv antibodi sampl od valu consid doubt anim sampl week quot