Name: detection of endotoxin in nano-formulations using limulus amoebocyte lysate (lal) assays
Uploaded: 2019-01-30T14:30:00
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Undersøgelsen blev støttet af føderale midler fra National Cancer Institute, National Institutes of Health, under kontrakt HHSN261200800001E. Indholdet af denne publikation afspejler ikke nødvendigvis synspunkter eller politikker af Department of Health og Human Services, og heller ikke nævner af firmanavne, kommercielle produkter, eller organisationer indebærer godkendelse af den amerikanske regering.

1. forberedelse af nanopartikel prøver
<ol><li>Forberede undersøgelse prøven i LAL grade vand.</li>
	<li>Hvis prøven pH-værdien er uden for området 6-8, Juster pH-værdien ved hjælp af pyrogen-fri natriumhydroxid eller saltsyre.</li>
	<li>Ved hjælp af LAL grade vand forberede flere fortyndinger af eksemplet undersøgelse. Sørg for, at den højeste fortynding ikke overskrider maksimale gyldig fortynding (MVD). Henvise til afsnittet diskussion for detaljer om MVD skøn.</li>
</ol>2. forbere...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+method+for+endotoxin+detection+depends+on+nanoformulation.">Choice of method for endotoxin detection depends on nanoformulation.</a> Nanomedicine (London). 9 (12), 1847-1856 (2014).</li><li>Neun, B. W., Dobrovolskaia, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+and+Some+Practical+Solutions+to+Overcome+Nanoparticle+Interference+with+LAL+Assays+and+to+Avoid+Endotoxin+Contamination+in+Nanoformulations.">Considerations and Some Practical Solutions to Overcome Nanoparticle Interference with LAL Assays and to Avoid Endotoxin Contamination in Nanoformulations.</a> Methods in Molecular Biology. 1682, 23-33 (2018).</li><li>Boratynski, J., Szermer-Olearnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+Removal+from+Escherichia+coli+Bacterial+Lysate+Using+a+Biphasic+Liquid+System.">Endotoxin Removal from Escherichia coli Bacterial Lysate Using a Biphasic Liquid System.</a> Methods in Molecular Biology. 1600, 107-112 (2017).</li><li>Li, H., Hitchins, V. M., Wickramasekara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+bacterial+endotoxins+in+ophthalmic+viscosurgical+device+materials+by+direct+analysis+in+real+time+mass+spectrometry.">Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry.</a> Analytica Chimica Acta. 943, 98-105 (2016).</li><li>Uhlig, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+3-hydroxy+fatty+acids+as+environmental+markers+of+endotoxin+using+liquid+chromatography+coupled+to+tandem+mass+spectrometry.">Profiling of 3-hydroxy fatty acids as environmental markers of endotoxin using liquid chromatography coupled to tandem mass spectrometry.</a> Journal of Chromatography A. 1434, 119-126 (2016).</li><li>Smulders, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contamination+of+nanoparticles+by+endotoxin:+evaluation+of+different+test+methods.">Contamination of nanoparticles by endotoxin: evaluation of different test methods.</a> Particle and Fibre Toxicology. 9, 41 (2012).</li><li>. NCL assay cascade Available from: <a target="_blank" href="https://ncl.cancer.gov/resources/assay-cascade-protocols">https://ncl.cancer.gov/resources/assay-cascade-protocols</a> (2015)</li><li>Dobrovolskaia, M. A., Germolec, D. R., Weaver, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+nanoparticle+immunotoxicity.">Evaluation of nanoparticle immunotoxicity.