i - ABSTRACT -
Production ofrecombinantmonoclonalantibodiesagainst HSET
HSET (Human Spleen Embryo Testis), belonged to the kinesin 14A family, is a protein required for centrosome clustering during cell division. Since cancer cells with extra-centrosomes could lead to multi-polar division, resulting in genomic instability and cell death, these cancer cells depend on the centrosome clustering activity of HSET that helps normal chromosome segregation by focusing several centrosomes into two poles. As the first step of the strategy to inhibit the function of HSET in cancer cells, we produced mouse monoclonalantibodies (mAbs) against HSET using a hybridoma technique and characterized them. Peptide antigens of 12 amino acid (aa)-length, designed from the regions corresponding to N-terminus (1-134th aa) and C-terminus (626-672th aa) that do not have homology with other kinesin molecules, were used for mouse immunization. Total 28 of mAbs against the peptides from N-terminus HSET were obtained, but no mAb against C-terminus of HSET. In ELISA with the Abs in ascitic fluids against yeast surface displayed HSET, clone 8C346 showed high affinity. In ELISA using Abs purified from hybridoma culture supernatants, clone 1C274, 2C280, 2C281, 6C407, 9C352, and 9C353 showed relatively strong binding to Whole HSET protein. We expect that Abs with high binding to HSET could inhibit HSET function, resulting in selective death of cancer cells.
Kyoung-Hui Kong, Myung-Joo Oh and Wi-Sik Kim †
Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University, Yeosu 59626, Korea
We developed and subsequently characterized mouse monoclonalantibodies (mAbs) against marine birnavirus (MABV). Eight hybridoma clones secreting mAbs against MABV were established. All eight mAbs (8G6, 11C3, 15E3, 17H6, 32A6, 35A7, 38B5, and 47E3) were reacted with viral protein 3 of MABV in MABV-infected CHSE-214, whereas, no reactivity was observed in normal CHSE-214 by western blot analysis. Moreover, these eight mAbs were strongly reacted with MABV, and no cross-reactivity has been observed against other five fish viruses (hirame rhabdovirus, infectious hema- topoietic necrosis virus, nervous necrosis virus, spring viraemia of carp virus, and viral hemorrhagic septicemia virus), although five mAb (11C3, 15E3, 17H6, 32A6, and 38B5) reacted with both MABV and infectious pancreatic necrosis virus by enzyme linked immunosorbent assay (ELISA). These results indicate that the mAbs can be of value in MABV detection.
*Indirect fluorescence antibody assay, Whole: IBDV-infected CEF cells, rVP2: IBDV VP2 recombinant baculovirus-infected Sf9 cells, rVP234: IBDV VP234 recombinant baculovirus-infected Sf9 cells, **Neutralizing activity, ***Not determined.
간접형광항체법과 면역조직화학염색법(immunohistoche- mistry assay, IHC)을 다음과 같이 실시하였다. IHC를 위하여 감염된 조직을 10% neutral buffered formalin 용액으로 48시간 고정한 후 일반적인 조직처리 과정 을 거쳐 조직절편을 준비하였다. 염색 전에 조직절편 은 xylene을 사용하여 wax를 제거하였으며 3% 과산 화수소가 함유된 methanol로 30분간 처리하여 조직 내의 peroxidase 활성을 제거하고 PBS로 3회 세척하 였다. 조직 내의 항원을 노출하기 위해서 2N HCl을 37 o C에서 20분간 처리 후 PBS로 3회 세척한 후 0.1%
46 방지형 · 김위식 · 김춘섭 · 김종오 · 오명주
이용한 면역학적인 방법(enzyme-linked immun- osorbent assay (ELISA), indirect fluorescent anti- body test, immunohistochemistry 등), 병리조직학적 방법 등이 사용되고 있다 (OIE, 2017). 이들 검사 방법 중, PCR과 병리조직학적 검사 방법은 국내 에서 WSSV를 검출하거나 WSD를 진단하는데 사 용되고 있다. 그러나 면역학적인 방법은 WSSV에 대한 특이 항체가 보급되어 있지 않아 사용되지 않고 있다. 본 연구에서는 양식 현장에서 신속한 진단에 용이하게 적용될 수 있는 면역학적 검사법 의 개선과 보급을 위한 목적으로 WSSV에 대한 단클론 항체(monoclonal antibody, MAb)를 제작 한 후 항체의 특성을 평가하였다. WSSV의 VP28은 다양한 면역학적 검사법에 유용하게 사용될 수 있 음이 보고되어 있어 (Sithigorngul et al., 2006;
