جستجوی مقالات مرتبط با کلیدواژه « نانوبادی » در نشریات گروه « پزشکی »

The development of antibody fragments is growing due to problems associated with commercially available antibodies including their large size and expensive and time-consuming production processes. Nanobodies are fragmented antibodies that are found naturally in the serum of the Camelidae family. Nanobodies, as the smallest functional part of antibodies, have many applications in the diagnosis and treatment of various diseases, especially in tumors. However, their small size causes them to be quickly removed from the blood circulation after administration, which restricted their use in the clinics. To solve this issue, various methods have been proposed to increase their plasma half-life, generally using two mechanisms: (1) the endosomal recycling of neonatal Fc receptor and (2) the increase of the hydrodynamic volume. The first mechanism naturally occurs in the body and leads to an increase in the plasma half-life of immunoglobulins and albumin. The chemical conjugation of nanobodies to the synthetic polymers, genetic fusion to polymer-mimetics peptides, and dimerization of the nanobodies increase the plasma half-life of nanobodies via the second mechanism. In this review, first, we will introduce the nanobodies, and in the following, we will discuss various methods that have been developed to increase their plasma half-lives.

Background and Monoclonal antibodies market has grown since approval of Muromonab-CD3 (trade name Orthoclone OKT3) in 1986 as immuno- suppressor. Nevertheless, the undesirable effects of these large specific biomolecules calls for further attempts for more effective alternatives. Variable domain of heavy chain (VHH) are the smallest antibody fragments called Nanobody (Nb), derived from camelid heavy chain-only antibodies (HcAb) by DNA recombinant gene technology. In this study, some of the unique structural and functional features of Nbs are summarized. The main context of this review speculate about several experimental therapeutic applications of Nbs against a wide range of diseases ranging from infectious, animal toxin, autoimmune, and cancer disease in three different functional platforms. Data about structural and functional features of Nbs and their therapeutic effects were retrieved from articles indexed in PubMed, Science direct, and Google Scholar. Development and employment of Nbs in different researches show that their unique physicochemical properties make them a useful tool for biomedical applications and drug discovery. This is because of some exceptional feathers such as small size, extraordinary stability, high affinity even for occluded epitopes, and cost-effective production. This review provides an overview of efficacy and success of Nbs as more promising therapeutic agents over conventional antibodies against multiple diseases.

Staphylococcus aureus bacteria causes many different diseases by secretion of various enterotoxins. Therefore, it is necessary to develop ways that facilitate the detection of enterotoxins. Nowadays, immunochemical methods which are based on monoclonal antibody technology are used. The heavy chain antibodies that are called VHH or Nano body were found in blood serum of the Camelidae family. The unique properties of this antibody such as their binding to small molecules like toxins make them attractive candidates for the development of immunodiagnostic tests. The present study was done to achieve a VHH molecules against Staphylococcus enterotoxin B.
Freighting phage library for isolate private Nano bodies against enterotoxin B was done in previous works. Next, pCANTAB 5E vector that consists VHH, extracted from E.coli bacteria strain xl1blue, and after doing PCR process with relative primers, sub cloning in pET21a() as an expression vector with cut sites NdeI and XhoI was done. Transformation in E.coli bacteria strain BL21(DE3) was done. Then, the cells effected with IPTG and producing time, and other terms were optimized. Finally, the expression of the protein with SDS-PAGE and western blot techniques was evaluated.
For proving cloning of nano body gene in pET21a () vector, nucleotide sequence of gene was analyzed, and transforming to E.coli bacteria strain BL21(DE3) was successful. After inspiration, active protein in cell was seen by SDS-PAGE technique and proved by western blot.
cloning, sub cloning, and nonabody expression were surveyed in this research. Production of this protein can help to develop new therapeutic methods and produce vaccine against enterotoxin B of Staphylococcus aureus

VHH (Nanobody) is a camel antibody and is the smallest unit binding to antigen. The small size of nanobodies is the greatest advantage that makes easy genetic manipulation of them. The main aim of this study was construction of a single domain antibody library from camel immunized with human breast adenocarcinoma-SKBR3 cell line. At first, in this experimental study, SKBR3 cell extract was subcutaneously injected to a camel in three rounds. Then, total RNA was extracted from spleen of camel and VHH fragments synthesized and amplified using RT-PCR method. The VHH fragments were inserted into Pcomb3X phagemid and recombinant constructs were electroporated into DH5α bacteria. Diversity of provided library was studied by enzymatic fingerprinting techniques. Finally, VHH expression was analyzed by SDS-PAGE. In this study, a camel antibody library with 105 colonies was constructed. Also, the enzymatic fingerprinting showed a high diversity in this antibody library. Primary studies by SDS-PAGE showed that VHH protein with a molecular weight of 15 kDa is expressed in transformant bacteria. Providing an immune antibody library against SKBR3 cell line makes an option for isolation of specific VHH antibodies against different breast cancer antigens.

Harnessing immune system and its powerful arm، T lymphocytes، against tumor cells are yielding promising results in cancer immunotherapy. Using two arms of immune system in the designing of engineered T cells expressing chimeric receptors with anti-HER2 nanobody (camelid single domain antibody) seems to be an effective strategy in the targeted cancer therapy. HER2 specific nanobodies were used as a recognition site for constructing chimeric receptors NbHER2-HHIgG3-CD28-OX40-CD3ζ (elongated) and NbHER2-HIgG3-CD28-OX40-CD3ζ (short) on the membrane of T cells. Expression of chimeric receptors was evaluated by RT-PCR. Function of engineered T cells against cancer cells was assessed by the secretion of interleukin 2 and LDH cytotoxicity assay. Elongated chimeric receptors with nanobodies RR6 and RR16 that target juxtamembranal domain of HER2 antigen functioned better than elongated constructs containing nanobodies RR4 and RR10. In contrast، nanobodies RR4 and RR10 used in the short chimeric receptors showed substantial results. Based on binding properties of nanobody، functional chimeric receptors can be designed and engineered T lymphocytes containing the mentioned receptors can be generated that substantially recognize and kill breast cancer cells.

