المرجع الالكتروني للمعلوماتية
المرجع الألكتروني للمعلوماتية

علم الاحياء
عدد المواضيع في هذا القسم 10456 موضوعاً
النبات
الحيوان
الأحياء المجهرية
علم الأمراض
التقانة الإحيائية
التقنية الحياتية النانوية
علم الأجنة
الأحياء الجزيئي
علم وظائف الأعضاء
المضادات الحيوية

Untitled Document
أبحث عن شيء أخر المرجع الالكتروني للمعلوماتية
القيمة الغذائية للثوم Garlic
2024-11-20
العيوب الفسيولوجية التي تصيب الثوم
2024-11-20
التربة المناسبة لزراعة الثوم
2024-11-20
البنجر (الشوندر) Garden Beet (من الزراعة الى الحصاد)
2024-11-20
الصحافة العسكرية ووظائفها
2024-11-19
الصحافة العسكرية
2024-11-19

Theory of chromatography
2-2-2020
الكذب
14-4-2016
مناة
2023-03-24
مقيم السنة بعد الفترة
27-9-2019
Myosins Genes
17-10-2020
معنى كلمة دحض
4-06-2015

Hybridomas  
  
2157   11:34 صباحاً   date: 19-5-2016
Author : I. Roitt, J. Brostoff, and D. Male
Book or Source : Immunology, 4th ed. Mosby, London, p. 28.9.
Page and Part :

Hybridomas

 

Classical seminal studies by Köhler and Milstein in 1975 (1) led to the generation of monoclonal antibodies with defined specificity. Hybridoma methodology was based primarily on fusing murine immune lymphocytes with a malignant myeloma lymphocyte cell line that possessed certain important genetic and biochemical properties. This important methodology was necessitated by the fact that immune antibody-producing B lymphocytes were difficult to maintain as stable cell lines under tissue culture conditions. However, fusion of antibody-producing cells with a malignant myeloma cell line resulted in proliferative cells that can be maintained indefinitely.

Spontaneous fusions between lymphocytes are rare events, so the frequency of fusion was increased to generate large numbers of fused cells by using surface-active agents such as inactivated Sendai virus or the chemical polyethylene glycol (PEG). Upon mixing immune lymphocytes with myeloma cells and the fusing reagent, a selection system was mandatory to eliminate unfused parental cells and to assay for appropriately fused cells that synthesized antibodies with the desired specificity. The tissue culture selection process was dependent on the procurement of myeloma cell lines that no longer synthesized an immunoglobulin and lacked genes for thymidine kinase (TK) and/or hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). These enzymes catalyze important steps in the salvage pathway used in DNA synthesis by dividing cells. Unfused myeloma cells are unable to proliferate in the HAT selection medium (containing hypoxanthine, aminopterin, and thymidine) because aminopterin inhibited the de novo DNA biosynthetic pathway, forcing the cells to use the nonfunctional salvage pathway. Fused myeloma cells proliferate in HAT medium because the immune lymphocytes (fusion partner) furnish the essential TK and HGPRT enzymes for the salvage pathway. Thus, cells that survived incubation with HAT medium represented a successful immune B-cell–myeloma cell fusion, usually on a 1:1 cellular basis. The second phase in hybridoma production was the selection of those fused cells that synthesized and secreted antibody of the desired specificity. This was generally achieved by diluting fused cell populations to a single cell and then generating clones by proliferation of the single cell. The extracellular fluid or media from the single cell and resulting progeny were assayed for the appropriate antigen binding. The methods and techniques used to detect monoclonal antibody activity are usually based upon some variation of the solid-phase immunoassay (eg, ELISA or radioimmunoassay formats).

 Hybridoma methodology and consistent generation of relatively large amounts of monoclonal antibodies with defined specificities has proven to be a source of important immunological reagents.

1. The provision of adequate quantities of monoclonal antibody has facilitated the routine determination of primary structures of both the H and L chains at the protein level.

2. Various monoclonal antibodies can be crystallized on a regular basis, providing atomic, 2- to 3-Å resolution structures by X-ray crystallographic procedures. In general, Fab fragments (four-domain substructures of antibodies, including the variable domains of both chains) with bound ligand crystallize most efficiently. Approximately 100 resolved structures of monoclonal antibodies (Fab fragments) have now been reported.

3. This methodology provides large homogeneous hybridoma cell populations from which messenger RNA responsible for the synthesis of H and L chains can be obtained. Through development of polynucleotide primers, polymerase chain reaction (PCR) technology can be used to generate complementary DNA copies for sequencing at the gene level.

4. Gene cloning experimentation with antibody genes has been important in successful site-directed mutagenesis studies. Such studies have been critical in deciphering structure–function relationships governing antibody activity.

