<?xml version="1.0" encoding="UTF-8"?><rss version="2.0" xmlns:content="http://purl.org/rss/1.0/modules/content/" xmlns:wfw="http://wellformedweb.org/CommentAPI/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:atom="http://www.w3.org/2005/Atom" xmlns:sy="http://purl.org/rss/1.0/modules/syndication/" xmlns:slash="http://purl.org/rss/1.0/modules/slash/" > <channel> <title>shark antibody – Creative Biolabs Blog</title> <atom:link href="https://www.creative-biolabs.com/blog/index.php/tag/shark-antibody/feed/" rel="self" type="application/rss+xml" /> <link>https://www.creative-biolabs.com/blog</link> <description>Specialized in providing custom biotechnology and pharmaceutical services</description> <lastBuildDate>Wed, 18 Apr 2018 06:09:09 +0000</lastBuildDate> <language>en-US</language> <sy:updatePeriod> hourly </sy:updatePeriod> <sy:updateFrequency> 1 </sy:updateFrequency> <generator>https://wordpress.org/?v=6.3.1</generator> <image> <url>https://www.creative-biolabs.com/blog/wp-content/uploads/2020/02/favicon.png</url> <title>shark antibody – Creative Biolabs Blog</title> <link>https://www.creative-biolabs.com/blog</link> <width>32</width> <height>32</height> </image> <item> <title>Definition and Production of Recombinant Antibody</title> <link>https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/</link> <comments>https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/#comments</comments> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Fri, 28 Apr 2017 08:47:01 +0000</pubDate> <category><![CDATA[Antibody Engineering Research]]></category> <category><![CDATA[Antibody Discovery]]></category> <category><![CDATA[antibody fragment]]></category> <category><![CDATA[antibody isotypes]]></category> <category><![CDATA[antibody production]]></category> <category><![CDATA[IgG]]></category> <category><![CDATA[phage display]]></category> <category><![CDATA[recombinant antibody]]></category> <category><![CDATA[ScFv]]></category> <category><![CDATA[shark antibody]]></category> <category><![CDATA[single-chain antibody]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=808</guid> <description><![CDATA[What is antibody (conventional antibody)? Before we begin to introduce recombinant antibody, we should learn about antibody. Antibody is known as immunoglobulins which are generally Y-shaped. They are peptide molecule secreted by<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/">Read More...</a>]]></description> <content:encoded><![CDATA[<h2>What is antibody (conventional antibody)?</h2> <p>Before we begin to introduce <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creativebiolabs.net/Hi-Affi-TM-Recombinant-Antibodies.htm" target="_blank">recombinant antibody</a></span>, we should learn about antibody. Antibody is known as immunoglobulins which are generally Y-shaped. They are peptide molecule secreted by b cells, mostly by differentiated B cells called plasma cells. Basically the function of antibodies is that control and stop pathogens and to assist in an immune response. They can protect us against infection and intoxication by mechanism of action for antibody functionality, such as antagonism, agonism, or cell killing via ADCC, ADCP, CDC, apoptosis PCD, or lysosomal-related PCD.</p> <p>Much has been learned about the antibody structure (Fig. 1). IgG is a heterotetrameric protein assembled from two identical heavy and light chains (H, L), assembled by disulfide bonds. Each chain has two regions, the constant region (C) and the variable region (V). There are two parts on the right chain. The top is the variable region of the light chain (VL) and the bottom is the constant region of light chain (VH). Similarly, the heavy chain is divided into four parts. The top one-fourth of it is the variable region of the heavy chain and the other three parts are the constant regions (CH1, CH2, CH3). The “arms” of the “Y” bind antigens. The tail of the “Y” is responsible for biological activity, such as C’ activity or binding to cells. The antigen binding region also called paratope is a small part of variable region which recognizes and binds to an antigen. Each arm of the Y shape of an antibody monomer is tipped with a paratope, which is a set of complementarity determining regions that short for CDR.