Name: generation of murine monoclonal antibodies by hybridoma technology
Uploaded: 2017-01-02T12:30:00
Description: Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

The authors acknowledge the German Federal Ministry of Education and Research (BMBF, Grant No: 03IPT7030X, 03IPT703A, and 03IP703) for funding our projects, "Artificial immune reactions," "Camelid antibodies," and "Antibody technologies." We thank Prof. Burkhard Micheel for proofreading the manuscript and for the helpful comments.

Balb / c eller NMRI-mus (Mus musculus) fra vores ynglekoloni ved universitetet i Potsdam (Potsdam, Tyskland) blev anvendt til fremstilling af monoklonale antistoffer. Dyret arbejde blev udført i overensstemmelse med relevante nationale og internationale retningslinjer. Undersøgelsen blev godkendt af Brandenburg Miljøministeriet, Sundhed og Forbrugerbeskyttelse (referencenummer V3-2347-A16-4-2012). 1. Forberedelse af immunokonjugater <ol><li> Til kobling af antigenet til en bær...

<ol>
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Frembringelsen af ​​monoklonale antistoffer ved hybridomteknologi kræver en intens og detaljeret epitop analyse, navnlig med hensyn til den endelige anvendelse, når antistoffet bør anerkende målet. Dette er ofte undervurderet af brugere og fører til antistoffer med svage præstationer. Fusionsprocessen er altid tilfældig, hvilket betyder, at resultatet af specifikke hybridomaer er meget afhængige af den celle-forhold og vitaliteten på dette punkt. Efter begrænset fortynding, cellerne er meget ustabile og kr...