</a> Nature Nanotechnology. 4 (7), 411-414 (2009).</li><li>Borton, L. K., Coleman, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material-mediated+pyrogens+in+medical+devices:+Applicability+of+the+in+vitro+Monocyte+Activation+Test.">Material-mediated pyrogens in medical devices: Applicability of the in vitro Monocyte Activation Test.</a> Altex. , (2018).</li><li>Stoppelkamp, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speeding+up+pyrogenicity+testing:+Identification+of+suitable+cell+components+and+readout+parameters+for+an+accelerated+monocyte+activation+test+(MAT).">Speeding up pyrogenicity testing: Identification of suitable cell components and readout parameters for an accelerated monocyte activation test (MAT).</a> Drug Testing and Analysis. 9 (2), 260-273 (2017).</li><li>Vipond, C., Findlay, L., Feavers, I., Care, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+the+rabbit+pyrogen+test+for+assessing+meningococcal+OMV+based+vaccines.">Limitations of the rabbit pyrogen test for assessing meningococcal OMV based vaccines.</a> Altex. 33 (1), 47-53 (2016).</li><li>Barenholz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxil(R)--the+first+FDA-approved+nano-drug:+lessons+learned.">Doxil(R)--the first FDA-approved nano-drug: lessons learned.</a> Journal of Controlled Release. 160 (2), 117-134 (2012).</li><li>Neun, B. W., Dobrovolskaia, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantitative+evaluation+of+endotoxin+contamination+in+nanoparticle+formulations+by+LAL-based+assays.">Detection and quantitative evaluation of endotoxin contamination in nanoparticle formulations by LAL-based assays.</a> Methods in Molecular Biology. 697, 121-130 (2011).</li><li>FDA, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. , (2005).</li><li>Mohan, P., Rapoport, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+as+a+molecular+nanotheranostic+agent:+effect+of+doxorubicin+encapsulation+in+micelles+or+nanoemulsions+on+the+ultrasound-mediated+intracellular+delivery+and+nuclear+trafficking.">Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking.</a> Molecular Pharmaceutics. 7 (6), 1959-1973 (2010).</li><li>Dabbagh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-melting-point+polymeric+nanoshells+for+thermal-triggered+drug+release+under+hyperthermia+condition.">Low-melting-point polymeric nanoshells for thermal-triggered drug release under hyperthermia condition.</a> International Journal of Hyperthermia. 31 (8), 920-929 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+use+of+commercial+LAL+assays+for+the+analysis+of+endotoxin+contamination+in+metal+colloids+and+metal+oxide+nanoparticles.">Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles.</a> Nanotoxicology. 9 (4), 462-473 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+endotoxin+(lipopolysaccharide)+binds+to+the+surface+of+gold+nanoparticles,+interferes+with+biocorona+formation+and+induces+human+monocyte+inflammatory+activation.">Bacterial endotoxin (lipopolysaccharide) binds to the surface of gold nanoparticles, interferes with biocorona formation and induces human monocyte inflammatory activation.</a> Nanotoxicology. 11 (9-10), 1157-1175 (2017).</li></ol>

