Name: generation of murine monoclonal antibodies by hybridoma technology
Uploaded: 2017-01-02T12:30:00.000+00:00
Duration: 9 min 42 s
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.

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The authors Pamela Holzl&#246;hner and Katja Hanack are the co-founders of new/era/mabs GmbH, a company that offers the customized generation of monoclonal antibodies.

توليد الأجسام المضادة وحيدة النسيلة من ورم هجين يتطلب تحليلا مكثفة ومفصلة حاتمة، وخاصة فيما يتعلق بتطبيق النهائي، عندما ينبغي أن تعترف الضد المستهدف. في كثير من الأحيان التقليل من شأنها من قبل المستخدمين ويؤدي إلى الأجسام المضادة مع ضعف الأداء. عملية الانصهار دائما...

تقنية هجين الواردة في هذا البروتوكول وصفت لأول مرة من قبل كولر وميلشتاين 1 في عام 1975، وباستثناء بعض التحسينات التقنية، الإجراء الرئيسي لم يتغير بشكل كبير خلال السنوات ال 40 الماضية 2. والهدف من هذا البروتوكول هو شرح استراتيجية أكثر ملائمة للتحصين، طريقة موحدة لتوليد الأجسام المضادة وحيدة النسيلة، ومثال لطريقة التحقق من صحة (ELISA). الأجسام المضادة هي أدوات لا يصدق وتسهم في مجموعة واسعة من الأساليب التكنولوجية، مثل التدفق الخلوي، فرز الخلايا المغناطيسي، أو المناعي، فضلا عن خيارات التشخيصية والعلاجية لرصد وعلاج 3 المرض. وتتجلى التوافر التجاري من الاجسام المضادة للأهداف المرجوة من وجود أكثر من 24 قواعد بيانات الويب المختلفة مع كميات لا حصر لها تقريبا من الأجسام المضادة أو المنتجات ذات الصلة الأجسام المضادة 4. في عام 2015، لوكانت جزيئات ntibody جزءا من المناقشات الدولية 5-7 بسبب مشاكل خطيرة في التحقق من صحة الصحيح وتوصيف الأجسام المضادة المتاحة تجاريا. ويمكن أن يكون صعبا ومكلفا للعثور على أجسام مضادة محددة للمستضد المستهدفة، وكثير من الأحيان، لم يكن لديهم تقارب أو خصوصية حاجة. على الرغم من توليد الأجسام المضادة لا تزال تستغرق وقتا طويلا ويتطلب الموظفين المهرة لتطوير والتحقق من صحة الأجسام المضادة، وإنتاج الأجسام المضادة بشكل فردي قد أفضل من شراء واحدة. يرجع ذلك إلى حقيقة أن إنتاج الأجسام المضادة وتستغرق وقتا طويلا ويتطلب خبرة، وتطوير أساليب بديلة لإنتاج جزيئات ملزمة للتغلب على هذه المشاكل. الطريقة البديلة الأكثر شيوعا هو إنتاج المؤتلف من الأجسام المضادة سلسلة واحدة عن طريق عرض فج. يتم استخراج الجينات في المنطقة ملزمة متغير من الخلايا وجنبا إلى جنب مع البروتين طلاء من فج. لياليثم أعرب إنجل السلسلة على سطح الجراثيم وعرض في أكثر من بالغسل 8 خطوات. إنتاج الأجسام المضادة سلسلة واحدة هو أسرع قليلا، ولكنه يتطلب أيضا التجريبي المهرة. مساوئ بعض الأجسام المضادة سلسلة واحدة المؤتلف والاستقرار الفقراء وعدم صلاحيتها للتشخيص في المختبر. في معظم الاختبارات التشخيصية، ومستقبلات التيسير للكشف ضروري، والتي