Den hybridomteknologi præsenteres i denne protokol blev først beskrevet af Köhler og Milstein 1 i 1975 og med undtagelse af nogle tekniske forbedringer, har den vigtigste procedure ikke ændret sig dramatisk i løbet af de sidste 40 år 2. Formålet med denne protokol er at forklare et mere passende immunisering strategi, en standard fremgangsmåde til generering af monoklonale antistoffer, og et eksempel for en validering fremgangsmåde (ELISA). Antistoffer er utrolig værktøjer og bidrager til en bred vifte af teknologiske metoder, såsom flowcytometri, magnetisk cellesortering, eller immunfluorescens, samt til diagnostiske og terapeutiske muligheder for overvågning og behandling 3 sygdom. Den kommercielle tilgængelighed af monoklonale antistoffer for ønskede mål påvises ved tilstedeværelsen af flere end 24 forskellige web databaser med næsten utallige mængder antistoffer eller antistof-relaterede produkter 4. I 2015, etntibody molekyler var en del af en international diskussion 5-7 på grund af alvorlige problemer i ordentlig validering og karakterisering af kommercielt tilgængelige antistoffer. Det kan være vanskeligt og dyrt at finde specifikke antistoffer for et målrettet antigen, og ofte har de ikke har behov for affiniteten eller specificiteten. Selvom generere et antistof er stadig tidskrævende og kræver uddannet personale til at udvikle og validere antistoffet, der producerer et antistof individuelt måske bedre end at købe en. På grund af det faktum, at antistofproduktion er tidskrævende og kræver erfaring, blev alternative metoder til fremstilling af bindingsmolekyler udviklet for at overvinde disse problemer. Den mest almindeligt anvendte alternativ metode er den rekombinante produktion af enkeltkædede antistoffer via fag-display. Generne for den variable bindingsregion er ekstraheret fra celler og kombineret med belægningen proteinet af en fag. sIngle kæde udtrykkes derefter på overfladen af en bakteriofag og screenet i flere panorering 8 trin. Produktionen af ​​single-antistoffer er en smule hurtigere, men det kræver også en dygtig eksperimentator. Ulemperne ved nogle rekombinante enkeltkædede antistoffer er dårlige stabilitet og manglende egnethed til in vitro diagnostik. I de fleste diagnostiske test, er det nødvendigt med en Fc-receptor for påvisning, som skal tilsættes til en rekombinant enkeltkædet antistof bagefter. Igen er dette tidsrøvende og endnu mere kompleks end hybridomteknikken. I in vitro-diagnostik, har fuld længde monoklonale muse- og kanin-antistoffer blevet påvist at være det bedste valg. Et af de store skridt i at generere monoklonale antistoffer skal gøres, før arbejdet i laboratoriet starter: design af immunokonjugatet. Spørgsmål, der skal behandles, er: Hvad er den fysiske sammensætning af målet i den endelige APPLICATion, og hvilke matricer er til stede? Hvilken koncentration vil målet har i ansøgningen? Hvad er den endelige ansøgning, og hvad er kravene, at antistoffet skal opfylde? tage altid hensyn til, at hvis der anvendes et lineært peptid fragment, har det også at være lineær i den endelige epitop i målet af valg; ellers vil antistoffet ikke binde. Selvfølgelig, uafhængig af screeningsmetode, kunne udvælges antistoffer til at genkende forskellige antigene formater i forskellige programmer, men dette skal valideres meget præcist. Disse er grundene til, at antistof-udvikling og validering er så ambitiøse processer. Valget af den antigene format til immunisering er grundlæggende for antistofudvikling og afgørende for succes eller fiasko af denne proces. Når mus udtrykker et relevant antistoftiter miltcellerne isoleres og fusioneres med myelomaceller. De mest almindelige myeloma- cellelinier tilmurint monoklonalt antistof udvikling er X63-Ag 8.653 9 og Sp2 / 0-Ag 14 10 fra en Balb / c-mus stamme. Cellerne ned fra en malign B-celle-lymfom og blev udvalgt, fordi de ikke udskiller nogen af ​​deres egne tunge eller lette kæder. Cellerne kan tilpasses til et forhold mellem 1:10 og 10: 1 (splenocytter versus myelomaceller). I denne protokol blev cellerne tilpasset til et forhold på 3: 1 og fusioneres ved polyethylenglycol (PEG) og elektrosvejsning, ifølge Stoicheva og Hui 11. Fusion af B-celler og myelomaceller er en tilfældig proces. Derfor kunne hybrider af to B-lymfocytter eller to myelomceller blive genereret, men de hybrider ville ikke være i stand til at overleve i lang tid i kultur. Cellerne underkastes en hypoxanthin, aminopterin og thymidin (HAT) udvælgelse, ved hvilken kun fusionerede hybridomceller kan overleve på grund af muligheden for at anvende de novo vejen for pyrimidin-syntese. Til generering af monoclonal antistoffer, er det nødvendigt at opnå en cellelinie, der stammer fra en mor celle. Den monoklonalitet sikres ved at begrænse fortyndingsteknikker og den mikroskopiske analyse af cellevækst. De hybridoma kultursupernatanter screenes for specifik antistofproduktion, for det meste ved ELISA eller flowcytometri, og de bedste bindere vælges. Efter massekultur og oprensning, kan antistofmolekylet endelig karakteriseres og valideret til den ønskede anvendelse.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">glutaraldehyde</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">G5882</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC)</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">39391-10ML</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Sulfo-GMBS</td>
			<td style="width:81px;">Perbio Science Germany</td>
			<td style="width:136px;">22324</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">ovalbumin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A5503</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">bovine serum albumin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A2153</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">keyhole limpet hemocyanin</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">H8283</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Falcon tubes 15 mL</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P91015</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">reaction vials, 1.5 mL</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">CNT2.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">hollow needle</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">C724.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">glass wool</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">6574.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Sephadex G25 coarse</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">GE-17-0034-02</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Freund&#180;s adjuvant, complete</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">F5881-10ML</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">ELISA plates, 96 well</td>