<br /> <a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg"><img decoding="async" fetchpriority="high" class="aligncenter size-full wp-image-809" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg" alt="Structure of Antibody (Immunoglobulins)" width="876" height="382" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins.jpg 876w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-1-Structure-of-Antibody-Immunoglobulins-300x131.jpg 300w" sizes="(max-width: 876px) 100vw, 876px" /></a></p> <p style="text-align: center;">Fig. 1 Structure of Antibody (Immunoglobulins)</p> <h2>Antibody Isotypes</h2> <p>The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The sequence of the heavy chain defines the class of Ig, such that α, δ, ε, γ and μ heavy chains define the immunoglobulins A (IgA), immunoglobulins D (IgD), immunoglobulins E (IgE), immunoglobulins G (IgG), and immunoglobulins M (IgM) classes, respectively, each with a distinct role in the human adaptive immune system (Fig. 2). The light chains are either kappa (κ) or lambda (λ) isoforms for all classes.</p> <p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg"><img decoding="async" class="aligncenter wp-image-810" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg" alt="Fig. 2 Antibody Isotypes" width="404" height="505" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes.jpg 567w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-2-Antibody-Isotypes-240x300.jpg 240w" sizes="(max-width: 404px) 100vw, 404px" /></a></p> <p> </p> <p style="text-align: center;">Fig. 2 Antibody Isotypes</p> <p>Human IgA constitutes only 13% (2.1 mg/ml) of the antibody in human serum, but it is the predominant class of antibody in extravascular secretions. They are monmeric form in blood. The J chain is a 15-kD polypeptide consisting of 129 amino acid residues and one carbohydrate group. IgA1 is the most prevalent form in serum, but IgA2 is slightly more prevalent in secretions. Human IgE (190 kD) makes up less than 0.003% (0.4mg/ml) of the antibody in serum. IgE binds mast cells or basophils through its Fc part. IgE protects against parasites by releasing mediators that attract eosinophils. IgE is made up of about 13% carbohydrate. IgG is the most thoroughly studied in all five isotypes and recombinant antibody engineering is based on it. IgG constitutes about 80% (12.5 mg/ml) of the antibody in serum. Human IgG consists of four subclasses (isotypes), which are numbered in order of their serum concentrations (IgG1, IgG2, IgG3, and IgG4). The chief distinguishing characteristic among the four IgG subclasses is the pattern of inter chain linkages in the hinge region. IgM, primarily induced by polysaccharide antigens, is a 950-kD pentamer that makes up about 8% (1.25mg/ml) of the antibody in the serum. The five monomeric IgM molecules are arranged radially, the fab fragments pointing outward and the Fc fragments pointing to the center of the circle. IgM can quickly clump antigen and efficiently activate complement. IgM acts as one of the main receptors on the surface of mature B cells, along with IgD. When IgM is a surface receptor, it is in its monomeric form.</p> <h2>Recombinant Antibody (rAb) Definition</h2> <p>What are recombinant antibodies? Recombinant antibodies are antibody fragments generated by using recombinant antibody coding genes as a source and display technology, delivering high reproducibility, specificity and scalability. Unlike monoclonal antibodies (mAbs) which are produced using traditional hybridoma technologies, rAbs do not need hybridomas and animals in the production process if you only use synthetic genes.</p> <h2><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/recombinant-igG-production.html">Production of Recombinant Antibody</a></span></h2> <p>How is recombinant antibody produced? Let me show you the process of recombinant antibody production (Fig.3). As we know, antibody is composed of two heavy chains (VH) and two light chains (VL).</p> <p>Step 1: Variable genes of heavy chain and light chain antibody should be cloned by PCR and designed primers. About the detailed strategy of PCR, <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/scfv-library-construction-protocol.html">scfv library construction protocol</a> </span>is introduced in resource of Creative Biolabs. By the recombinant DNA technology, link purified genes of VH and VL with prokaryotic expression vector which requires select in advance based on your target recombinant antibody fragments. If you need scfv fragment antibody, linker must be designed in your PCR strategy.</p> <p>Step 2: Transformation: Electroporate ligation product of VH and VL received in previous step into cloned expression host cells. In this step, it is very important that highly competent of commercial source be recommended to obtain high transformation efficiency.