تحتاج إلى أن تضاف إلى الأجسام المضادة سلسلة واحدة المؤتلف بعد ذلك. مرة أخرى، وهذا هو مضيعة للوقت وأكثر تعقيدا من تقنية هجين. في التشخيص في المختبر، وقد أظهرت كامل طول وحيدة النسيلة الماوس والأرنب الأجسام المضادة ليكون أفضل خيار. ويجب أن يتم واحدة من الخطوات الرئيسية في توليد الأجسام المضادة وحيدة النسيلة قبل العمل في المختبر يبدأ: تصميم immunoconjugate. الأسئلة التي تحتاج إلى التعامل معها هي: ما هو التكوين الجسمي لهدف في تطبيق و النهائيأيون، والتي المصفوفات هي موجودة؟ التي تركز فان الهدف يكون في التطبيق؟ ما هو التطبيق النهائي، وما هي الشروط التي يجب أن تتوفر الأجسام المضادة؟ تأخذ دائما بعين الاعتبار أنه إذا تم استخدام الببتيد شظية الخطي، كما أن لديها أن تكون خطية في حاتمة النهائية في الهدف من الاختيار؛ على خلاف ذلك، فإن الأجسام المضادة لا يلزم. وبطبيعة الحال، بغض النظر عن طريقة الفحص، يمكن اختيار الأجسام المضادة ليتعرف على تنسيقات مختلفة مولدة في تطبيقات مختلفة، ولكن هذا يجب أن يتم التحقق من صحة بدقة متناهية. هذه هي الأسباب التي تطور الأجسام المضادة والمصادقة عليها هي تلك العمليات الطموحة. اختيار شكل الأنتيجين للتحصين أمر أساسي لتطوير الأجسام المضادة ويحدد نجاح أو فشل هذه العملية. وبمجرد أن الفئران تعبر عن الأجسام المضادة ذات الصلة، وعزل خلايا الطحال وتنصهر مع خلايا المايلوما. خطوط الخلايا المايلوما الأكثر شيوعا لالفئران تطوير الأجسام المضادة وحيدة النسيلة هي X63-حج 8.653 9 و SP2 / 0-AG 14 10 من / ج الماوس سلالة BALB. الخلايا تنحدر من خبيث سرطان الغدد الليمفاوية الخلية البائية واختيرت لأنها لا تفرز أي من السلاسل الثقيلة أو الخفيفة الخاصة بها. الخلايا يمكن أن تتكيف مع نسبة بين 1:10 و 10: 1 (splenocytes مقابل خلايا الورم النقوي). في هذا البروتوكول، تم تكييف الخلايا إلى نسبة 3: 1 وتنصهر التي كتبها البولي ايثيلين جلايكول (PEG) والكهربائي، وفقا لStoicheva وهوى 11. اندماج الخلايا البائية والخلايا المايلوما هو عملية عشوائية. لذلك، الهجينة من اثنين ب الخلايا الليمفاوية أو اثنين خلايا المايلوما يمكن أن تتولد، ولكن تلك الهجينة لن تكون قادرة على البقاء على قيد الحياة لفترة طويلة في الثقافة. الخلايا الخضوع لاختيار هيبوزانتين، أمينوبترين، والثيميدين (HAT)، الذي فقط خلايا هجين تنصهر يمكن البقاء على قيد الحياة نظرا لإمكانية استخدام دي نوفو مسار تخليق بيريميدين. لتوليد مونالأجسام المضادة oclonal، فمن الضروري للحصول على خط الخلية التي تنشأ من خلية أم واحدة. يتم ضمان monoclonality عن طريق الحد من تقنيات التخفيف والتحليل المجهري للنمو الخلايا. يتم فحص وsupernatants ثقافة هجين لإنتاج الأجسام المضادة المحددة، ومعظمهم من ELISA أو قياس التدفق الخلوي، ويتم اختيار أفضل المواد اللاصقة. بعد الثقافة الجماهيرية وتنقية، ويمكن للجزيء الضد أخيرا أن توصف والتحقق من صحتها عن التطبيق المطلوب.

