Molecular biology / m ə ˈ l ɛ k j ʊ l ər / is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells , including biomolecular synthesis, modification, mechanisms, and interactions.
107-719: 1CDH , 1CDI , 1CDJ , 1CDU , 1CDY , 1G9M , 1G9N , 1GC1 , 1JL4 , 1Q68 , 1RZJ , 1RZK , 1WIO , 1WIP , 1WIQ , 2B4C , 2JKR , 2JKT , 2KLU , 2NXY , 2NXZ , 2NY0 , 2NY1 , 2NY2 , 2NY3 , 2NY4 , 2NY5 , 2NY6 , 2QAD , 3B71 , 3CD4 , 3JWD , 3JWO , 3LQA , 3O2D , 3S5L , 3T0E , 4JM2 , 1WBR , 3S4S , 4H8W , 4P9H , 4Q6I , 4R2G , 4R4H , 4RQS , 3J70 , 5A7X , 5A8H , 5CAY 920 12504 ENSG00000010610 ENSMUSG00000023274 P01730 P06332 NM_001382705 NM_001382706 NM_001382707 NM_001382714 NM_013488 NP_001369634 NP_001369635 NP_001369636 NP_001369643 NP_038516 In molecular biology , CD4 ( cluster of differentiation 4)
214-446: A 2D gel electrophoresis . The Bradford assay is a molecular biology technique which enables the fast, accurate quantitation of protein molecules utilizing the unique properties of a dye called Coomassie Brilliant Blue G-250. Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein. In its unstable, cationic state, Coomassie Blue has a background wavelength of 465 nm and gives off
321-417: A Greek key topology . CD4 interacts with the β 2 -domain of MHC class II molecules through its D 1 domain. T cells displaying CD4 molecules (and not CD8 ) on their surface, therefore, are specific for antigens presented by MHC II and not by MHC class I (they are MHC class II-restricted ). MHC class I contains Beta-2 microglobulin . The short cytoplasmic / intracellular tail (C) of CD4 contains
428-736: A plasmid ( expression vector ). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker (usually antibiotic resistance ). Additionally, upstream of the MCS are the promoter regions and the transcription start site, which regulate the expression of cloned gene. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means
535-554: A 0.45 μm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is produced by a low-secreting cell line. The sample is therefore concentrated by ultrafiltration or dialysis . Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These can be separated by ion exchange chromatography . Either cation exchange chromatography
642-421: A co-receptor expressed on the host cell. These co-receptors are chemokine receptors CCR5 or CXCR4 . Following a structural change in another viral protein ( gp41 ), HIV inserts a fusion peptide into the host cell that allows the outer membrane of the virus to fuse with the cell membrane . HIV infection leads to a progressive reduction in the number of T cells expressing CD4 . Medical professionals refer to
749-448: A density gradient, which separated the DNA molecules based on their density. The results showed that after one generation of replication in the N medium, the DNA formed a band of intermediate density between that of pure N DNA and pure N DNA. This supported the semiconservative DNA replication proposed by Watson and Crick, where each strand of the parental DNA molecule serves as a template for
856-461: A given antigen . In 1973, Jerrold Schwaber described the production of monoclonal antibodies using human–mouse hybrid cells. This work remains widely cited among those using human-derived hybridomas . In 1975, Georges Köhler and César Milstein succeeded in making fusions of myeloma cell lines with B cells to create hybridomas that could produce antibodies, specific to known antigens and that were immortalized. They and Niels Kaj Jerne shared
963-526: A host's immune system cannot recognize the bacteria and it kills the host. The other, avirulent, rough strain lacks this polysaccharide capsule and has a dull, rough appearance. Presence or absence of capsule in the strain, is known to be genetically determined. Smooth and rough strains occur in several different type such as S-I, S-II, S-III, etc. and R-I, R-II, R-III, etc. respectively. All this subtypes of S and R bacteria differ with each other in antigen type they produce. The Avery–MacLeod–McCarty experiment
1070-453: A labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression
1177-541: A level of 350 cells per microliter in Europe but usually around 500/μL in the US; people with less than 200 cells per microliter are at high risk of contracting AIDS defined illnesses. Medical professionals also refer to CD4 tests to determine efficacy of treatment. Viral load testing provides more information about the efficacy for therapy than CD4 counts. For the first 2 years of HIV therapy, CD4 counts may be done every 3–6 months. If
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#17328489771331284-495: A media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. Cell culture sample contaminants consist primarily of media components such as growth factors, hormones and transferrins . In contrast, the in vivo sample is likely to have host antibodies, proteases , nucleases , nucleic acids and viruses . In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as
1391-518: A mixture of proteins. Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression. In western blotting , proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE . The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane. This membrane can then be probed with solutions of antibodies . Antibodies that specifically bind to