</p> <p>Step 3: Choose appropriate antibody display technology, such as phage display (which can screening small antibody fragment to obtain large antibody library); ribosome display (which can get a library of a capacity of 10<sup>14 </sup>without limitation of transformation efficiency and acquire mutant antibody library); yeast display (which make human antibody expression more superior because that yeast expression system is similar with mammalian cells). According to these above procedures, some recombinant antibodies can be constructed successfully, such as scfv antibody fragments, fab antibody fragments, and single domain antibodies.</p> <p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg"><img decoding="async" class="aligncenter size-full wp-image-811" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg" alt="Production of Recombinant Antibody" width="1174" height="464" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody.jpg 1174w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody-300x119.jpg 300w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-3-Production-of-Recombinant-Antibody-1024x405.jpg 1024w" sizes="(max-width: 1174px) 100vw, 1174px" /></a></p> <p> </p> <p style="text-align: center;">Fig. 3 Production of Recombinant Antibody</p> <h2>Common Types of Recombinant Antibody</h2> <p>There are 3 common types of recombinant antibody fragments listed: Fab, scfv, single domain antibody (sdab) (Fig. 4).</p> <p>Fab fragment was originally defined as one of the cleavage products after treatment of rabbit IgG with papain, which cleaves the core hinge, resulting in two identical fab fragments and the intact Fc as products. The molecular weight of Fab fragment is ~50 kDa. The nonspecific protease pepsin cuts below the first disulfide bond in the hinge region giving rise to a F(ab’)<sub>2</sub> fragment. The molecular weight of F(ab’)<sub>2</sub> fragment is ~100 kDa. The Fab contains four domains: the heavy chain variable domain (VH) linked to constant domain 1 (CH1), and the light chain variable domain (VL) linked to a constant domain (CL). Due to the hinge region which allows for flexibility of fabs in relation to the Fc, intact IgGs have proven difficult to crystallize in forms suitable for diffraction studies, and thus, to this time, only a few structures for intact IgGs have been determined.</p> <p>Single-chain fragment variable (scfv) molecules combine the coding sequence of the variable heavy (VH) and sequence of the variable light chain (VL) domains of an antibody in a single-gene encoded format. The resulting polypeptides, with the variable light (VH ) and heavy chain (VH ) domains connected by a flexible peptide linker, were assemble into functional antigen-binding sites. The linker technology is a key step of success of constructing scfv antibody library. Scfvs have been developed as possible drug candidates in their own right, as well as components or domains of drug candidates.</p> <p>Single domain antibody (sdAb) is discovered in in camelids and nurse sharks that consisted of a lone VH domain, lacking a paired VL, attached to a constant region. The primary advantages of domain antibodies as compared with scFvs are generally better folding and stability characteristics, the absence of the linker, and size.</p> <p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg"><img decoding="async" loading="lazy" class="aligncenter size-full wp-image-812" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg" alt="Common Types of Recombinant Antibody" width="847" height="451" srcset="https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody.jpg 847w, https://www.creative-biolabs.com/blog/wp-content/uploads/2017/04/Fig.-4-Common-Types-of-Recombinant-Antibody-300x160.jpg 300w" sizes="(max-width: 847px) 100vw, 847px" /></a></p> <p style="text-align: center;">Fig. 4 Common Types of Recombinant Antibody</p> <p>Compared with conventional antibody, recombinant antibody (rAb) possesses some advantages, such as smaller size, monovalency, ease of engineering and manufacture, improved tissue penetration, no animal immunization and broader biodistribution, as well as lack of potentially deleterious Fc effector function. However, there are some drawbacks in recombinant antibodies, such as lower antibody yield, highly training and experienced technician. Most of scientists need to get them from outsourced companies because of complexity and intensive high technology of recombinant antibody production.