			<td style="width:81px;">Greiner bio-one</td>
			<td style="width:136px;">655101</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">neonatal calf serum</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">S1025</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">TipOne Tips 1000 &#181;L</td>
			<td style="width:81px;">Starlab</td>
			<td style="width:136px;">S1111-2021</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Pipette tips 200 &#181;L</td>
			<td style="width:81px;">Greiner bio-one</td>
			<td style="width:136px;">739291</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">HRP-conjugated goat-anti-mouse IgG antibody</td>
			<td style="width:81px;">Dianova</td>
			<td style="width:136px;">115-035-003</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">tetramethylbenzidine</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">6350.2</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">Natriumdihydrogenphosphat</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">K300.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">peroxide/urea</td>
			<td style="width:81px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">sulphuric acid</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">4623.3</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">RPMI 1640</td>
			<td style="width:81px;">Life technologies GmbH</td>
			<td style="width:136px;">31870074</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">L-glutamine</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">HN08.2</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">beta-mercaptoethanol</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:247px;">fetal calf serum</td>
			<td style="width:81px;">Invitrogen</td>
			<td style="width:136px;">10270106</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-flask 25 cm2</td>
			<td style="width:81px;">Peske GmbH</td>
			<td style="width:136px;">86-V025</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-flask 75 cm2</td>
			<td style="width:81px;">Peske GmbH</td>
			<td style="width:136px;">86-V075</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">ethanol, 96%</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">P075.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">cell strainer</td>
			<td style="width:81px;">VWR international</td>
			<td style="width:136px;">734-0002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Falcon tubes 50 mL</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P91050</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">PEG 8000</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">1546605</td>
			<td></td>
		</tr>
		<tr height="97">
			<td height="97" style="height:97px;width:247px;">electroporation cuvette, 2mm</td>
			<td style="width:81px;">Biodeal Handelsvertretung Edelmann e.K.</td>
			<td style="width:136px;">EKL2,25</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">hypoxanthine</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">H9636-25G</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">azaserine</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">A4142</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">thymidine</td>
			<td style="width:81px;">USB Europa GmbH</td>
			<td style="width:136px;">22305 1 GM</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-plates 96 well</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P92696</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">TC-plates 24 well</td>
			<td style="width:81px;">Biochrom GmbH</td>
			<td style="width:136px;">P92424</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">cryotubes, 1mL</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">V7384-1CS</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">dimethylsulfoxid</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">4720.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">protein A sepharose</td>
			<td style="width:81px;">Sigma Aldrich</td>
			<td style="width:136px;">P3391-1G</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">SDS sample loading buffer, Roti-Load 1</td>
			<td style="width:81px;">Carl Roth GmbH &#38; C0.KG</td>
			<td style="width:136px;">K929.1</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">unstained protein ladder</td>
			<td style="width:81px;">BioRad Laboratories</td>
			<td style="width:136px;">161-0363</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:247px;">comassie brilliant blue R-250</td>
			<td style="width:81px;">BioRad Laboratories</td>
			<td style="width:136px;">161-0406</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of murine monoclonal antibodies by hybridoma technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Figur 1 viser et eksempel på antigen-specifikke serum titer for en immunisering udført i laboratoriet. I denne figur blev immunsera A og B titreret fra 1:50 til 1: 5.000.000 i sammenligning med en serum fra en naiv mus. Antigenet blev belagt på den faste fase, og de specifikke antistoffer blev påvist med et POD-konjugeret gede-anti-muse-Ig-antistof. Både sera af de immuniserede mus viste en signifikant højere antigenspecifik titer sammenlignet med naive mus. Signal...

Watch this Scientific Journal Video about Generation of Murine Monoclonal Antibodies by Hybridoma Technology at JoVE.com

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Frembringelse af murine monoklonale antistoffer ved hybridomteknologi

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Research

JoVE Journal

Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Påvisning af Invasiv pulmonal aspergillose hos maligne hæmatologiske lidelser patienter ved hjælp af lateral strømning og Teknologi

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Fremstilling af rekombinant humant IgG monoklonale antistoffer fra Immortaliserede Sorteret B-celler

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generation af undslippe varianter af neutraliserende Influenza Virus monoklonale antistoffer

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

En metode til at vurdere Fc-medieret effektor funktion induceret af Influenza Hemagglutinin specifikke antistoffer

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Generation af diskriminerende menneskelige monoklonale antistoffer fra sjældne Antigen-specifikke B celler cirkulerer i blodet

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

Generation af monoklonale antistoffer mod naturprodukter

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antigene Liposomer for Generation af sygdoms-specifikke antistoffer

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...