The aim of this study was preparation of polyethyleneimine (PEI) nanoparticles targeted with anti-TAG72 nanobody for delivery of t-Bid gene construct into the human colonic adenocarcinoma cells. First of all، purified recombinant nanobody was verified by SDS-PAGE، western blotting and ELISA. Targeted polyplexes were constructed by reacting polyethylene glycol (PEG) with PEI and covalent bonding of nanobody to nanoparticles. Surface charge and size of the resulting nanoparticles were evaluated by Malvern zeta sizer and nanosight instruments. Efficiency of constructed gene vector for killer gene delivery into the colonic adenocarcinoma cells was assessed using real time PCR assay. Production of nanoparticles with the average size of 168 nm and +7. 18 surface charge was confirmed by nanosight and Malvern zeta sizer. Gel retardation assay verified the efficiency of carrier for pDNA complexation. Real time PCR results confirmed the targeted gene expression in the cancerous cell lines. Results of this study confirmed the efficiency of targeted polyplexes in gene delivery and their potency for targeted gene delivery into the cells.

Chimeric antigen T cell receptors provide a good approach for adoptive immunotherapy of cancer. In this new kind of chimeric T cell receptor, nanobodies are replaced as variable fragment of T cell receptor. Nanobodies (VHH) are the smallest fragments of antibodies that have great homology to human VH and low immunogenic potential. VHH-hing-CD28-CD3و construct was made in our laboratory. Tumor cells rarely provide costimulatory signals and hence CAR receptors that transmit just a CD3و signal can only initiate target cell killing and interferon γ release and fail to induce full activation. Although incorporation of a CD28 component results in IL-2 (Interlukin 2) release and limited proliferation, T cell activation remains incomplete. To optimize CAR signaling, tripartite endodomains were constructed which consist of CD28-OX40-CD3ζ. During T cell activation, OX40 transmits a potent and prolonged T cell activation signal and it is crucial for maintaining an immunological response. We designed overlapping primers that contain 60% of OX40 sequence, then, CD28-Ox40-CD3ζ region was synthesized by SOE-PCR, consequently, it was replaced with CD28-CD3ζ region in VHH-Hing-CD28-CD3ζ construct. Then, this construct was transfected in Jurkat T cells, so IL-2 secretion and T cells proliferation were increased. CD28-OX40-CD3ζ tripartite cytoplasmic domain provided a full complement of activation, proliferation, and survival signals for enhanced anti-tumor activity. With IL-2 and semi-quantitative RT PCR, it is concluded that mRNA expression of pCDNA-1-hinge-CAR-OX40 and pCDNA-2-hinge-CAR-OX40 constructs are the same but the function of pCDNA-2-hinge-CAR-OX40 construct is more than the others.

The human papillomavirus (HPV) is the main cause of cervical cancer. The symptoms of the disease are non-specific and it can be transferred from one person to another one. In order to prevent the spread of HPV virus and reducing mortality rate of cervical cancer, early detection programs are needed. Accurate and highly sensitive test is essential for screening examination. So, in this study, our aim was enrichment of nanobody library against HPVs L1 proteins to isolate clones which produce specific nanobody against L1 proteins. In the future, these nanobodies can be used in highly sensitive diagnostic kits for early detection of cervical cancer. The phagmids of polyclonal library were amplified in Escherichia coli strain TG1. Panning of library and selection of clones were carried out against HPV L1 protein. The titer of output phagmids and optical density in Phage-ELISA were evaluated. The best clones were isolated by monoclonal phage ELISA method and checked by sequencing and colony PCR. An increase in the number of output clones in consecutive rounds of panning and the rise in the difference of OD450 in phage ELISA shows the accuracy of the enrichment process. At last, 4 clones were isolated as best ones. This study proved that after three rounds of panning, the phagmid library was enriched for anti HPVs L1 nanobodies and isolation of specific clones against this antigen were accomplished. By use of these anti HPV L1 nanobodies in laboratory kits, new hopes for early diagnosis of precancerous lesions with high reliability can be achieved.

The antigen binding fragments of camelids heavy chain antibodies are comprised in one single domain (VHH). The crystal structure of an isolated VHH indicated that it is a problate particle of 2.5 nm in diameter and <4 nm high, and has been referred to as a nanobody. The very close similarity of these molecules to human VH3 illustrates the potential application of these novel products as an immunodiagnostic immunotherapeutic reagent. One of the key components in roduction, characterizationand application of nanobodies in detection is the anti-nanobody HRP conjugate. In this study, we reported high expression and purification of some nanobodies against tumor markers. The nanobodies genes were sub-cloned into a pSJF9 vector to over-express the protein coupled with fusion tags in E. coli TG1. The expressed nanobodies were purified by immobilized metal affinity romatography (IMAC). Described here is the preparation, purification and haracterization of anti-nanobody antibody HRP conjugate for use in the various nanobody detection systems. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the purified nanobodies. Because of application of several biochemical modifications, the produced anti-nanobodies HRP conjugate have efficient sensitivity and specificity.