</p> <p>Application of Recombinant Antibody (rAb):</p> <ul> <li>desire or requirement for a short circulating half-life in serum;</li> <li>a smaller biologic that would have broader tissue distribution or the ability to penetrate tumors;</li> <li>a molecule that can be manufactured in either yeast or <em>E. coli </em>to potentially reduce cost of goods or increase scale of manufacturing;</li> <li>a molecule lacking an Fc effector functionality to eliminate both cellular responses against the target and potential for dimerization of receptors due to bivalency;</li> <li>a <span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creativebiolabs.net/bispecific-antibody-production.htm" target="_blank">bispecific antibody </a></span>fragment, such as has been demonstrated by BiTEs (bispecific T cell engagers; diabodies and most recently, DARTs;</li> <li>a molecule lacking an Fc effector functionality to eliminate both cellular responses against the target and potential for dimerization of receptors due to bivalency; a molecule that can be manufactured in either yeast or <em>E. coli </em>to potentially reduce cost of goods or increase scale of manufacturing.</li> </ul> <p>Creative Biolabs is a professional custom service provider with extensive experience in antibody engineering. With over ten years of efforts, we have extended our services to over 26 countries. We provide recombinant antibody product, such as scfv antibody fragments, fab antibody fragments, single domain antibody, rabbit monoclonal antibodies, as well as recombinant antibody construction and expression services. If you are interested in learning more about how we can help with your projects, please don’t hesitate to contact us.</p> ]]></content:encoded> <wfw:commentRss>https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/feed/</wfw:commentRss> <slash:comments>50</slash:comments> </item> <item> <title>Single Domain Antibody Overview</title> <link>https://www.creative-biolabs.com/blog/index.php/single-domain-antibody-overview/</link> <comments>https://www.creative-biolabs.com/blog/index.php/single-domain-antibody-overview/#comments</comments> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Wed, 18 Jan 2017 02:40:15 +0000</pubDate> <category><![CDATA[Antibody Engineering Research]]></category> <category><![CDATA[Antibody Discovery]]></category> <category><![CDATA[shark antibody]]></category> <category><![CDATA[single domain antibodies]]></category> <category><![CDATA[single domain antibody]]></category> <category><![CDATA[single-chain antibody]]></category> <category><![CDATA[Therapeutic Antibody]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=765</guid> <description><![CDATA[Introduction Single domains represent the smallest known fragment still capable of binding antigen that can be isolated from a full-sized immunoglobulin. In work on the cloning of antibody genes and construction of<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/single-domain-antibody-overview/">Read More...</a>]]></description> <content:encoded><![CDATA[<h2>Introduction</h2> <p>Single domains represent the smallest known fragment still capable of binding antigen that can be isolated from a full-sized immunoglobulin. In work on the cloning of antibody genes and construction of human antibody libraries, Greg Winter and his colleagues found that single domains comprised of either V<sub>H</sub> or V<sub>L</sub> derived from human antibodies could also be stabilized as stand-alone, antibody fragments. Shortly thereafter, heavy-chain-only antibodies (HCAbs) were discovered to occur naturally in camelids where the binding domains constitute paired VHs with no light chains. HCAbs are thought to represent up to 50–80% of the antibody repertoire in camels and up to 10–25% of the antibody repertoire in other members of the Camelidae family such as llamas. Similar antibodies also lacking a CH1 domain and a light chain termed Ig-NAR have also been identified in nurse sharks, although these will not be discussed further in this overview (Figure 1). More recently, a new type of “domain antibody” was constructed using the CH2 domain of an IgG as the base scaffold into which CDR loops were grafted. These antibodies with unpaired antigen binding domains, and the single domain versions derived from them, have unique properties and have already shown promise as potential therapeutic antibodies. For the purposes of this review, these molecules (whether derived from V<sub>H</sub> or V<sub>L</sub> variable regions or isolated from camelids) will be described as <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Nanobody-Single-Domain-Antibody.html">single-domain antibodies</a></span></strong> (sdAbs).