1498-499: A monoclonal antibody drug, received an emergency use authorization from the US FDA for use as pre-exposure prophylaxis to protect certain moderately to severely immunocompromised individuals against COVID-19. Several monoclonal antibodies, such as bevacizumab and cetuximab , can cause different kinds of side effects. These side effects can be categorized into common and serious side effects. Some common side effects include: Among
1605-421: A patient's viral load becomes undetectable after 2 years then CD4 counts might not be needed if they are consistently above 500/mm. If the count remains at 300–500/mm, then the tests can be done annually. It is not necessary to schedule CD4 counts with viral load tests and the two should be done independently when each is indicated. CD4 continues to be expressed in most neoplasms derived from T helper cells . It
1712-445: A protein has no net charge. When the pH > pI, a protein has a net negative charge, and when the pH < pI, a protein has a net positive charge. For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. Thus, at a pH between 4.8 and 6.1, the average charge of albumin molecules is likely to be more negative, while mAbs molecules are positively charged and hence it
1819-436: A reddish-brown color. When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595 nm and the dye gives off a bright blue color. Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it is recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation. The concentration of protein in
1926-410: A result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture and thus the contaminants, one or the other method ( in vivo or in vitro ) may be preferable. The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with
2033-408: A single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence . A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified ( expression profiling ). In this technique the RNA in a tissue is isolated and converted to labeled complementary DNA (cDNA). This cDNA is then hybridized to the fragments on
2140-455: A special sequence of amino acids that allow it to recruit and interact with the tyrosine kinase Lck . CD4 is a co-receptor of the T cell receptor (TCR) and assists the latter in communicating with antigen-presenting cells . The TCR complex and CD4 bind to distinct regions of the antigen-presenting MHC class II molecule. The extracellular D 1 domain of CD4 binds to the β2 region of MHC class II. The resulting close proximity between
2247-553: A substance in either frozen tissue section or live cells. Antibodies can also be used to purify their target compounds from mixtures, using the method of immunoprecipitation . Therapeutic monoclonal antibodies act through multiple mechanisms, such as blocking of targeted molecule functions, inducing apoptosis in cells which express the target, or by modulating signalling pathways. One possible treatment for cancer involves monoclonal antibodies that bind only to cancer-cell-specific antigens and induce an immune response against
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#17328489771332354-474: A transplant, the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight. Like many cell surface receptors/markers, CD4 is a member of the immunoglobulin superfamily . It has four immunoglobulin domains (D 1 to D 4 ) that are exposed on the extracellular surface of the cell: The immunoglobulin variable (IgV) domain of D 1 adopts an immunoglobulin-like β-sandwich fold with seven β-strands in two β-sheets, in
2461-462: A viewpoint on the interdisciplinary relationships between molecular biology and other related fields. While researchers practice techniques specific to molecular biology, it is common to combine these with methods from genetics and biochemistry . Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as bioinformatics and computational biology . Molecular genetics ,
2568-468: Is protein A/G affinity chromatography . The antibody selectively binds to protein A/G, so a high level of purity (generally >80%) is obtained. The generally harsh conditions of this method may damage easily damaged antibodies. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause protein A/G itself to leak off the column and appear in
2675-541: Is a glycoprotein that serves as a co-receptor for the T-cell receptor (TCR). CD4 is found on the surface of immune cells such as helper T cells , monocytes , macrophages , and dendritic cells . It was discovered in the late 1970s and was originally known as leu-3 and T4 (after the OKT4 monoclonal antibody that reacted with it) before being named CD4 in 1984. In humans, the CD4 protein
2782-657: Is about $ 100,000 higher in oncology and hematology than in other disease states", comparing them on a per patient basis, to those for cardiovascular or metabolic disorders, immunology, infectious diseases, allergy, and ophthalmology. Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. Proteins can be detected using the Western blot and immuno dot blot tests. In immunohistochemistry , monoclonal antibodies can be used to detect antigens in fixed tissue sections, and similarly, immunofluorescence can be used to detect