Production stability is one of the most important criteria for the selection of rCHO cell lines in industry due to the inherent instability of CHO cells 22,35 . For the DHFR-based system, causes for the production instabil- ity during long-term cultures have been extensively studied, and the two major mechanisms are a loss of gene copy number 5,6 and epigenetic gene silencing such as methylation 4,36 . For the GS-based system, information on production instability is somewhat limited. Possible causes for production instability in the GS-based system have been attributed to gene copy number and methylation 21 , lower cumulative cell time values and increased sensitivity to cellular stress 37 , and a gradual appearance of a secondary less producing population 20 . In this study, to examine the production stability of the 24 high producing clones selected at various MSX concentrations, cells were continuously sub-cultured for 30 passages, which was determined by considering the manufacturing processes of scale up from a frozen vial to a production-scale manufacturing process 38 . Regardless of the host cell lines and the MSX concentration up to 50 μM used for selection, the q mAb of most clones decreased during 30 passages. Despite the decreased q mAb , 13 clones showed no change in the relative gene copy number, indicating a mechanism other than loss of gene copy number is responsible for the production instability in those clones. Such a weak correlation between the changes of the relative gene copy numbers and q mAb also suggest that these high producing clones did not undergo GS/MSX-mediated gene amplification in a single round of selection.
TGEV has an antigenic determinant site capable of producing a major neutralizing antibody in the spike protein among the N, M, and S proteins. ELISA test made only with S protein will present consistent results with the commonly used serum neutralization test will reduce confusion in applying the results. Therefore, in this study, the recombinant S protein of TGEV pro- duced in the baculovirus expression system was used to develop the MAC-ELISA with high sensitivity and spe- cificity compared with the serum neutralization test.
Tordo, N., Benmansour, A., Calisher, C., Dietgen, R., Fang, R.-X., Jackson, A.O., Kurath, G., Nadin-Davis,
S., Tesh, R.B. and Walker, P.J.: Family Rhabdovir- idae. In Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U and Ball, L. A., editors. Virus taxon- omy: Eighth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, London, United Kingdom. pp. 623-644, 2005.
Therefore, we produced MAbs targeting viral proteins of a Korean field isolate to decrease the mismatches be- tween MAbs and viral proteins. IFA results showed that total 6 NP- and NS1-specific MAbs reacted to all other subtypes of AIVs in this study. Non-structural viral pro- teins commonly shared higher amino acid sequence sim- ilarities among AIV subtypes compared to surface struc- tural proteins, HA and NA. Therefore, these 6 MAbs could be used as new materials for AIV diagnostics such as ELISA and rapid immunochromatographic assay (RIA).
6) 단클론항체를 생산하는 세포군 선택
단클론항체(monoclonal antibody)를 생산하는 단세포군(monoclone)의 선택은 제한개수희석법(limiting dilution)을 시행하였다. 75-cm 2 culture flask에서 자란 세포들을 수확하여 96-well plate의 well당 0.25개씩 되도록 세포개수를 희석하여 96-well plate에 넣고 37°C, 5% CO 2 항온기에서 배양하였다. 그 후 well당 1개의 세포만 들어간 것을 확인하고 충분히 자란 well을 선택하였다. 그 세포배양액을 이용하여 ELISA를 시행하였고 항체분비가 확인된 세포는 24-well plate로, 그 다음 75-cm 2 culture flask로 계대배양하여 37°C, 5% CO 2 항온기에서 배양하였다.