<br /> <img decoding="async" class=" size-full wp-image-751 aligncenter" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2017/01/Single-Domain-Antibody-Overview.png" alt="Single-Domain-Antibody-Overview" /></p> <p style="text-align: center;"><strong>Figure 1</strong> Single-domain antibodies(sdabs). Sdabs can be derived from either conventional mAbs or from HCAbs or IgNARs. Camelid V<sub>H</sub>s andV<sub>H</sub>Hs differ in four hallmark amino acid residues found in the V<sub>H</sub>/V<sub>L</sub> interface. (de Marco, A. 2011)</p> <p>Since these initial researches, there has been an explosion of interest aimed at understanding the unique properties of sdAbs. The primary advantages of sdAbs as compared with scFvs are generally better folding and stability characteristics, the absence of the linker, and size. Table 1 compares some of the high-level similarities and differences of Fab fragments, scFvs, and single domain antibodies. With a molecular weight of around 15 kDa, sdAbs are amenable to applications that require enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. Because of their small size, sdAbs are below the renal clearance cutoff, with a resulting half-life that is significantly shorter than full-sized mAbs. This can be extended using modifications such as PEGs (polyethylene glycols), by Fc-fusion, or by binding to long-lived serum components, for example, albumin. Fc-fusion has the additional benefit of allowing the exploitation of mAb-like properties such as sdAb-directed antibody-dependent cell-mediated cytotoxicity (ADCC) and FcRn recycling. In addition, sdAbs allow higher molar doses for the same (mg kg<sup>−1</sup>) amount when compared to monoclonal antibodies.</p> <p><strong>Table 1</strong> Comparison of general properties of different forms of antibody fragments</p> <table class="size-full aligncenter" style="margin: auto; width: 100%; text-align: center;" border="1" cellspacing="0" cellpadding="0"> <tbody> <tr> <td style="text-align: center; border-left: 0px;">Property</td> <td style="text-align: center;">FAb fragments</td> <td style="text-align: center;">ScFvs</td> <td style="text-align: center; border-right: 0px;">sdAbs **</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Size</td> <td style="text-align: center;">~50 kDa (FAbs)</td> <td style="text-align: center;">~25 kDa</td> <td style="text-align: center; border-right: 0px;">~12 kDa</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Proven commodity</td> <td style="text-align: center;">+++ (severalmarketed)</td> <td style="text-align: center;">+/– (late phaseClinical trials)</td> <td style="text-align: center; border-right: 0px;">+/– (early phase clinical trials)</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">High affinity</td> <td style="text-align: center;">+</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">+/– (depends ontechnology used)</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Manufacturing</td> <td style="text-align: center;">CHO, <em>E. coli</em>, yeasts</td> <td style="text-align: center;"><em>E. coli</em>, yeasts</td> <td style="text-align: center; border-right: 0px;"><em>E. coli</em></td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Stability (e.g. pH, tempera xidation, shear stress)</td> <td style="text-align: center;">+</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">++</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Ease of purification</td> <td style="text-align: center;">+/–</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">+/–</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Potential for ambient temperaturestorage formulation</td> <td style="text-align: center;">+/–</td> <td style="text-align: center;">–</td> <td style="text-align: center; border-right: 0px;">+</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Alternative routes ofadministration (e.g. intranasal,inhalation)</td> <td style="text-align: center;">–</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">+</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Potential for blocking enzyme active sites</td> <td style="text-align: center;">+/–</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">+ (particularly nanobodies and IgNARs)</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Tissue penetration</td> <td style="text-align: center;">++</td> <td style="text-align: center;">+++</td> <td style="text-align: center; border-right: 0px;">++++</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Cavity binding</td> <td style="text-align: center;">–</td> <td style="text-align: center;">–</td> <td style="text-align: center; border-right: 0px;">++</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Manufacturable bi-,tri-,and multi-specificity</td> <td style="text-align: center;">–</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">++</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Effector