2889-439: Is also a long tradition of studying biomolecules "from the ground up", or molecularly, in biophysics . Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector. This recombinant DNA technology was first developed in the 1960s. In this technique, a DNA sequence coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes , into
2996-551: Is an antibody produced from a cell lineage made by cloning a unique white blood cell . All subsequent antibodies derived this way trace back to a unique parent cell. Monoclonal antibodies can have monovalent affinity, binding only to the same epitope (the part of an antigen that is recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are usually made by several different antibody-secreting plasma cell lineages. Bispecific monoclonal antibodies can also be engineered, by increasing
3103-407: Is an inhibitory receptor, is upregulated in activated T cells as a kind of negative feedback loop. CD4 has also been shown to interact with SPG21 , and Uncoordinated-119 (Unc-119) . HIV-1 uses CD4 to gain entry into host T-cells and achieves this through its viral envelope protein known as gp120 . The binding to CD4 creates a shift in the conformation of gp120 allowing HIV-1 to bind to
3210-439: Is becoming more affordable and used in many different scientific fields. This will drive the development of industries in developing nations and increase accessibility to individual researchers. Likewise, CRISPR-Cas9 gene editing experiments can now be conceived and implemented by individuals for under $ 10,000 in novel organisms, which will drive the development of industrial and medical applications. The following list describes
3317-413: Is called transfection . Several different transfection techniques are available, such as calcium phosphate transfection, electroporation , microinjection and liposome transfection . The plasmid may be integrated into the genome , resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection. DNA coding for a protein of interest
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3424-410: Is centrifuged and the pellet which contains E.coli cells was checked and the supernatant was discarded. The E.coli cells showed radioactive phosphorus, which indicated that the transformed material was DNA not the protein coat. The transformed DNA gets attached to the DNA of E.coli and radioactivity is only seen onto the bacteriophage's DNA. This mutated DNA can be passed to the next generation and
3531-547: Is encoded by the CD4 gene . CD4+ T helper cells are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T helper cells or T4 cells. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells , which then destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following immune suppression prior to
3638-433: Is found in a cDNA library . PCR has many variations, like reverse transcription PCR ( RT-PCR ) for amplification of RNA, and, more recently, quantitative PCR which allow for quantitative measurement of DNA or RNA molecules. Gel electrophoresis is a technique which separates molecules by their size using an agarose or polyacrylamide gel. This technique is one of the principal tools of molecular biology. The basic principle
3745-430: Is helpful to quantify the amount of T-cells on fresh-frozen tissue with CD4+, CD8+, and CD3+ T-cell markers (which stain different markers on a T-cell – giving different results). Molecular biology Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until
3852-454: Is known as horizontal gene transfer (HGT). This phenomenon is now referred to as genetic transformation. Griffith's experiment addressed the pneumococcus bacteria, which had two different strains, one virulent and smooth and one avirulent and rough. The smooth strain had glistering appearance owing to the presence of a type of specific polysaccharide – a polymer of glucose and glucuronic acid capsule. Due to this polysaccharide layer of bacteria,
3959-472: Is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in
4066-456: Is occurring by measuring how much of that RNA is present in different samples, assuming that no post-transcriptional regulation occurs and that the levels of mRNA reflect proportional levels of the corresponding protein being produced. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues. A western blot is a technique by which specific proteins can be detected from
4173-921: Is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support. Product heterogeneity is common in monoclonal antibodies and other recombinant biological products and is typically introduced either upstream during expression or downstream during manufacturing. These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. These seemingly minute structural changes can affect preclinical stability and process optimization as well as therapeutic product potency, bioavailability and immunogenicity . The generally accepted purification method of process streams for monoclonal antibodies includes capture of