Oplysningerne i denne protokol er blevet beskrevet før15,26 og bygger på flere lovgivningsmæssige dokumenter udgivet af US Food and Drug Administration (US FDA eller FDA) og Amerikas Forenede Stater farmakopé (USP)4 , 5 , 6 , 27, og er også tilgængelig på NCL hjemmeside20 i protokoller STE1.2 (turbiditet L...

En endotoxin er en byggesten af Gram-negative bakterielle cellevæg1,2. Det kan aktivere immunceller på meget lav (pikogram) koncentrationer1,2. Proinflammatoriske mæglere (cytokiner, leukotriener, eicosanoider, etc.) produceret af celler som reaktion på en endotoxin er ansvarlig for feber, hypotension, hypertension og mere alvorlige sundhedsmæssige problemer, herunder flere organsvigt 1 , 2 , 3. sværhedsgraden af immun-medieret bivirkninger udløst af endotoxin afhænger dens potens bestemmes af endotoxin sammensætning og struktur og måles i internationale endotoxin enheder (IUs eller EUs)3. Antallet af disse enheder pr. kg kropsvægt bruges til at indstille en tærskel pyrogen dosis af endotoxin. Denne dosis er 5 EU/kg for lægemidler administreres via alle ruter men ruten Intratekal. Lægemidler doseres pr kvadratmeter af kroppens overflade, intraokulært væsker, radioaktive lægemidler og produkter administreres via Intratekal rute har en forskellige pyrogen tærskeldosis, som er 100 EU/m2, 0,2 EU/mL, 175 EU/V (hvor V er den volumenet af det produkt, der er beregnet til administration), 0,2 EU/kg, henholdsvis4. Flere detaljer om den pyrogen tærskeldosis for forskellige lægemidler og enheder leveres og diskuteret andetsteds4,5,6.
Dyr varierer meget i deres følsomhed over for endotoxin-medierede reaktioner. Mennesker, ikke-menneskelige primater og kaniner er blandt de mest ekstremt følsomme over for endotoksiner3arter. For at undgå endotoxin-medieret bivirkninger hos patienter og forebygge urigtige konklusioner af prækliniske toksicitet og effekt studier, er det nødvendigt at præcist at detektere og kvantificere endotoksiner i både kliniske og prækliniske grade formuleringer. Flere i øjeblikket tilgængelige metoder kan opnå denne opgave. En af dem er Limulus Amoebocyte Lysate (LAL) analysen, som er almindeligt anvendt over hele verden til skærmen biomedicinske produkter for den potentielle endotoxin forurening samt at opdage bakterieinfektioner7,8,9. Den lysate er fremstillet af amoebocytes, cellerne i blod fra horseshoe krabber Limulus Polyfem bosiddende i øst-kysten af det europæiske kontinent Nordamerika7. Interessant, der er et par forskellige arter af horseshoe krabber (Tachypleus gigas og Tachypleus tridentatus) i Asien10. Tachypleus Amoebocyte Lysate (TAL) er brugt i flere asiatiske lande til påvisning af endotoxin svarende til hvordan LAL bruges i andre cuntries10. Lysates (LAL og TAL) indeholder en gruppe af proteiner, der ved aktivering giver protease aktivitet. En af disse proteiner, den såkaldte faktor C aktiveres ved kontakt med endotoxin. Aktiveret faktor C kløver Factor B, som til gengæld også bliver en protease