Rotaviruses have been known to be a major etiological agent of gastroenteritis in both infants and young animals. Subsequently new rotaviruses, which were morphologically indistinguishable but antigenically and electrophoretically distinct with each other, were reported from several animals throughout world including Korea. These new rotaviruses were named as non-group A or group B or group C rotaviruses and so on. It has been very difficult to isolate and grow the non-group A rotaviruses in vitro, and this has greatly limited the characterizations of non-group A rotaviruses and serological studies. In this study, monoclonalantibodies (MAbs) against porcine non-group A rotavirus were produced and characterized. The VP6 gene of porcine group C rotavirus Korean isolate(#06-52-1) was cloned and expressed. For expression of VP6 gene, baculovirus expression system was applied. The VP6 gene and expressed protein in the recombinant virus were confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test and Western blot, respectively. The expressed VP6 was used for MAbs production. The MAbs produced in this study would be promising as diagnostic reagents for detection of group C rotavirus infection.
Departments of Microbiology, a Pathology, c Pediatrics, f and Genetics, g School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA; Department of Pediatrics, School of Medicine, Ewha Womans University, Seoul, South Korea b ; University of Texas Health Science Center, School of Public Health, Houston, Texas, USA d ; School of Pharmacy, Sungkyunkwan University, Suwon, South Korea e
The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface pro- tein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant ␣-helical or proline- rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels ofantibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonalantibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibod- ies to PspA.
Song et al., 2015; Huang et al., 2016; Banerjee and Jaiswal, 2018). 신속 진단키트는 간단하게 구성되
어 있고 크기가 작아 쉽게 휴대할 수 있으며, 측정 장비가 요구되지 않고 현장에서 누구나 쉽게 빠른 시간 내에 (10분 이내) 질병을 진단 할 수 있는 장 점을 가지고 있다. 본 연구에서는 현장검사용 VHS 신속 진단키트 개발을 위한 기초연구로서 VHSV 에 대한 단클론 항체(monoclonal antibody, MAb)를 생산하고자 한다.
Abstract Antibodies are powerful and versatile tools to play a critical role in the diagnosis and treatment of many diseases.
Their application has been enhanced significantly with the advanced recombinant DNA and heterologonous expression technologies, allowing to produce immunotherapeutic proteins with improved biofunctional properties. However, with currently available technologies, mammalian cell-based therapeutic antibody production, as an alternative for production in humans and animals, is often not plentiful for passive immunotherapeutics in treatment of many diseases. Recently, plant expression systems for therapeutic antibodies have become well-established. Thus, plants have been considered to provide an attractive alternative production system for therapeutic antibodies, as plants have several advantages such as the lack of human pathogens, and low cost of upstream production and flexible scale-up of highly valuable recombi- nant glycoproteins. Recent advances in modification of post- translational processing for human-like glycosylation in transgenic plants will make it possible that plant can become a suitable protein expression system over the animal cell-
MAbs have been broadly utilized as medicine, as well as diagnostic tools for virus infections. MAbs are generated by hybridoma technology where mice are injected with an im- munogenic antigen and then the splenocytes are utilized to fuse with myeloma cells . In this regard, antigens of high purity and with native protein folding are a pre-requisite for generating neutralizing antibodies that can target a native form of viral surface proteins. DNA vaccines are thought to be useful at generating the native from of viral antigens as they tend to express their proteins in cells in a manner similar to real viral infection. However, DNA vaccines are less effective at producing IgG-secreting hybridomas, possibly due to the presence of un-methylated CpG sequences in the DNA vac- cines that stimulate polyclonal B cell activation . In the Lee et al. (2017) study , use of protein boosting immuni- zation was effective at reversing the preference for produc- tion of IgM-secreting hybridomas over IgG-secreting hybrid- omas. Moreover, multiple immunizations with GP after GP DNA vaccination might be useful at increasing the chance of generating antibodies with higher antigen binding affinity. In this context, it is highly likely that an appropriate application
혈액으로부터 IgM을 검출할 수 있다면 백신의 효 능을 평가할 수 있을 뿐만 아니라 병원체에 감염되 지 않은 어류를 선별하거나 병원체에 대한 감염 이력 등을 확인할 수 있다 (LaPatra, 1996; Watanabe et al., 1998; 2000; Kim et al., 2008; Kole et al., 2019). 하지만 혈액에 존재하는 IgM을 검출하기 위 해서는 IgM을 인식하는 항체가 필요하다. 현재까 지 능성어 IgM에 대한 항체는 보급되어 있지 않아 본 연구에서는 능성어 IgM에 대한 단클론 항체 (monoclonal antibody, mAb)를 생산하고자 하였다.
