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2.
Article in English | WPRIM | ID: wpr-186607

ABSTRACT

Minimal residual disease (MRD) following B-lymphoblastic leukemia (B-ALL) treatment has gained prognostic importance. Clonal immunoglobulin heavy chain (IGH) gene rearrangement is a useful follow-up marker in B-ALL owing to its high positivity rate. We evaluated the performance and clinical applicability of a next-generation sequencing (NGS) assay for IGH rearrangement in B-ALL MRD monitoring. IGH rearrangement was tested by using fluorescence PCR-fragment analysis and the NGS assay in eight B-ALL patients. The NGS assay was run on two platforms: the Ion Torrent PGM (Thermo Fisher Scientific, USA) (18 samples from 1st to 7th patients) and the MiSeq system (Illumina, USA) (four samples from 8th patient). All initial diagnostic samples and four follow-up samples were positive for clonal IGH rearrangement with fluorescence PCR-fragment analysis and the NGS assay, and six follow-up samples were positive only with NGS. In one case with BCR-ABL1 translocation, BCR-ABL1 quantitative PCR was negative but the NGS IGH assay was positive just prior to full-blown relapse, suggesting the high sensitivity and clinical utility of the NGS assay. The NGS assay is proposed for MRD monitoring in B-ALL Additional studies are needed to confirm the clinical implications of cases showing positive results only in NGS.


Subject(s)
Fluorescence , Follow-Up Studies , Gene Rearrangement , Humans , Immunoglobulin Heavy Chains , Immunoglobulins , Leukemia , Neoplasm, Residual , Polymerase Chain Reaction , Recurrence
3.
Chinese Medical Journal ; (24): 135-142, 2017.
Article in English | WPRIM | ID: wpr-303185

ABSTRACT

<p><b>BACKGROUND</b>The established clinical staging systems (Rai/Binet) of chronic lymphocytic leukemia (CLL) cannot accurately predict the appropriate treatment of patients in the earlier stages. In the past two decades, several prognostic factors have been identified to predict the outcome of patients with CLL, but only a few studies investigated more markers together. To predict the time to first treatment (TTFT) in patients of early stages, we evaluated the prognostic role of conventional markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system, the CLL prognostic index (CLL-PI).</p><p><b>METHODS</b>Taking advantage of a population of 406 untreated Chinese patients with CLL at early and advanced stage of disease, we identified the strongest prognostic markers of TTFT and, subsequently, in a cohort of 173 patients who had complete data for all 3 variables, we integrated the data of traditional staging system, cytogenetic aberrations, and mutational status of immunoglobulin heavy chain variable region (IGHV) in CLL-PI. The median follow-up time was 45 months and the end point was TTFT.</p><p><b>RESULTS</b>The median TTFT was 38 months and the 5-year overall survival was 80%. According to univariate analysis, patients of advanced Rai stages (P < 0.001) or with 11q- (P = 0.002), 17p- (P < 0.001), unmutated IGHV (P < 0.001), negative 13q- (P = 0.007) and elevated lactate dehydrogenase levels (P = 0.001) tended to have a significantly shorter TTFT. And subsequently, based on multivariate Cox regression analysis, three independent factors for TTFT were identified: advanced clinical stage (P = 0.002), 17p- (P = 0.050) and unmutated IGHV (P = 0.049). Applying weighted grading of these independent factors, a CLL-PI was constructed based on regression parameters, which could categorize four different risk groups (low risk [score 0], intermediate low [score 1], intermediate high [score 2] and high risk [score 3-6]) with significantly different TTFT (median TTFT of not reached (NR), 65.0 months, 36.0 months and 19.0 months, respectively, P < 0.001).</p><p><b>CONCLUSIONS</b>This study developed a weighted, integrated CLL-PI prognostic system of CLL patients which combines the critical genetic prognostic markers with traditional clinical stage. This novel modified PI system could be used to discriminate among groups and may help predict the TTFT and prognosis of patients with CLL.</p>


Subject(s)
Adult , Aged , Aged, 80 and over , China , Chromosome Aberrations , Chromosomes, Human, Pair 17 , Genetics , DNA Mutational Analysis , Female , Humans , Immunoglobulin Heavy Chains , Genetics , Metabolism , In Situ Hybridization, Fluorescence , Leukemia, Lymphocytic, Chronic, B-Cell , Diagnosis , Genetics , Metabolism , Male , Middle Aged , Mutation , Prognosis
4.
Chinese Journal of Virology ; (6): 14-18, 2016.
Article in Chinese | WPRIM | ID: wpr-296223

ABSTRACT

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.


Subject(s)
Animals , Antibodies, Monoclonal , Genetics , Allergy and Immunology , Cloning, Molecular , Ebolavirus , Genetics , Allergy and Immunology , Hemorrhagic Fever, Ebola , Allergy and Immunology , Virology , Humans , Immunoglobulin Heavy Chains , Genetics , Allergy and Immunology , Mice , Nucleoproteins , Genetics , Allergy and Immunology , Viral Proteins , Genetics , Allergy and Immunology
5.
Chinese Journal of Pathology ; (12): 100-105, 2015.
Article in Chinese | WPRIM | ID: wpr-298143

ABSTRACT

<p><b>OBJECTIVE</b>To study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL).</p><p><b>METHODS</b>Seven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study.</p><p><b>RESULTS</b>All the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up.</p><p><b>CONCLUSIONS</b>PCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.</p>