og kløver et pro-koagulation enzym til at producere en blodpropper enzym. Resultatet af denne kæde af reaktioner er dannelsen af en gel, en stigning i stikprøven Turbiditet og i overværelse af et kromogent substrat, udseendet af en farvet produkt, der tjener som et fundament for gel-blodprop, turbiditet og kromogent assays, henholdsvis. Mens der er ingen obligatoriske LAL format, US Food and Drug Administration (FDA) forklarer i retningslinjer for baseret industrien dokument, at beslutningen om foretages i tilfælde af uoverensstemmelse i testresultater mellem forskellige LAL formater, på gel-blodprop assay5 .
Mange almindeligt anvendte laboratoriekemikalier (fx., EDTA) og kendte lægemiddel produkter (fx penicillin) forstyrrer LAL-undersøgelser,11. Indblanding identificeres normalt ved at vurdere inddrivelse af endotoxin standard aks på en kendt koncentration i en løsning, der indeholder testmateriale. Hvis spike opsving er mindre end 50% eller mere end 200%, så resultatet af LAL assay for er den givne prøvemateriale ugyldig på grund af hæmning eller ekstraudstyr, henholdsvis4. Nanoteknologi-baserede formuleringer er ofte komplekse og forstyrre LAL gennem en række mekanismer12,13,14. Mange tilgange er blevet beskrevet til at overvinde indblanding: prøve rekonstituering i specifikke buffere og overfladeaktive stoffer, protein inaktiveres ved opvarmning, ødelæggelse af lipid-baserede hule materialer ved opvarmning og supplere prøve med overskud divalent kationer5,12,13,14,15. Alternative metoder til situationer, hvor LAL indblanding ikke kan overvindes er også blevet beskrevet: ELISA, en HEK-TLR4 reporter celle line assay og massespektrometri16,17,18, 19.
Heri, er eksperimentelle procedurer for udførelse af gel-blodprop, turbiditet og kromogent LAL assays beskrevet. Disse analyser er også tilgængelige på nanoteknologi karakterisering Lab (NCL) hjemmeside20 i protokoller STE1.2 (turbiditet LAL), STE1.3 (gel-blodprop LAL) og STE1.4 (kromogent LAL). Det anbefales at foretage mindst to forskellige formater for at karakterisere den samme nano-formulering. Når resultaterne af Turbiditet og kromogent LAL er uenige, anses gel-blodprop resultater5. Når resultaterne af to LAL formater er uenige, gennemført yderligere undersøgelser, enten monocyt aktivering test (MAT) eller kanin pyrogen test (RPT) til at kontrollere LAL resultater er21. Det er vigtigt at bemærke, at hver metode bruges til registrering af endotoxin og sikkerhedsforsøg vurdering har fordele og begrænsninger21,22,23,24. Erkendelse af begrænsninger af den procedure, der anvendes til at karakterisere en given nanoteknologi formulering er afgørende for at opnå videnskabelig begrundelse for anvendelse af proceduren for optimal for at nano-formulering.
I denne undersøgelse, blev pegyleret liposomal doxorubicin brugt som en model nanopartikel formulering. Denne formulering er godkendt af det amerikanske FDA i 1995 og anvendes til behandling af kræft patienter på verdensplan25.