5. Hybridoma methodology has led to the development of antibody derivatives, such as single-chain antibodies (2). The latter represent ~25,000–Da structures containing only the variable domains of the H and L chains of a specific antibody molecule. Variable domains constituting single-chain antibodies are usually attached covalently through the use of flexible polylinkers (10 to 15 amino acid residues) that are encoded in the single-chain synthetic gene. Single-chain genes are subsequently incorporated into the appropriate plasmids and expressed in either prokaryote or eukaryote cell lines.

6. Antigen–antibody interactions can now be studied on a homogeneous basis, yielding important information regarding the thermodynamics of binding.

7. The provision of monoclonal antibodies by hybridoma technology has led to the development of standard diagnostic procedures as well as therapeutically useful immunochemical reagents.

Monoclonal antibodies of defined specificity generated through hybridoma methodology have been used to solve many issues related to structure–function relationships within the antibody molecule. Based on studies with monoclonal antibodies, the property of Fab (active site) segmental flexibility within the IgG class of antibodies was solved. Homogeneous binding at both active sites within a bivalent molecule ruled out allosteric effects within antibody molecules. Thus, binding at one active site does not influence antigen binding at the adjacent site. Conformational changes transmitted throughout the molecule subsequent to binding of antigen were dismissed as an explanation for such important phenomena as complement binding and fixation. In all cases, the availability of monoclonal antibodies proved important to examine definitively these important questions.

 Although hybridoma technology has been the method of choice to produce monoclonal antibodies of defined specificity, new techniques have now emerged. Similar to the construction of single-chain antibodies, gene segments encoding the H- and L-chain variable domains are genetically fused to genes encoding a bacteriophage coat protein (3). The engineered bacteriophage infect bacteria, and the resulting phage particles express active antibody products on their surface. The resulting phage display library expresses many different antigen-binding domains. Phage that specifically bind antigen are selected and used to infect bacteria in a second cycle. Each selected phage produces a monoclonal antigen-reactive particle. Primary structures of the variable domains can be determined, and those genes can be fused to the antibody constant region genes to reconstruct a monoclonal antibody. Such genes transfected into myeloma cell lines are expressed, and the antibody products are secreted. Phage display methodology has important implications for the future.

 

References

1. G. Köhler and C. Milstein (1975). Nature 256, 495–497

2. R. E. Bird, K. D. Hardman, J. W. Jacobson, S. Johnson, B. M. Kaufman, S.-M. Lee, T. Lee, S. H. Pope, G. S. Riordan, and M. Whitlow (1988) Science 242, 423–426

3. I. Roitt, J. Brostoff, and D. Male (1996) Immunology, 4th ed. Mosby, London, p. 28.9. 




علم الأحياء المجهرية هو العلم الذي يختص بدراسة الأحياء الدقيقة من حيث الحجم والتي لا يمكن مشاهدتها بالعين المجرَّدة. اذ يتعامل مع الأشكال المجهرية من حيث طرق تكاثرها، ووظائف أجزائها ومكوناتها المختلفة، دورها في الطبيعة، والعلاقة المفيدة أو الضارة مع الكائنات الحية - ومنها الإنسان بشكل خاص - كما يدرس استعمالات هذه الكائنات في الصناعة والعلم. وتنقسم هذه الكائنات الدقيقة إلى: بكتيريا وفيروسات وفطريات وطفيليات.



يقوم علم الأحياء الجزيئي بدراسة الأحياء على المستوى الجزيئي، لذلك فهو يتداخل مع كلا من علم الأحياء والكيمياء وبشكل خاص مع علم الكيمياء الحيوية وعلم الوراثة في عدة مناطق وتخصصات. يهتم علم الاحياء الجزيئي بدراسة مختلف العلاقات المتبادلة بين كافة الأنظمة الخلوية وبخاصة العلاقات بين الدنا (DNA) والرنا (RNA) وعملية تصنيع البروتينات إضافة إلى آليات تنظيم هذه العملية وكافة العمليات الحيوية.



علم الوراثة هو أحد فروع علوم الحياة الحديثة الذي يبحث في أسباب التشابه والاختلاف في صفات الأجيال المتعاقبة من الأفراد التي ترتبط فيما بينها بصلة عضوية معينة كما يبحث فيما يؤدي اليه تلك الأسباب من نتائج مع إعطاء تفسير للمسببات ونتائجها. وعلى هذا الأساس فإن دراسة هذا العلم تتطلب الماماً واسعاً وقاعدة راسخة عميقة في شتى مجالات علوم الحياة كعلم الخلية وعلم الهيأة وعلم الأجنة وعلم البيئة والتصنيف والزراعة والطب وعلم البكتريا.