4280-674: Is possible to separate them. Transferrin, on the other hand, has a pI of 5.9, so it cannot be easily separated by this method. A difference in pI of at least 1 is necessary for a good separation. Transferrin can instead be removed by size exclusion chromatography . This method is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low capacity and low elution times. A much quicker, single-step method of separation
4387-479: Is susceptible to influence by strong alkaline buffering agents, such as sodium dodecyl sulfate (SDS). The terms northern , western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting , after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as
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4494-400: Is that DNA fragments can be separated by applying an electric current across the gel - because the DNA backbone contains negatively charged phosphate groups, the DNA will migrate through the agarose gel towards the positive end of the current. Proteins can also be separated on the basis of size using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as
4601-412: Is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR , to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice or in
4708-401: Is then selected for future use. The hybridomas can be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity , surrounding the gut). There, they produce tumors secreting an antibody-rich fluid called ascites fluid. The medium must be enriched during in vitro selection to further favour hybridoma growth. This can be achieved by
4815-412: Is therefore possible to use CD4 immunohistochemistry on tissue biopsy samples to identify most forms of peripheral T cell lymphoma and related malignant conditions. The antigen has also been associated with a number of autoimmune diseases such as vitiligo and type I diabetes mellitus . T-cells play a large part in autoinflammatory diseases. When testing a drug's efficacy or studying diseases, it
4922-412: Is used at a low enough pH that the desired antibody binds to the column while anions flow through, or anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated along with the anions based on their isoelectric point (pI). In proteins, the isoelectric point (pI) is defined as the pH at which
5029-516: Is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates. A DNA microarray is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragments. Arrays make it possible to put down large quantities of very small (100 micrometre diameter) spots on
5136-453: Is useful in treating moderate-to-severe allergic asthma . Monoclonal antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine like CiteAb . Below are examples of clinically important monoclonal antibodies. casirivimab/imdevimab In 2020, the monoclonal antibody therapies bamlanivimab/etesevimab and casirivimab/imdevimab were given emergency use authorizations by
5243-553: The Medical Research Council Unit, Cavendish Laboratory , were the first to describe the double helix model for the chemical structure of deoxyribonucleic acid (DNA), which is often considered a landmark event for the nascent field because it provided a physico-chemical basis by which to understand the previously nebulous idea of nucleic acids as the primary substance of biological inheritance. They proposed this structure based on previous research done by Franklin, which
5350-514: The Nobel Prize in Physiology or Medicine in 1984 for the discovery. In 1988, Gregory Winter and his team pioneered the techniques to humanize monoclonal antibodies, eliminating the reactions that many monoclonal antibodies caused in some patients. By the 1990s research was making progress in using monoclonal antibodies therapeutically, and in 2018, James P. Allison and Tasuku Honjo received
5457-425: The genetic code is a triplet code, where each triplet (called a codon ) specifies a particular amino acid. Furthermore, it was shown that the codons do not overlap with each other in the DNA sequence encoding a protein, and that each sequence is read from a fixed starting point. During 1962–1964, through the use of conditional lethal mutants of a bacterial virus, fundamental advances were made in our understanding of
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#17328489771335564-422: The northern blot , actually did not use the term. Named after its inventor, biologist Edwin Southern , the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action . The membrane
5671-401: The 1908 Nobel Prize for Physiology or Medicine for providing the theoretical basis for immunology. By the 1970s, lymphocytes producing a single antibody were known, in the form of multiple myeloma – a cancer affecting B-cells . These abnormal antibodies or paraproteins were used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to
5778-796: The 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in the biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury , who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observations of so-called classical biology, which instead studies biological processes at larger scales and higher levels of organization. In 1953, Francis Crick , James Watson , Rosalind Franklin , and their colleagues at