<table>
	<tbody>
		<tr>
			<td>Turbidity LAL Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S2770</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H9892</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>LAL Reagent</td>
			<td>Associates of Cape Cod</td>
			<td>T0051</td>
			<td>This reagent can be used with turbidity assay only</td>
		</tr>
		<tr>
			<td>Control Endotoxin Standard</td>
			<td>Associates of Cape Cod</td>
			<td>E0005</td>
			<td>This reagent can be used with turbidity and gel-clot assays</td>
		</tr>
		<tr>
			<td>LAL grade water</td>
			<td>Associates of Cape Cod</td>
			<td>WP0501</td>
			<td>This reagent can be used with any LAL format</td>
		</tr>
		<tr>
			<td>Glucashield Buffer</td>
			<td>Associates of Cape Cod</td>
			<td>GB051-25</td>
			<td>Used to prevent false-positive response from beta-glucans</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TB240</td>
			<td>These tubes can be used with all three assays</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass reaction tubes 8 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TK100</td>
			<td>These tubes can be used with turbidity and chromogenic assays</td>
		</tr>
		<tr>
			<td>Pyrogen-free tips with volumes 0.25 and 1.0 mL</td>
			<td>RAININ</td>
			<td>PPT25, PPT10</td>
			<td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td>
		</tr>
		<tr>
			<td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td>
			<td>Eppendorf</td>
			<td>22600044</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Pyrogen-fee combitips, 5mL</td>
			<td>Eppendorf</td>
			<td>30089669</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Repeat pipettor</td>
			<td>Eppendorf</td>
			<td>4982000020</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Microcetrifuge</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Refrigerator, 2-8 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Freezer, -20 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Pyros Kinetix or Pyros Kinetix Flex reader</td>
			<td>Associates of Cape Cod</td>
			<td>PKF96</td>
			<td>Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions</td>
		</tr>
		<tr>
			<td>Chromogenic LAL Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrochrome LAL Reagent</td>
			<td>Associates of Cape Cod</td>
			<td>CG1500-5</td>
			<td>This reagent is specific to the Chromogenic Assay</td>
		</tr>
		<tr>
			<td>Control Endotoxin Standard</td>
			<td>Associates of Cape Cod</td>
			<td>EC010</td>
			<td>This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S2770</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H9892</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>LAL grade water</td>
			<td>Associates of Cape Cod</td>
			<td>WP0501</td>
			<td>This reagent can be used with any LAL format</td>
		</tr>
		<tr>
			<td>Glucashield Buffer</td>
			<td>Associates of Cape Cod</td>
			<td>GB051-25</td>
			<td>Used to prevent false-positive response from beta-glucans</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TB240</td>
			<td>These tubes can be used with all three assays</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass reaction tubes 8 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TK100</td>
			<td>These tubes can be used with turbidity and chromogenic assays</td>
		</tr>
		<tr>
			<td>Pyrogen-free tips with volumes 0.25 and 1.0 ml</td>
			<td>RAININ</td>
			<td>PPT25, PPT10</td>
			<td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td>
		</tr>
		<tr>
			<td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td>
			<td>Eppendorf</td>
			<td>22600044</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Pyrogen-fee combitips, 5mL</td>
			<td>Eppendorf</td>
			<td>30089669</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Repeat pipettor</td>
			<td>Eppendorf</td>
			<td>4982000020</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Microcetrifuge</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Refrigerator, 2-8 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Freezer, -20 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Pyros Kinetix or Pyros Kinetix Flex reader</td>
			<td>Associates of Cape Cod</td>
			<td>PKF96</td>
			<td>Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions</td>
		</tr>
		<tr>
			<td>Gel-Clot LAL Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LAL Reagent</td>
			<td>Associates of Cape Cod</td>
			<td>G5003</td>
			<td>This reagent is specific to the gel-clot assay</td>
		</tr>
		<tr>
			<td>Control Endotoxin Standard</td>
			<td>Associates of Cape Cod</td>
			<td>E0005</td>
			<td>This reagent can be used with turbidity and gel-clot assays</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S2770</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma</td>
			<td>H9892</td>
			<td>When needed, it is used to adjust sample pH to be between 6-8</td>
		</tr>
		<tr>
			<td>LAL grade water</td>
			<td>Associates of Cape Cod</td>
			<td>WP0501</td>
			<td>This reagent can be used with any LAL format</td>
		</tr>
		<tr>
			<td>Glucashield Buffer</td>
			<td>Associates of Cape Cod</td>
			<td>GB051-25</td>
			<td>Used to prevent false-positive response from beta-glucans</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TB240</td>
			<td>These tubes can be used with all three assays</td>
		</tr>
		<tr>
			<td>Disposable endotoxin-free glass reaction tubes 10 x 75 mm</td>
			<td>Associates of Cape Cod</td>
			<td>TS050</td>
			<td>These tubes are for use with the gel-clot assay</td>
		</tr>
		<tr>
			<td>Pyrogen-free tips with volumes 0.25 and 1 mL</td>
			<td>RAININ</td>
			<td>PPT25, PPT10</td>
			<td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td>
		</tr>
		<tr>
			<td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td>
			<td>Eppendorf</td>
			<td>22600044</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Pyrogen-fee combitips, 5mL</td>
			<td>Eppendorf</td>
			<td>30089669</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Repeat pipettor</td>
			<td>Eppendorf</td>
			<td>4982000020</td>
			<td>Other equivalent supplies can be used</td>
		</tr>
		<tr>
			<td>Microcetrifuge</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Refrigerator, 2-8 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Freezer, -20 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Water bath, 37 C</td>
			<td>any brand</td>
			<td></td>
			<td>Any brand can be used, however, it is important either to switch off water circulation or use non-circualting water bath because water flow will affect clot formation and lead to false-negative results</td>
		</tr>
	</tbody>
</table>

