T. gondii, an intracellular protozoan parasite, is the etiological agent of the worldwide human and animal toxoplasmosis. Until now, there is no vaccine to prevent toxoplasmosis in humans. Clinical signs of toxoplasmosis are non-specific and are not sufficiently characteristic for a definite diagnosis. In fact, toxoplasmosis mimics several other infectious diseases. Therefore, diagnosis of the infection is important not only for treatment but from the point of view epidemiology and prevention. But diagnosis of any infectious disease depends on isolation of the organism and demonstration of an antibody response to the infection. In the case of toxoplasmosis the organism is difficult to isolate because it is very difficult to culture. The number of organism seen in histological sections or isolated from enlarged lymph nodes is very small. Isolation of organisms from other tissues is also a difficult and time consuming procedure. Diagnosis therefore depends on serological tests (Fleck, 1989).
i - ABSTRACT -
Production of recombinant monoclonalantibodies against 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.
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;
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.
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 forproduction 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.
It is now possible to produce various lactate-containing polyesters by one-step fermentation of recombinant
microorganisms equipped with the engineered PLA bio- synthesis system. When we consider the key reactions for the biosynthesis oflactate-containing PHAs in recom- binant microorganisms, two major problems should be solved for in vivo PLA biosynthesis system to become more competitive. The first major problem is related with lactyl-CoA biosynthesis using acetyl-CoA as a CoA donor. It is not an efficient way in a metabolic perspective for three reasons. First, consumption of acetyl-CoA for lactyl-CoA synthesis is not preferred for optimized cell growth since it is one of the most important central metabolites used in diverse cellular metabolism. Second, conversion of pyruvate into both acetyl-CoA and lactate hinders maximizing the lactyl-CoA pool, which ultimately affects PLA biosynthesis efficiency. Third, acetate is pro- duced when using acetyl-CoA as a CoA donor for lactyl- CoA synthesis. Acetate, at high concentration, is known to be detrimental to cell growth. Indeed, it was found to be a problem in fed-batch culture for the high-level pro- duction oflactate-containing polyesters (Jung and Lee, 2011).
☯ These authors contributed equally to this work.
Osteoclasts seem to be metabolic active during their differentiation and bone-resorptive activation. However, the functional role oflactatedehydrogenase (LDH), a tetrameric enzyme consisting of an A and/or B subunit that catalyzes interconversion of pyruvate to lactate, in RANKL-induced osteoclast differentiation is not known. In this study, RANKL treatment induced gradual gene expression and activation of the LDH A 2 B 2 isotype during osteoclast differentiation as well as the LDH A 1 B 3 and B 4 isotypes during osteoclast matura- tion after pre-osteoclast formation. Glucose consumption and lactateproduction in growth media were accelerated during osteoclast differentiation, together with enhanced expres- sion of H + -lactate co-transporter and increased extracellular acidification, demonstrating that glycolytic metabolism was stimulated during differentiation. Further, oxygen consump- tion via mitochondria was stimulated during osteoclast differentiation. On the contrary, depletion of LDH-A or LDH-B subunit suppressed both glycolytic and mitochondrial metabo- lism, resulting in reduced mature osteoclast formation via decreased osteoclast precursor fusion and down-regulation of the osteoclastogenic critical transcription factor NFATc1 and its target genes. Collectively, our findings suggest that RANKL-induced LDH activation stim- ulates glycolytic and mitochondrial respiratory metabolism, facilitating mature osteoclast for- mation via osteoclast precursor fusion and NFATc1 signaling.
This study investigated the effect of LDH-to-albumin ratio on prognosis in CRC, based on the high LDH and low albumin levels associated with poor prognosis in many cancers. The results indicated that high LDH-to-albumin ratio had an adverse effect on the prognosis in CRC patients undergoing curative resection. To our knowledge, this is the first study in the literature investigating the relationship between LDH-to-albumin ratio and prognosis in CRC patients. The oxidoreductase LDH, which converts pyruvate to lactate when oxygen is absent or in short supply, plays a crucial role in the metabolism of cancer cells. LDH-A is overexpressed in hypoxic carcinomas as well as metastatic cancer cells, and its levels correlate with tumor viability. In numerous tumor types, serum LDH levels are indirect markers of tumor hypoxia, neo-angiogenesis, and poor prognosis [12,24]. Additionally,
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 forproduction 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
portant for the prevention of disease and should be care- fully monitored.