functionality (e.g. ADCC,ADPC, CDC)</td> <td style="text-align: center;">–</td> <td style="text-align: center;">–</td> <td style="text-align: center; border-right: 0px;">–</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Serum half-life (without half-lifeextension technology)</td> <td style="text-align: center;"><1 h</td> <td style="text-align: center;">< 1 h</td> <td style="text-align: center; border-right: 0px;"><1 h***</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Ability to extend half-life</td> <td style="text-align: center;">+++ (up to ~10–12d with PEGylation)*</td> <td style="text-align: center;">+++ (up to ~10–12 d with PEGylation)*</td> <td style="text-align: center; border-right: 0px;">+++ (up to ~10–12 d with PEGylation)*,***</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Potential for intracellular targeting</td> <td style="text-align: center;">–</td> <td style="text-align: center;">+/–</td> <td style="text-align: center; border-right: 0px;">+ (especially if disulfide bondsare removed)</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Ease of manipulation</td> <td style="text-align: center;">+</td> <td style="text-align: center;">+/– (some aggregation issues)</td> <td style="text-align: center; border-right: 0px;">+</td> </tr> <tr> <td style="text-align: center; border-left: 0px;">Ability to dimerize or concatenate</td> <td style="text-align: center;">–</td> <td style="text-align: center;">+</td> <td style="text-align: center; border-right: 0px;">++</td> </tr> </tbody> </table> <p>**General principles covering both human domain antibodies, camelid sdAbs, and IgN antibodies.<br /> ***Ability to make albumin binding domains that can be fused to domain antibodies, yielding better half-life with only modest increase in size.</p> <h2>Generation of sdAbs</h2> <p>Single domain antibodies are generally either isolated from Camelidae or based on human frameworks. A diverse sdAb (V<sub>H</sub>H) library can be prepared in two methods: by amplification of V<sub>H</sub>H genes from isolated lymphocytes of naïve or immunized members of Camelidae, or by introducing diversity into a V<sub>H</sub>H scaffold synthetically. Libraries of human sdAbs (V<sub>K </sub>or V<sub>H</sub>) are mainly based on synthetic libraries where diversity is introduced into one or more scaffolds.The recent generation of rodents expressing human HCAbs, devoid of rodent immunoglobulin chains, now provides an alternative route for isolating human single V<sub>H</sub> (but not V<sub>L</sub>) domains.</p> <p>Isolating sdAbs from immune camelid libraries is a popular way over the last decade. The most commonly used method involves the immunization of a member of the Camelidae family with the antigen of interest, recovery of lymphocytes from the immunized animal, preparation of the cDNA, generation of a <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/phage-display-service.html">phage display library</a></span></strong> using standard cloning protocols, and finally, three to four rounds of phage screening to enrich antigen-specific binders. Because of the naturally occurring affinity maturation process of antibodies <em>in vivo</em>, driven by somatic hypermutation, the repertoires of sdAbs obtained from immunized animals frequently contain a large proportion of high-affinity binders to the antigen used for immunization. Antigen-specific sdAbs can be also selected from naïve libraries isolated from nonimmunized animals which can be used for selections against multiple antigens. This approach is often at the cost of affinity, yielding clones with lower affinities to the cognate antigen because the library consists of naïve antibodies that have not undergone affinity maturation <em>in vivo</em>. The majority of the diversity present in camelid-derived sdAb libraries is generated <em>in vivo</em>; by contrast, the diversity present in synthetic libraries is achieved through the use of diversifying oligonucleotides in conjunction with combinatorial approaches. Synthetic libraries can be of use when there is a need for a sdAb against a target that is poorly immunogenic, for example, amyloid β fibrils. Diversity can be introduced into the human antibody scaffold through the use of both diversifying oligonucleotides and recombination of CDRs or by grafting CDRs from pre-existing naïve or immune human Fab antibody library repertoires. This allows the identification of specific binders with low nanomolar affinities to components of insulin-like growth factor (IGF) system that had not been screened from an earlier library based on this framework. Several approaches have been taken to generate human sdAbs that demonstrate both high affinity and good biophysical properties, since the stability of human sdAbs is variable and depends on both the framework used and the CDR sequences. Some organizations have developed transgenic mice and rats capable of expressing fully functional human HCAbs. Advances in methods of stably introducing artificial chromosomes into rodents, combined with silencing the endogenous IgG loci, have proved capable of allowing the generation of mice and rats that can produce these functional human sdAbs.