detection of endotoxin in nano-formulations using limulus amoebocyte lysate (lal) assays

Når de findes i farmaceutiske produkter, en Gram-negative bakterielle cellevæg komponent endotoxin (ofte også kaldet LPS) kan forårsage betændelse, feber, hypo- eller hypertension, og i ekstreme tilfælde kan føre til væv og orgel skader, der kan blive fatal. Mængder af endotoxin i farmaceutiske produkter, derfor er strengt reguleret. Blandt de metoder, der er til rådighed for endotoxin påvisning og kvantificering, er Limulus Amoebocyte Lysate (LAL) assay almindeligt anvendt over hele verden. Mens enhver farmaceutisk produkt, der kan forstyrre LAL assay, udgør nano-formuleringer en særlig udfordring på grund af deres kompleksitet. Formålet med dette dokument er at give en praktisk vejledning for forskere uerfarne i estimeringen af endotoksiner i nanomaterialer og nanopartikel-formuleret narkotika. Heri, diskuteres praktiske anbefalinger til at udføre tre LAL formater herunder turbiditet, kromogent og gel-blodprop assays. Disse assays kan bruges til at bestemme endotoxin forurening i nanoteknologi-baserede lægemidler, vacciner og adjuvanser.

Eksempel på data genereret efter afprøvning denne formulering i LAL assays er vist i tabel 1. Pegyleret liposomal doxorubicin blandede sig med kromogent LAL ved fortynding 5. Men denne indgriben blev overvundet af større fortyndinger. Spike opsving var mellem 50 og 200%, da denne formulering blev testet på fortyndinger 50 og 500 i Turbiditet og kromogent LAL samt på fortynding 5 i turbiditet LAL. Når justeret med fortyndingsfaktoren, var resultaterne konsekvent mell...

Watch this Scientific Journal Video about Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays at JoVE.com

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Påvisning af endotoksiner i industrielt fremstillet nanomateriale repræsenterer en af de store udfordringer inden for Nanomedicin. Vi præsenterer her, et casestudie, der beskriver rammerne består af tre forskellige LAL formater til at vurdere potentielle endotoxin forurening i nanopartikler.

