ultrasensitive detection talk 13-5-13 fv

7/30/2019 Ultrasensitive Detection TALK 13-5-13 FV

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Acute phase proteins, homeostasis regulators ApoH a tool to reach unmet ultrasensitive diagnostics needs

to detect pern icious m icroorganisms

1

FranciscoVeas,PhD

LaboratoiredImmuno-PhysiopathologieMolculaireCompare

UMR-MinistryofDefense3-IRD-AMU

&CSOofApoH-Technologies

FacultyofPharmacy-UM1

MontpellierFrance

[email protected](mobilephone+33681416506)


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2

Acutephaseproteins&homeosta1cfunc1ons

The acute phase response consists in a change in the concentra0on ofseveralplasmaproteins,mostlysynthesizedintheliver,including:

GroupIincreaseupto5%(ceruloplasminandcomplementfactor-3(C3))

Group II from 2 to 5 0mes (haptoglobin, fibrinogen, -globulins withan0protease-ac0vityandlipopolysaccharidebindingprotein)

Group III from 5 to more than 1 0mes (CRP and SAA) (Dowton &

Colten,1988).


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ApoHcaptureaproprietarytechnology


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ApoHcaptureaproprietarytechnology

molecularmassof5kDaHighstabilityofsolidsupport-coatedApoHformsplasma0cconcentra0oncloseto2mg/LApoHcomprises5domains:4SCR(shortconsensusrepeats)fromCCP(complementcontrolprotein)moduletypeandafiVhlysinerich

domain(withalargepatchof14posi0velychargedresidues)

unusualcomposi0onwith6.2%cysteineand8.3%proline.Hydrophobicinterac0onwithanionicphospholipids(PS,Cardiolipin,someofwhicharepresentinHI,HC..)Protein-Proteininterac0on(Staphylococcusaureus)Highmicroorganismcaptureaffinityandefficiencyofthroughnovelphysico-chemicalcondi1ons


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ApoHcapturetechnology


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FunctionalApoH-coatednano-magneticbeads

Work8low


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capture Magnetic

separation

Puri8ication

washes

concentrationonApoHbeads

Virus/baxteriaPCR

serum+

beadsApoH

elution Beads

elimination

pathogenss

magnetApoHbeads

pathogen

Bacteria

CultureInhibitor

ApoHtechnology


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ApoHaforclinicaldiagnos1cofviralinfec1ons

Currentblood(orotherscomplextargetmatricesassputum,urineetc)pathogendetec0on&iden0fica0onmethods(including

moleculartechniques)s0llgeneratenumerousfalsenega1ve

resultswhenviralloadislowand/orthepresenceofinhibitorsof

detec0on.

ApoHtechnology,isthetoolpermi^ngtobeusefulin:

personalizedmedicine:byshortendelayinthediagnosis&earlystagemanagementofviralinfec0ons(isola0onofpa0ents)

inPublicHealthissues:regardingmanagementofepidemics,aswellassafertransplanta0onsandtransfusions


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An1-HBsAgenrichedsucrose

frac1on&layereddirectly

onEMgrids

SupernatantApoH-unbound

ofcentrifuged460KG(DNA

NEG)=HbsAg

ApoH-enrichedsucrose

frac1on

An1-HBsAgenriched&layered

onan1-ApoHApoHcomplex

HEPATOLOGY2001;33:207-217.)


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Highly positive Plasma Suspected Plasma

0

500000

000000

500000

2000000

2500000

23

4

5 6 78

Viralload(copies/ml)

withoutApoH-beads

withApoH-beads

Serum dilution

Low positive Plasma


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82samplesincludingpa0ents:- withlowviralload- suspectedofHCinfec0onbutnega0velydiagnosed.


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ApoHaforclinicaldiagnos1cofsuspectedDengueinfec1ons

DENVcopies/mL

Differentgenera1onsofApoH-coatedbeadstodetectDENV

withPCRinserafromCambodianindividuals

withnega1vediagnos1c

: Sample S522168 was previously (IPC) weakly positive & ** : Sample S5606185 previously (IPC) positive blue bars were obtained without ApoHa but with a new qPCR for dengue)>70% of false negatives were solved !!!!!!