5885-631: The Bradford assay can then be measured using a visible light spectrophotometer , and therefore does not require extensive equipment. This method was developed in 1975 by Marion M. Bradford , and has enabled significantly faster, more accurate protein quantitation compared to previous methods: the Lowry procedure and the biuret assay. Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride. However, it
5992-400: The CD4 count to decide when to begin treatment during HIV infection, although recent medical guidelines have changed to recommend treatment at all CD4 counts as soon as HIV is diagnosed. A CD4 count measures the number of T cells expressing CD4. While CD4 counts are not a direct HIV test—e.g. they do not check the presence of viral DNA, or specific antibodies against HIV—they are used to assess
6099-456: The DNA model was Phoebus Levene , who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast. In 1950, Erwin Chargaff expanded on the work of Levene and elucidated a few critical properties of nucleic acids: first, the sequence of nucleic acids varies across species. Second, the total concentration of purines (adenine and guanine) is always equal to
6206-475: The Nobel Prize in Physiology or Medicine for their discovery of cancer therapy by inhibition of negative immune regulation, using monoclonal antibodies that prevent inhibitory linkages. The translational work needed to implement these ideas is credited to Lee Nadler. As explained in an NIH article, "He was the first to discover monoclonal antibodies directed against human B-cell–specific antigens and, in fact, all
6313-526: The TCR complex and CD4 allows the tyrosine kinase Lck bound to the cytoplasmic tail of CD4 to phosphorylate tyrosine residues of immunoreceptor tyrosine activation motifs (ITAMs) on the cytoplasmic domains of CD3 to amplify the signal generated by the TCR. Phosphorylated ITAMs on CD3 recruit and activate SH2 domain -containing protein tyrosine kinases (PTK), such as ZAP70 , to further mediate downstream signalling through tyrosine phosphorylation. These signals lead to
6420-497: The US Food and Drug Administration to reduce the number of hospitalizations, emergency room visits, and deaths because of COVID-19 . In September 2021, the Biden administration purchased US$ 2.9 billion worth of Regeneron monoclonal antibodies at $ 2,100 per dose to curb the shortage. As of December 2021, in vitro neutralization tests indicate monoclonal antibody therapies (with
6527-438: The activation of transcription factors , including NF-κB , NFAT , AP-1 , to promote T cell activation. CD4 is closely related to LAG-3 , and together they form an evolutionary conserved system from the level of sharks competing for binding Lck by conserved motifs in their cytoplasmic tails: CD4 through a Cys-X-Cys/His motif and LAG-3 through an immunoreceptor tyrosine-based inhibition motif like (ITIM-like) motif. LAG-3, which
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#17328489771336634-407: The antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide , it is commonly synthesized with a terminal cysteine , which allows selective attachment to a carrier protein, such as KLH during development and to support purification. The antibody-containing medium is then incubated with the immobilized antigen, either in batch or as the antibody
6741-437: The array and visualization of the hybridization can be done. Since multiple arrays can be made with exactly the same position of fragments, they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. There are many different ways to fabricate microarrays;
6848-473: The atomic level. Molecular biologists today have access to increasingly affordable sequencing data at increasingly higher depths, facilitating the development of novel genetic manipulation methods in new non-model organisms. Likewise, synthetic molecular biologists will drive the industrial production of small and macro molecules through the introduction of exogenous metabolic pathways in various prokaryotic and eukaryotic cell lines. Horizontally, sequencing data
6955-403: The bacteriophage's protein coat with radioactive sulphur and DNA with radioactive phosphorus, into two different test tubes respectively. After mixing bacteriophage and E.coli into the test tube, the incubation period starts in which phage transforms the genetic material in the E.coli cells. Then the mixture is blended or agitated, which separates the phage from E.coli cells. The whole mixture
7062-632: The binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of this " chimeric " or "humanised" DNA through cell culture yielded part-mouse, part-human antibodies. Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of fully human products to reduce the side effects of humanised or chimeric antibodies. Several successful approaches have been proposed: transgenic mice , phage display and single B cell cloning. Monoclonal antibodies are more expensive to manufacture than small molecules due to
7169-539: The complex processes involved and the general size of the molecules, all in addition to the enormous research and development costs involved in bringing a new chemical entity to patients. They are priced to enable manufacturers to recoup the typically large investment costs, and where there are no price controls, such as the United States, prices can be higher if they provide great value. Seven University of Pittsburgh researchers concluded, "The annual price of mAb therapies