The S protein of the TGEV known to be involved in cell adhesion is about 20 nm in size and highly glyco- sylated, which plays an important role to induce neutral- izing antibodies in pigs (Suñé et al, 1990; Godet et al, 1994), containing at least five antigenic determinants (Delmas et al, 1986; Correa et al, 1988; Laude et al, 1990). It could effectively be used to diagnose anti- bodies to TGEV through those antigenic determinant sites (Gebauer et al, 1991). Paired serological tests be- tween 2 and 6 months could inform when the mater- nally derived antibodies decay and the endemic TGEV or PRCV start to cause a problem in farms (Derbyshire et al, 1969; Saif and Wesley, 1999). Several serological assays found to be useful for the detection ofantibodies to TGEV, including the serum neutralization test (Harada et al, 1967) and indirect fluorescent antibody assays (Benfield et al, 1978). However, the agar gel immuno- diffusion test has been complained about discrepancies with a neutralizing antibody titration due to the too high specificity (Bohac and Derbyshire, 1976; Stone et al, 1976). Fluorescent antibody assays are more sensitive but the results could be subjective. Although the serum neutralization test is widely used as the most reliable method, it requires expertise and facilities, and it is dif- ficult to test a large amount of serum.
Geddes, 2016); however, recent research has identified a new intrinsic protein fluorescence pattern in the visible range, even if excited in the UV region (Breydo & Uversky, 2014). This kind of fluorescence emission pattern is commonly observed in amyloid aggregates which have a high proportion of β-sheets (Chan, Kaminski Schierle, Kumita, Bertoncini, Dobson, & Kaminski, 2013). The fluorescence was emitted in LDH treated with the different freezing rates, but was not observed in the untreated sample (Fig. 11). At the fastest freezing rate, the fluorescence intensity was significantly increased, indicating that a substantial amount of aggregates were formed. The aggregation degree of LDH increased as the freezing rate increased. This result demonstrated that aggregates rich in β -sheets were formed after freeze- thawing. However, it is unclear whether only amyloids were formed, or whether other aggregates also exist, because proteins can form aggregations with different structures such as amorphous aggregates, amyloid fibrils, and oligomers.
이번 연구에서 국내에서 조사된 여러 연구결과와 마찬가지로 경남 북부지역의 한우 및 젖소 사육농가 사육되는 소에서도 상당수가 N. caninum과 T. gondii 에 노출되어 있음이 밝혀졌다. 이 질병들에 대하여서 는 많은 연구가 이루어졌음에도 지금까지 효율적인 예방약 또는 치료제가 개발되어 있지 않은 상황으로, 농장에서는 혈청학적으로 항체를 보유한 개체는 도 태를 우선으로 고려하여야 하며, 예방 차원에서 축사 에 설치류의 접근을 막고, 사육하는 개와 야생에 방 치된 고양이의 우사나 사육소로의 접근을 차단하는 것이 농장의 경제적 피해를 줄이는 한 방안으로 생각 된다. 또한, 정확한 감염경로와 감염양상을 파악하기 위해 더 많은 연구가 체계적으로 이루어져야 할 것이 며, 효과적인 치료법 및 예방법 개발에 대한 연구도 수행돼야 할 것으로 판단된다.
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.
Introduction: Kakkalide have been isolated from the flower of Pueraria thunbergiana is metabolized into irisolidone by human in- testinal microflora. Pueraria thunbergiana is a plant of Leguminosae family contain significant amount of kakkalide. Kakkalide have been well known for their bioactivities including hepatoprotective, anti-inflammatory and antihyperlipidemic activity.