* **


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A

B

C

IgM IgG

Ctrl

IgM +

IgG -

IgG +/-IgM +

Sera Neg CTRL

PCR Neg CTRL

Early diagnosis of Hantavrius could play a critical role in prognostic of patients


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Lab strain

No virus

isolated strain #1

isolated strain #2


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102

10-2

dilution series

103

104

105

106

101

100

10-3 10-4 10-5 10-610-1

copies/r

xn

spiked with cell cultured virusesstored for 24 hr @ room tempdiluted in 4 mL MEMwithout ApoHwith ApoH-beads

Results

Pa1entsamplecopies/rxn

withoutApoH withApoH

.7E+05 .8E+06


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Apo-H captured H3N2 using an

anti-H3N2 MAb

H3N2 infection detection using an

anti-H3N2 MAb


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ApoHaforclinicaldiagnos1cofbacterialinfec1ons


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Currentbacterialdetec0on&iden0fica0onmethodsare:(i)tooslowand/or(ii)notsensi1veenough

topromptlydrivean0-biotherapyforlife-threateninginfec0ons

(sep0cemia..)or,fornosocomialscreening.

Bloodculturebaseddiagnosistakes3daysormore(>0days) ,hasalackofsensi1vity(falsenega0veduetopresenceofan0bio0cs)

Molecularmethodss1lldofaceasensi1vityissuedueto:

thechallengetoconcentrateafewpathogensoutofseveralmlofblood, thepresenceofinhibitors


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Bacillus sp. Ochrobactrum anthropi

Bacteroides fragilis Prevotella orisBacteroides ureolyticus Porphyromonas endodontalis

Acinetobacter baumannii Propionibacterium acnes

Candida albicans Proteus mirabilis

Capnocytophaga canimorsus Providencia stuarti

Chlamydia trachomatis Pseudomonas aeruginosa

Citrobacter freundi Pseudomonas sp.

Coagulase-negative Staphylococcus Salmonella arizonae

Corinebacterium sp. Salmonella enteritidesEnterobacter aerogenes Salmonella typhirium

Enterococcus faecalis Serratia marcescens

Enterobacter cloacae Staphylococcus aureus

Escherichia coli Staphylococcus epidermidis

Fusobacterium nucleatum Staphylococcus hominis

Klebsiella pneumoniae Streptococcus bovis

Listeria monocytogenes Streptococcus mitis

Micrococcus sp Streptococcus pyogenesMycobacter *Tropheryma whipplei (difficult to cultivate)

Mycobacterium chelonae * *spores & ***fungi

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ApoH:exhibitahighaffinityforinfec0ousbacteriabutalowaffinityforcommensalbacteriafromthegut&fromcollec0ons(ATCC).

0

10

20

30

40

0 10 20 30 40

Cp

Time(min)

Swab Urine


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27 hemocultivation false positive(CO2 positive but negative sub cultivation)

147 CO2-producing ApoH-tested hemocultures

64positives ApoH -

Subcultivation &PCR

64 positives ApoH+Subcultivation & PCR

0 positive ApoH-

Subcultivation &PCR9 positives ApoH

+

Subcultivation & PCR

0 positive ApoH-Subcultivation &PCR

11 positives ApoH+

Subcultivation & PCR

64 hemocultivation positives

Clinicaldata

ApoHtest&diagnostic

56 hemocultivation negatives


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1: K. Pneumoniae sans billes ApoH

2: K. Pneumoniae avec billesApoH

3: S. aureus sans billes ApoH4: S. aureus avec billesApoH

5: Coagulase-ngative Staphylococcus sans billes ApoH

6: Coagulase-ngative Staphylococcus avec billesApoH

7: P. acnes sans billes ApoH

8: P. acnes avec billesApoH9: Contrle positif l (ADN bactrien)

10: H2O

1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10


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Generalconclusion()


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Generalconclusion(2)


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Specialthanks&contacts:

EuropeanUSDEPproject UniversityMontpellier,France TheFrenchResearchIns1tuteforDevelopmentOSEO,theFrenchinnova1onagencyTheRegionLanguedocRoussillonFranceLaboratoiredImmuno-PhysiopathologieMolculaireCompareTheApoH-Technologiestech-team&colls:GregorDubois,EstelleLucarz,SylviaTigre

&IliasStefas(CEO)

Biotechnologies-Dveloppement-Conseil (France,USA,Israel&Japan),Chris1anPolicardCEO(formerCEOofPasteurIns0tuteBusinessDev&formerCEOofSanofi-PasteurDiagnos0cs )

[email protected]

ultrasensitive detection talk 13-5-13 fv

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