7276-550: The de novo pathway and become fully auxotrophic for nucleic acids , thus requiring supplementation to survive. The selective culture medium is called HAT medium because it contains hypoxanthine , aminopterin and thymidine . This medium is selective for fused ( hybridoma ) cells. Unfused myeloma cells cannot grow because they lack HGPRT and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells referred to as hybridomas, are able to grow indefinitely in
7383-493: The development of new technologies and their optimization. Molecular biology has been elucidated by the work of many scientists, and thus the history of the field depends on an understanding of these scientists and their experiments. The field of genetics arose from attempts to understand the set of rules underlying reproduction and heredity , and the nature of the hypothetical units of heredity known as genes . Gregor Mendel pioneered this work in 1866, when he first described
7490-431: The differences between them were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the blood, as well as systemic inflammatory effects and the production of human anti-mouse antibodies (HAMA). Recombinant DNA has been explored since the late 1980s to increase residence times. In one approach called "CDR grafting", mouse DNA encoding
7597-426: The eluted sample. Gentle elution buffer systems that employ high salt concentrations are available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized protein A/G is a more expensive resin. To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide specificity for the antibody. In this method,
7704-401: The engineering of gene knockout embryonic stem cell lines . The northern blot is used to study the presence of specific RNA molecules as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis , and a blot . In this process RNA is separated based on size and is then transferred to a membrane that is then probed with
7811-574: The exception of sotrovimab and tixagevimab/cilgavimab ) were not likely to be active against the Omicron variant. Over 2021–22, two Cochrane reviews found insufficient evidence for using neutralizing monoclonal antibodies to treat COVID-19 infections. The reviews applied only to people who were unvaccinated against COVID‐19, and only to the COVID-19 variants existing during the studies, not to newer variants, such as Omicron. In March 2024, pemivibart ,
7918-412: The experiment involved growing E. coli bacteria in a medium containing heavy isotope of nitrogen ( N) for several generations. This caused all the newly synthesized bacterial DNA to be incorporated with the heavy isotope. After allowing the bacteria to replicate in a medium containing normal nitrogen ( N), samples were taken at various time points. These samples were then subjected to centrifugation in
8025-399: The extract. They discovered that when they digested the DNA in the extract with DNase , transformation of harmless bacteria into virulent ones was lost. This provided strong evidence that DNA was the genetic material, challenging the prevailing belief that proteins were responsible. It laid the basis for the subsequent discovery of its structure by Watson and Crick. Confirmation that DNA is
8132-504: The functions and interactions of the proteins employed in the machinery of DNA replication , DNA repair , DNA recombination , and in the assembly of molecular structures. In 1928, Frederick Griffith , encountered a virulence property in pneumococcus bacteria, which was killing lab rats. According to Mendel, prevalent at that time, gene transfer could occur only from parent to daughter cells. Griffith advanced another theory, stating that gene transfer occurring in member of same generation
8239-526: The genetic material which is cause of infection came from the Hershey–Chase experiment . They used E.coli and bacteriophage for the experiment. This experiment is also known as blender experiment, as kitchen blender was used as a major piece of apparatus. Alfred Hershey and Martha Chase demonstrated that the DNA injected by a phage particle into a bacterium contains all information required to synthesize progeny phage particles. They used radioactivity to tag
8346-407: The immune system of a patient. National Institutes of Health guidelines recommend treatment of any HIV-positive individuals, regardless of CD4 count Normal blood values are usually expressed as the number of cells per microliter (μL, or equivalently, cubic millimeter, mm) of blood, with normal values for CD4 cells being 500–1200 cells/mm. Patients often undergo treatments when the CD4 counts reach
8453-404: The implications of this unique structure for possible mechanisms of DNA replication. Watson and Crick were awarded the Nobel Prize in Physiology or Medicine in 1962, along with Wilkins, for proposing a model of the structure of DNA. In 1961, it was demonstrated that when a gene encodes a protein , three sequential bases of a gene's DNA specify each successive amino acid of the protein. Thus