Påvisning af Endotoxin i Nano-formuleringer bruger Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Denne metode kan hjælpe med at besvare på det medicinske område om sikkerheden af nanomedicin. Fordelene ved denne teknik er, at det er standard, og bruges over hele verden til evaluering af produkter, Til nano medicin. Demonstration af proceduren, vil blive Barry Neun, på Nano Technology Karakterisering Lab.Til kalibreringsstandardforberedelse anvendes 900 mikroliter limulus amebocyt lysat eller LAL-vand. Og 100 mikroliter af kontrolstandardenoksin, der skal forberede så mange mellemliggende fortyndinger som nødvendigt for at muliggøre fremstilling af en kalibreringsstandard med en koncentration af en endoksinenhed pr. milliliter. Derefter ved hjælp af 900 mikroliter lal-kvalitet vand, og 100 mikroliter af den ene endoksin enhed pr milliliter kalibrering standard, forberede en anden kalibrering standard, ved en koncentration på 0,1 endotoxin enhed per milliliter.Gentag derefter den serielle tidobbelte fortynding for at forberede to lavere kalibreringsstandarder. For at opnå i alt fire kalibreringsstandarder, der spænder fra 0001 til en endoksinenhed pr. milliliter. For at forberede en 05 endotoksin enhed pr. milliliter kvalitetskontrol, kombinerer 50 mikroliter af den ene endoksin enhed pr milliliter kontrol standard endoksin opløsning, med 950 mikroliter af LAL grade vand.Næste i softwaren skal du vælge instrumentindstillingerne, og oprette skabelonen. Vælg Indsamle data, og angiv test-id'et og datagruppen under fanen Generelt. Vælg instrumenttypen og LAL-metoden under fanen Hardware, og kontroller, at der vises et serienummer, system-id'et og de serielle portoplysninger på skærmen.Klik på okay to gange for at bekræfte instrumentets valg og kommunikationen med instrumentet, og indtast eksempel-id'et i samme rækkefølge, som prøverne vil blive testet. Brug derefter standardknapperne til at angive standardkurven for negativ kontrol og teste prøver. Der tilsættes en passende mængde negative kontrolkalibreringsstandarder og kvalitetskontrol i dublerede formærkede glasrør.Og tilføje den passende mængde LAL til den første test modbydelige. Vortex den modbydelige kort, og placere hætteglasset i de relevante test slots i instrumentet karrusel. Før prøverne indlæses, udføres der flere fortyndinger af testnanomaterialet inden for den maksimale gyldige fortynding som påvist.For at forberede en 05 endotoksinenhed pr. milliliter hæmningsforbedringskontrol skal der kombineres 25 mikroliter af den ene endoksinenhed pr. milliliter kontrolstandardenoksinopløsning med 475 mikroliter af testnanomaterialet ved en given fortynding. Efter vortexing indlæses de relevante styringskontroller til innovationsforbedring og de uspikerede undersøgelsesprøver i instrumentet. Derefter aliquot uspiked og de spidse nano materialer på en bestemt fortynding i præmærkede rør.Før vortexing, og lastning i instrumentet karrusel. For at udføre en Gel-Clot LAL, forberede kontrol standard endoksin til en endelig koncentration af fire lambda, og kombinere 100 mikroliter af standard, med 100 mikroliter vand, eller prøve for at opnå en endelig koncentration af kontrol standard endoksin to lambda. Tilsæt 100 mikroliter lysin til hver lambda fortynding.Vortex rørene kort, og placere rack af rør i en 37 grader Celsius vandbad i en time. Ved slutningen af inkubationen, invertere rørene med en jævn bevægelse, og manuelt registrere resultaterne ved hjælp af plus for en fast blodprop, og minus for ingen blodprop, eller en løs blodprop. I denne repræsentative analyse forstyrrede PEGylated liposomal doxorubicin krokron LAL ved fortynding fem, men denne interferens blev overvundet ved højere fortyndinger.Spike opsving var mellem 50 og 200%, når formuleringen blev testet ved fortyndinger 50 og 500 i turbiditet og kromogen LAL, samt ved fortynding fem, i turbiditet LAL. Når justeret af udvanding faktor, resultaterne var konsekvent mellem vrangforestillinger i begge essays. Desuden var resultaterne konsistente mellem de tre essayformater.Husk, at hver prøve har sit eget sortiment af fortyndinger, hver LAL format bruger sin kontrol standard i doxin og lysate, og at rørene bruges til at forberede fortyndinger, og til at udføre en reaktion på forskellige for hver LAL essay. Til denne procedure andre metoder som Kan udføres for at besvare yderligere spørgsmål om materiale Efter dens udvikling banede denne teknik vejen for suturer og inden for nanomedicin, for at udforske de medicinske anvendelser af nanoteknologi formulerede lægemidler hos mennesker og kliniske modeller, herunder men ikke begrænset til ikke-menneskelige primater, kaniner, hunde og rotter. Glem ikke, at arbejde med doxin kan være yderst farligt, og de forholdsregler, såsom at bære personlige værnemidler, bør altid tages, mens du udfører denne procedure.

Research

JoVE Journal

Bioengineering

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Påvisning og isolation af cirkulerende melanom celler ved hjælp af Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

Bakteriel Detection og identifikation Brug elektrokemiske sensorer

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

Anatomiske rekonstruktioner af Human Cardiac Venøs System ved hjælp Kontrast-computertomografi af Perfusion-faste Prøver

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Bead Aggregation Analyser til karakterisering af formodede celleadhæsionsmolekyler

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Øjenirritation Test (EIT) for Hazard Identifikation af Eye Irriterer Kemikalier hjælp rekonstrueret human Cornea-lignende Epithelial (RHCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Label-fri Single Molecule Detection Brug Microtoroid Optiske Resonatorer

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Multicolor Fluorescens Detection for Droplet Microfluidics Brug af optiske fibre

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...