</p> <p>Whatever the method used to generate the library or whether the camelid/human <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/single-domain-antibody-library-service.html">sdAb library</a></span></strong>, identification of a target-specific antibody suitable for therapeutic use needs <em>in</em> <em>vitro</em> screening and characterization. The most traditional method to enrich for antigen-specific binders from either camelid or human sdAb libraries is phage display, although a number of other display methods have been successfully investigated, including ribosome, yeast, and bacterial display. Phage display involves fusing libraries of sdAb genes to a phage coat protein and displaying the sdAbs on phage particle surface. The majority of sdAbs have been isolated in this way, owing to the relative simplicity and the ability to rapidly enrich binders of this technique. One potential shortcoming of phage display is the variable levels of sdAb display on the phage particle surface, which can lead to faster enrichment of clones with lower affinity but higher display levels. Alternative selection systems to phage display include both bacteria and yeast display, which are facilitated by fluorescent-activated cell sorting (FACS). SdAb affinity can be directly measured by using FACS, while it is still displayed on the cell surface can allow discrimination between clones differing by only a small degree in affinity. Another advantage of using yeast display is that the secretory pathways in yeast are similar to those of higher eukaryotes. However, the major limitation of sdAb display on the yeast has been its low transformation efficiency. All of the display techniques described above involve a cell transformation step, the efficiency of which limits the size of the library. Alternative display techniques hat do not require a cell transformation step, such as ribosome display, is viable alternatives to cell-based systems.</p> <h2>Outlook</h2> <p>Recently, a range of high-affinity sdAbs of both camelid and human origin have been isolated by <em>in vivo</em> and <em>in vitro </em>approaches against a broad scope of targets. These sdAbs have been found to applicate in both the therapeutic and diagnostic fields. Monomeric sdAbs have value as diagnostic tools and also therapeutically both in imaging, where their short life aids the production of high contrast images. sdAbs can also form the basis of a number of bi-specific formats either utilizing multiple sdAbs in tandem or through fusion to a more conventional mAb framework. sdAbs targeting tissue or cell-specific markers can be used to target payloads such as peptides, small molecules, and oligonucleotides.</p> <p>The modularity, good expression, and biophysical properties of sdAbs makes them an attractive option in exploring this broad range of formats and will aid their development from early stage clinical assets into medicines of value in the future.</p> <p> </p> ]]></content:encoded> <wfw:commentRss>https://www.creative-biolabs.com/blog/index.php/single-domain-antibody-overview/feed/</wfw:commentRss> <slash:comments>68</slash:comments> </item> <item> <title>Native™ Shark Antibody Discovery Used in Shark Monoclonal Antibody Production</title> <link>https://www.creative-biolabs.com/blog/index.php/native-shark-antibody-discovery-used-in-shark-monoclonal-antibody-production/</link> <comments>https://www.creative-biolabs.com/blog/index.php/native-shark-antibody-discovery-used-in-shark-monoclonal-antibody-production/#comments</comments> <dc:creator><![CDATA[biolabs]]></dc:creator> <pubDate>Thu, 08 Sep 2016 06:28:19 +0000</pubDate> <category><![CDATA[Antibody Engineering Research]]></category> <category><![CDATA[News]]></category> <category><![CDATA[antibody development]]></category> <category><![CDATA[Antibody Discovery]]></category> <category><![CDATA[phage display]]></category> <category><![CDATA[shark antibody]]></category> <guid isPermaLink="false">http://www.creative-biolabs.com/blog/?p=711</guid> <description><![CDATA[With years of experience in the field of antibody engineering, Creative Biolabs released Native™ Shark Antibody Discovery Service for shark antibody production. Creative Biolabs is glad to develop monoclonal shark antibody production<a class="moretag" href="https://www.creative-biolabs.com/blog/index.php/native-shark-antibody-discovery-used-in-shark-monoclonal-antibody-production/">Read More...