<table><tbody><tr><td>glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&lt;/em&gt;-ethylcarbodiimide (EDC)</td><td>Sigma Aldrich</td><td>39391-10ML</td><td></td></tr><tr><td>Sulfo-GMBS</td><td>Perbio Science Germany</td><td>22324</td><td></td></tr><tr><td>ovalbumin</td><td>Sigma Aldrich</td><td>A5503</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma Aldrich</td><td>A2153</td><td></td></tr><tr><td>keyhole limpet hemocyanin</td><td>Sigma Aldrich</td><td>H8283</td><td></td></tr><tr><td>Falcon tubes 15 mL</td><td>Biochrom GmbH</td><td>P91015</td><td></td></tr><tr><td>reaction vials, 1.5 mL</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>CNT2.1</td><td></td></tr><tr><td>hollow needle</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>C724.1</td><td></td></tr><tr><td>glass wool</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6574.1</td><td></td></tr><tr><td>Sephadex G25 coarse</td><td>Sigma Aldrich</td><td>GE-17-0034-02</td><td></td></tr><tr><td>Freund&amp;acute;s adjuvant, complete</td><td>Sigma Aldrich</td><td>F5881-10ML</td><td></td></tr><tr><td>ELISA plates, 96 well</td><td>Greiner bio-one</td><td>655101</td><td></td></tr><tr><td>neonatal calf serum</td><td>Biochrom GmbH</td><td>S1025</td><td></td></tr><tr><td>TipOne Tips 1,000 &amp;micro;L</td><td>Starlab</td><td>S1111-2021</td><td></td></tr><tr><td>Pipette tips 200 &amp;micro;L</td><td>Greiner bio-one</td><td>739291</td><td></td></tr><tr><td>HRP-conjugated goat-anti-mouse IgG antibody</td><td>Dianova</td><td>115-035-003</td><td></td></tr><tr><td>tetramethylbenzidine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>6350.2</td><td></td></tr><tr><td>Natriumdihydrogenphosphat</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K300.2</td><td></td></tr><tr><td>peroxide/urea</td><td></td><td></td><td></td></tr><tr><td>sulphuric acid</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4623.3</td><td></td></tr><tr><td>RPMI 1640</td><td>Life technologies GmbH</td><td>31870074</td><td></td></tr><tr><td>L-glutamine</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>HN08.2</td><td></td></tr><tr><td>beta-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td></td></tr><tr><td>fetal calf serum</td><td>Invitrogen</td><td>10270106</td><td></td></tr><tr><td>TC-flask 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V025</td><td></td></tr><tr><td>TC-flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Peske GmbH</td><td>86-V075</td><td></td></tr><tr><td>ethanol, 96%</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>P075.1</td><td></td></tr><tr><td>cell strainer</td><td>VWR international</td><td>734-0002</td><td></td></tr><tr><td>Falcon tubes 50 mL</td><td>Biochrom GmbH</td><td>P91050</td><td></td></tr><tr><td>PEG 8000</td><td>Sigma Aldrich</td><td>1546605</td><td></td></tr><tr><td>electroporation cuvette, 2 mm</td><td>Biodeal Handelsvertretung Edelmann e.K.</td><td>EKL2,25</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma Aldrich</td><td>H9636-25G</td><td></td></tr><tr><td>azaserine</td><td>Sigma Aldrich</td><td>A4142</td><td></td></tr><tr><td>thymidine</td><td>USB Europa GmbH</td><td>22305 1 GM</td><td></td></tr><tr><td>TC-plates 96 well</td><td>Biochrom GmbH</td><td>P92696</td><td></td></tr><tr><td>TC-plates 24 well</td><td>Biochrom GmbH</td><td>P92424</td><td></td></tr><tr><td>cryotubes, 1 mL</td><td>Sigma Aldrich</td><td>V7384-1CS</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>4720.1</td><td></td></tr><tr><td>protein A sepharose</td><td>Sigma Aldrich</td><td>P3391-1G</td><td></td></tr><tr><td>SDS sample loading buffer, Roti-Load 1</td><td>Carl Roth GmbH &amp;amp; C0.KG</td><td>K929.1</td><td></td></tr><tr><td>unstained protein ladder</td><td>BioRad Laboratories</td><td>161-0363</td><td></td></tr><tr><td>comassie brilliant blue R-250</td><td>BioRad Laboratories</td><td>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.