8560-405: The known human B-cell–specific antigens were discovered in his laboratory. He is a true translational investigator, since he used these monoclonal antibodies to classify human B-cell leukemia and lymphomas as well as to create therapeutic agents for patients. . . More importantly, he was the first in the world to administer a monoclonal antibody to a human (a patient with B-cell lymphoma)." Much of
8667-434: The laws of inheritance he observed in his studies of mating crosses in pea plants. One such law of genetic inheritance is the law of segregation , which states that diploid individuals with two alleles for a particular gene will pass one of these alleles to their offspring. Because of his critical work, the study of genetic inheritance is commonly referred to as Mendelian genetics . A major milestone in molecular biology
8774-472: The medium because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell). This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or antigen microarray assay) or immuno- dot blot . The most productive and stable clone
8881-415: The most common are silicon chips, microscope slides with spots of ~100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. Arrays can also be made with molecules other than DNA. Allele-specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without
8988-399: The need for PCR or gel electrophoresis. Short (20–25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA, hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the unhybridized probes are removed. The target DNA is then analyzed for the presence of
9095-400: The newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by PCR and produce in either bacterial or mammalian systems with recombinant technology. One of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals in the laboratory. After obtaining either
9202-463: The pharmaceutical industry, the activity of new drugs against the protein can be studied. Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. PCR has many applications, including
9309-639: The preclinical development phase is critical for enhanced product quality understanding and provides a basis for risk management and increased regulatory flexibility. The recent Food and Drug Administration's Quality by Design initiative attempts to provide guidance on development and to facilitate design of products and processes that maximizes efficacy and safety profile while enhancing product manufacturability. The production of recombinant monoclonal antibodies involves repertoire cloning , CRISPR/Cas9 , or phage display / yeast display technologies. Recombinant antibody engineering involves antibody production by
9416-405: The probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation. In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis ( agarose or polyacrylamide ), the size of DNA molecules
9523-440: The product target with protein A , elution, acidification to inactivate potential mammalian viruses, followed by ion chromatography , first with anion beads and then with cation beads. Displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pharmacokinetic studies. Knowledge gained during
9630-530: The protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence , or autoradiography . Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. The eastern blotting technique
9737-421: The study of gene expression, the detection of pathogenic microorganisms, the detection of genetic mutations, and the introduction of mutations to DNA. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis . PCR can also be used to determine whether a particular DNA fragment
9844-532: The study of gene structure and function, has been among the most prominent sub-fields of molecular biology since the early 2000s. Other branches of biology are informed by molecular biology, by either directly studying the interactions of molecules in their own right such as in cell biology and developmental biology , or indirectly, where molecular techniques are used to infer historical attributes of populations or species , as in fields in evolutionary biology such as population genetics and phylogenetics . There
9951-533: The success rate is low, so a selective medium in which only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for these cells unless the de novo purine synthesis pathway is also disrupted. Exposing cells to aminopterin (a folic acid analogue which inhibits dihydrofolate reductase ) makes them unable to use
10058-554: The synthesis of a new complementary strand, resulting in two daughter DNA molecules, each consisting of one parental and one newly synthesized strand. The Meselson-Stahl experiment provided compelling evidence for the semiconservative replication of DNA, which is fundamental to the understanding of genetics and molecular biology. In the early 2020s, molecular biology entered a golden age defined by both vertical and horizontal technical development. Vertically, novel technologies are allowing for real-time monitoring of biological processes at
10165-902: The target cancer cell. Such mAbs can be modified for delivery of a toxin , radioisotope , cytokine or other active conjugate or to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with its Fc region . MAbs approved by the FDA for cancer include: Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab , which are effective in rheumatoid arthritis , Crohn's disease , ulcerative colitis and ankylosing spondylitis by their ability to bind to and inhibit TNF-α . Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E (IgE) and