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Quanti ﬁcation of secretome costs. A genome-scale model of protein secretion was adapted for use here 24 using MATLAB 2018B [https://github.com/
LewisLabUCSD/CHOSecretoryKO]. Brieﬂy, this model was adapted to focus on the bioenergetic costs of protein synthesis and translocation of each host-cell secreted protein. Information on each secreted protein in CHO cells was obtained, including mRNA and protein sequence, signal peptides, etc. We then simulated the cost of producing each protein. Each cost was scaled by the measured mRNA levels using published RNA-Seq from CHO –S cells grown under similar conditions 25 , and this was used as a proxy for the relative resources allocated to each secreted protein. Entrez gene identiﬁers were matched to their corresponding entry number in the UniProt database to determine presence of signal peptide. For those genes without a UniProt match, we used the online tool PrediSi 45 to determine the presence of a signal peptide. To estimate ATP cost of secretion for all secreted genes identi ﬁed, we added the following costs, adapted from the genome-scale model of protein secretion 24 . First, the energy cost of protein translation was equal to 4xL ATP molecules where L is the length of the amino acid sequence. Next, the average cost of signal peptide degradation was equal to 22 ATP molecules. Finally, the energetic cost of translocation across ER membrane was equal to L/40 + 2.
the induction of multiple cytokines. 11 Similar to IRF-3 and IRF-7, IRF-5 is a direct transducer of virus-mediated signaling. However, this only occurs with specific viruses such as the Newcastle disease virus, vesiculostomatitis virus, and herpes simplex type 1 virus. 10,12 It also plays a role in the expression of cytokines and chemokines. 10,12,13 IRF- 5 is a direct target of p53. Its expression is modu- lated by p53, 14 and it has a role in the p53-indepen- dent proapoptotic signaling pathway. 15,16 Recent studies have reported the association between IRF-5 and systemic lupus erythematosus. 17,18 In a gene chip study using overexpressed B cells which contained IRF-5 or IRF-7, the presence of IRF-5 was related to a strong immune response and adhesion genes. The presence of IRF-7, however, selectively upregulated the expression of mitochondrial genes and DNA repair genes. 19 This suggests a distinct role for IRF-5. The IRF family of proteins resides in the cytoplasm of resting cells. They are activated by phosphorylation on the C terminus, and are transported to the nucleus after homo- or hetero- dimerization. 10,13 IRF-5 dimerizes either with itself or with IRF-3, and activates IFNA gene transcrip- tion. 10,13 However, the heterodimerization of IRF-5 with IRF-7 represses IFNA transcription in virus- infected cells which were cotransfected with IRF-5 and IRF-7. 10,13 Recently, IRF-5 was found to have an important role in TLR signaling and the induction of proinflammatory cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)- . It is impaired in cells from IRF-5-deficient α mice, 11 suggesting that IRF-5 is generally involved downstream of the TLR signaling pathway. IRF-5 associates with both MyD88 and TRAF-6, and is translocated to the nucleus in a MyD88-dependent fashion. 11 However, many of the downstream mediators of the IRF-5 pathway need further iden- tification.