ويبين الشكل 1 مثالا من عيار المصل مستضد معين للتحصين واحدة أجريت في المختبر. في هذا الرقم، والمناعة الأمصال ألف وباء ومعاير من 1:50 إلى 1: 5،000،000 بالمقارنة مع مصل الفأر السذاجة. والمغلفة للمستضد على المرحلة الصلبة، وتم الكشف عن الأجسام المضادة...

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.

جيل من الفئران وحيدة النسيلة الأجسام المضادة بواسطة تقنية هجين

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

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Research

JoVE Journal

Immunology and Infection

41.5K Views.  University of Potsdam. 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. 

Video: Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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 histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

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

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

الإدارة السلبي من الاجسام المضادة ضد H. المغمدة مسببات الأمراض الفطرية وآخرون

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 of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

الجيل المؤتلف الإنسان مفتش حيدة النسيلة من مخلد خلايا التصنيف B

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

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

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

جيل من التمييزية مونوكلونل البشرية من خلايا نادرة محددة مستضد ب المتداولة في الدم

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

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

جيل من الأجسام المضادة ضد المنتجات الطبيعية

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

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

إنتاج أجسام مضادة وحيدة النسيلة تستهدف أمينوببتيداز N في ظهارة الغشاء المخاطي المعوي الخنازير

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

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Generating Recombinant Monoclonal Antibodies From Mammalian Cell Cultures

Source: Nogales-Gadea, G. et al., <a href="https://app.jove.com/v/52830">Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.</a>&#160;J. Vis. Exp.&#160;(2015)
This video demonstrates a method of antibody generation from mammalian cells by introducing plasmid vectors with heavy and light chain genes. These vectors interact with polyethylenimine and enter the cells by forming polyplexes; later, they are translated into individual proteins and assembled into complete antibodies, which are secreted into the extracellular medium.

توليد الأجسام المضادة أحادية النسيلة المؤتلفة من مزارع خلايا الثدييات

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of Peripheral Blood ...

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

A Hybridoma Technology for Producing Monoclonal Antibodies against a Metallopeptidase

Source: Xia, P., et al. <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a> J. Vis. Exp. (2021).
This video demonstrates an assay to produce monoclonal antibodies against porcine aminopeptidase N (APN), a membrane-bound metallopeptidase, using a traditional hybridoma technology. Upon immunizing a mouse with APN, the spleen cells are harvested and fused with immortal myeloma cells to produce hybridomas. The hybridomas are then selected and proliferated using the HAT (hypoxanthine-aminopterin-thymidine) medium to produce monoclonal antibodies specific to different APN epitopes.

تقنية الورم الهجين لإنتاج الأجسام المضادة وحيدة النسيلة ضد ميتالوببتيداز

All animal model procedures have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

A Technique to Generate Monoclonal Antibodies from Antigen-Specific B Cells

Source: Devilder, M., et al. <a href="https://app.jove.com/v/56508">Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood.</a> J. Vis. Exp. (2018).
The video demonstrates a technique for generating monoclonal antibodies from antigen-specific B cells. Eukaryotic expression vectors encoding the heavy and light chains of antibodies, cloned from isolated antigen-specific B cells, are introduced into mammalian cells using polyplexes. Once inside the cells, these vectors undergo translation, and the resulting heavy and light chain peptides assemble into antibodies, which are subsequently secreted by the cells.

تقنية لتوليد الأجسام المضادة وحيدة النسيلة من الخلايا البائية الخاصة بالمستضد

1. Production of mAbs

	The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 &#181;L of Dulbecco&#39;s ...

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges K&#246;hler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques &#8212; electroporation, polyethylene glycol (PEG) mediated fusion, and the use of fusogenic viruses &#8212; generally produce hybridomas at a very low frequency. Fusion products thus have a high ratio of self-fused B-cells and myeloma cells, or unfused cells, with a low number of hybrid cells.
The hypoxanthine-aminopterin-thymidine (HAT) medium is used to select hybridomas by selectively allowing their growth. The aminopterin blocks the default nucleotide synthesis in cells. However, cells can utilize hypoxanthine and thymidine from the medium to synthesize nucleotides via the salvage pathway.
Myeloma cells deficient in the HGPRT enzyme cannot synthesize nucleotides via this pathway and thus, do not grow in HAT medium. Although B-cells produce functional HGPRT enzymes, they cannot divide indefinitely. Thus, only the hybrid cells with functional HGPT from the B-cells and immortality from the myeloma line can grow on the HAT selection medium. Such a culture of hybrid cells is called a hybridoma, which can be screened for monoclonal antibody production.
Hybridoma Screening
Hybridoma cultures from the HAT selection are plated onto a 96-well plate; each well containing only one cell. Using Enzyme-linked Immunosorbent Assay (ELISA), each cell is screened for the production of antibodies specific to the epitope of interest. Cells that test positive are then selected and grown in a larger culture vessel to establish hybridoma cell lines. These cell lines are a permanent source of unlimited monoclonal antibodies. Thus hybridoma technology is a convenient and cost-effective method for mass production of monoclonal antibodies.

تقنية الورم الهجين

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or ...