10272-482: The theory of Transduction came into existence. Transduction is a process in which the bacterial DNA carry the fragment of bacteriophages and pass it on the next generation. This is also a type of horizontal gene transfer. The Meselson-Stahl experiment was a landmark experiment in molecular biology that provided evidence for the semiconservative replication of DNA. Conducted in 1958 by Matthew Meselson and Franklin Stahl ,
10379-451: The therapeutic targets of one monoclonal antibody to two epitopes. It is possible to produce monoclonal antibodies that specifically bind to almost any suitable substance; they can then serve to detect or purify it. This capability has become an investigative tool in biochemistry , molecular biology , and medicine . Monoclonal antibodies are used in the diagnosis of illnesses such as cancer and infections and are used therapeutically in
10486-447: The total concentration of pyrimidines (cysteine and thymine). This is now known as Chargaff's rule. In 1953, James Watson and Francis Crick published the double helical structure of DNA, based on the X-ray crystallography work done by Rosalind Franklin which was conveyed to them by Maurice Wilkins and Max Perutz . Watson and Crick described the structure of DNA and conjectured about
10593-419: The treatment of e.g. cancer and inflammatory diseases. In the early 1900s, immunologist Paul Ehrlich proposed the idea of a Zauberkugel – " magic bullet ", conceived of as a compound which selectively targeted a disease-causing organism, and could deliver a toxin for that organism. This underpinned the concept of monoclonal antibodies and monoclonal drug conjugates. Ehrlich and Élie Metchnikoff received
10700-707: The use of viruses or yeast , rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. The phage antibody libraries are a variant of phage antigen libraries. These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy and their detectability in diagnostic applications. Fermentation chambers have been used for large scale antibody production. While mouse and human antibodies are structurally similar,
10807-593: The use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-media conditioned by macrophages can be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, ascites is considered unethical . Several monoclonal antibody technologies have been developed recently, such as phage display , single B cell culture, single cell amplification from various B cell populations and single plasma cell interrogation technologies. Different from traditional hybridoma technology,
10914-443: The use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine . Molecular biology sits at the intersection of biochemistry and genetics ; as these scientific disciplines emerged and evolved in the 20th century, it became clear that they both sought to determine the molecular mechanisms which underlie vital cellular functions. Advances in molecular biology have been closely related to
11021-399: The work behind production of monoclonal antibodies is rooted in the production of hybridomas, which involves identifying antigen-specific plasma/plasmablast cells that produce antibodies specific to an antigen of interest and fusing these cells with myeloma cells. Rabbit B-cells can be used to form a rabbit hybridoma . Polyethylene glycol is used to fuse adjacent plasma membranes, but
11128-467: Was a landmark study conducted in 1944 that demonstrated that DNA, not protein as previously thought, carries genetic information in bacteria. Oswald Avery , Colin Munro MacLeod , and Maclyn McCarty used an extract from a strain of pneumococcus that could cause pneumonia in mice. They showed that genetic transformation in the bacteria could be accomplished by injecting them with purified DNA from
11235-495: Was conveyed to them by Maurice Wilkins and Max Perutz . Their work led to the discovery of DNA in other microorganisms, plants, and animals. The field of molecular biology includes techniques which enable scientists to learn about molecular processes. These techniques are used to efficiently target new drugs, diagnose disease, and better understand cell physiology. Some clinical research and medical therapies arising from molecular biology are covered under gene therapy , whereas
11342-414: Was the discovery of the structure of DNA . This work began in 1869 by Friedrich Miescher , a Swiss biochemist who first proposed a structure called nuclein , which we now know to be (deoxyribonucleic acid), or DNA. He discovered this unique substance by studying the components of pus-filled bandages, and noting the unique properties of the "phosphorus-containing substances". Another notable contributor to
11449-487: Was typically determined by rate sedimentation in sucrose gradients , a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate. Monoclonal antibodies A monoclonal antibody ( mAb , more rarely called moAb )
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