</a>]]></description> <content:encoded><![CDATA[<p>With years of experience in the field of antibody engineering, Creative Biolabs released Native<img src="https://s.w.org/images/core/emoji/14.0.0/72x72/2122.png" alt="™" class="wp-smiley" style="height: 1em; max-height: 1em;" /> Shark Antibody Discovery Service for shark antibody production.</p> <p>Creative Biolabs is glad to develop monoclonal <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Native-TM-Shark-Antibody-Discovery-Service.html">shark antibody</a></span></strong> production service for antibody researchers from all over the world. With their shark immunization and <span style="color: #0000ff;"><strong><a style="color: #0000ff;" href="http://www.creative-biolabs.com/Phage-Display-Platform.html">phage display</a></strong></span> technology, the novel monoclonal shark antibodies can be obtained with high specificity and affinity.</p> <p><a href="http://www.creative-biolabs.com/blog/wp-content/uploads/2016/09/images.jpg"><img decoding="async" loading="lazy" class="alignleft size-full wp-image-712" src="http://www.creative-biolabs.com/blog/wp-content/uploads/2016/09/images.jpg" alt="images" width="275" height="183" /></a>As we know, sharks have been in existence for over 500 million years. From an evolutionary perspective of vies, they are among the oldest animals with a “modern” immune system not unlike that of humans. Shark blood contains large quantities of urea that makes their antibodies much more robust than that of other vertebrates. While urea protects sharks from dehydration, it can also destabilize sensitive protein molecules such as antibodies.</p> <p>Compared to the various immunoglobulin isotypes produced by other species, sharks, on the other hand, mainly produce IgM, IgW and IgNAR (immunoglobulin new antigen receptor).</p> <p>IgNAR is a heavy chain homodimer, which consists of five constant domains and one variable region (VNAR) for each heavy chain. Meanwhile, relevant researches also indicated that IgNAR has several disulfide bonds in unusual positions, an extended loop formed in CDR3, a deletion in the FR2-CDR2 region and smaller size than standard antibodies. These differences result in remarkable stability and penetrability of IgNAR and enable the function under extreme conditions.</p> <p>Meanwhile, it is much easier for V<sub>NAR</sub> to access to clefts, grooves or buried epitopes from the target surface that cannot be reached by larger antibody fragments, and it can also penetrate into dense tissues, such as tumors. In this way, V<sub>NAR</sub> is also considered a wonderful source for the development of single domain antibodies, which can contribute to various researches and pharmaceutical applications.</p> <p>With years of <strong><span style="color: #0000ff;"><a style="color: #0000ff;" href="http://www.creative-biolabs.com/custom-antibody-development.html">antibody development</a></span></strong> and research experience, scientists of Creative Biolabs have developed the Native<img src="https://s.w.org/images/core/emoji/14.0.0/72x72/2122.png" alt="™" class="wp-smiley" style="height: 1em; max-height: 1em;" /> Antibody Discovery Platform to generate monoclonal antibody with native and <em>in vivo</em> characteristics. This platform takes advantage of the antigen-specific B lymphocytes cytometry technology, and can generate large scale of candidate shark antibodies in one step.</p> <p>“Our scientists take pride in having earned great reputation from our global clients by satisfactorily completed thousands of relevant projects in the past decade. We are therefore confident in offering the best and most suitable service for our customer to meet each of their specific demands”, said Dr. Monika, scientific officer of Creative Biolabs.</p> <p><strong>About Creative Biolabs</strong></p> <p>Creative Biolabs is specialized in providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early antibody drug discovery and development.</p> ]]></content:encoded> <wfw:commentRss>https://www.creative-biolabs.com/blog/index.php/native-shark-antibody-discovery-used-in-shark-monoclonal-antibody-production/feed/</wfw:commentRss> <slash:comments>53</slash:comments> </item> </channel> </rss>