Subject(s)
Aged , Aged, 80 and over , Animals , Antigens, CD , Culicidae , Gene Rearrangement , Humans , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin kappa-Chains , Genetics , Immunophenotyping , Insect Bites and Stings , Leg , Leg Injuries , Lymphoma, Large B-Cell, Diffuse , Genetics , Metabolism , Pathology , Male , Middle Aged , Prognosis , Proto-Oncogene Proteins c-bcl-6 , Metabolism , Skin Neoplasms , Genetics , Pathology
6.
Article in English | WPRIM | ID: wpr-64359

ABSTRACT

Translocations leading to fusions between the immunoglobulin heavy chain gene (IGH) and various partner genes have been reported in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, submicroscopic deletions within IGH in B-ALL have not been rigorously assessed. In this study, we investigated characteristics of IGH submicroscopic deletions, by FISH, in B-ALL with IGH rearrangements. FISH was performed by using commercially available IGH dual-color break-apart rearrangement probes (Abbott/Vysis, Downers Grove, IL, USA; Kreatech, Amsterdam, Netherlands). The study group included seven B-ALL patients with IGH rearrangements, observed by FISH. Among them, two exhibited deletion of the 5' variable region of IGH by FISH. The B-ALL in these two patients included two kinds of abnormal cells; one had an IGH rearrangement without any IGH submicroscopic deletion, while the other had an IGH submicroscopic deletion, which showed that one normal fusion signal and one 3' IGH signal were detected. Thus, submicroscopic deletion of the IGH 5' variable region may have occurred in either the native or rearranged chromosome 14. These findings indicate that B-ALL with IGH rearrangements may be accompanied by submicroscopic deletions of the IGH 5' variable region, which can be detected by FISH. The clinical significance of such deletions is unclear, but the loss of part of the IGH gene in B-ALL warrants further study.


Subject(s)
Adult , Child , Female , Gene Deletion , Gene Rearrangement , Humans , Immunoglobulin Heavy Chains/genetics , In Situ Hybridization, Fluorescence , Infant , Male , Middle Aged , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Young Adult
8.
Article in Chinese | WPRIM | ID: wpr-265599

ABSTRACT

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of camels. Using nanobody-based probes, several imaging techniques such as radionuclide-based, optical and ultrasound have been employed for visualization of target expression in various disease models. Combined with application and clinical data of nanobody in molecular imaging in recent years, this paper introduces its application in the diagnosis of diseases and the future development as a novel molecular imaging tool.


Subject(s)
Humans , Immunoglobulin Heavy Chains , Molecular Imaging , Methods , Molecular Probes , Nanotechnology
9.
Chinese Journal of Hematology ; (12): 95-98, 2015.
Article in Chinese | WPRIM | ID: wpr-278902

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the specificity and sensitivity of immunoglobulin heavy/light chain (HLC) and serum free light chain (FLC) level in minimal residual disease monitoring of IgG type multiple myeloma (MM) patients during complete remission (CR).</p><p><b>METHODS</b>Immunoglobulin HLC was assessed in 20 IgG myeloma patients by immune turbidimetry using SPAplus Analyzer. The serum level of HLC and FLC was detected at same time. Combine with those obtained by serum protein electrophoresis (SPE) and immune fixation electrophoresis (IFE), the specificity and sensitivity of HLC in detection of serum immunoglobulin were analyzed. Combined with the clinical efficacy, kappa/lambda ratios of HLC (rHLC) and FLC (rFLC) were compared between the patients and normal controls.</p><p><b>RESULTS</b>Among 20 patients, there were 10 male and 10 female, the median age was 56 years (35-70). There were 6 patients with abnormal rHLC but normal rFLC; 3 patients with abnormal rFLC but normal HLC; and 11 patients with both normal rHLC and rFLC. During the mean follow-up time of 18 months, 4 of the6 patients with abnormal rHLC accepted intervention therapies, 1 case relapsed in 9 months,the other 2 untreated patients relapsed in 3 months. Among the 3 cases with abnormal rFLC, 2 patients are still in remission after intervention therapies,the other untreated patient relapsed in 1.5 months. Among the 11 untreated patients with both normal rHLC and rFLC, 3 relapsed with the disease free survival time of 3.5 months, 5.0 months and 5.5 months respectively.</p><p><b>CONCLUSION</b>The combined detection of HLC and FLC is helpful to assess the curative efficacy and the accuracy of minimal residual disease monitoring, and more effectively evaluate the prognosis of MM patients. Abnormal rHLC and rFLC are correlated with poor prognosis, while early intervention therapies can help to improve disease free survival.</p>


Subject(s)
Adult , Aged , Female , Humans , Immunoglobulin G , Immunoglobulin Heavy Chains , Immunoglobulin Light Chains , Male , Middle Aged , Multiple Myeloma , Neoplasm, Residual , Nephelometry and Turbidimetry , Prognosis , Remission Induction
10.
Article in English | WPRIM | ID: wpr-30790

ABSTRACT

BACKGROUND/AIMS: The gastrointestinal (GI) tract often becomes involved in patients with systemic amyloidosis. As few GI amyloidosis data have been reported, we describe the clinical features and outcomes of patients with pathologically proven GI amyloidosis. METHODS: We identified 155 patients diagnosed with systemic amyloidosis between April 1995 and April 2013. Twenty-four patients (15.5%) were diagnosed with GI amyloidosis using associated symptoms, and the diagnoses were confirmed by direct biopsy. RESULTS: Among the 24 patients, 20 (83.3%) had amyloidosis light chain (AL), three (12.5%) had amyloid A, and one (4.2%) had transthyretin-related type amyloidosis. Their median age was 57 years (range, 37 to 72), and 10 patients were female (41.7%). The most common symptoms of GI amyloidosis were diarrhea (11 patients, 45.8%), followed by anorexia (nine patients, 37.5%), weight loss, and nausea and/or vomiting (seven patients, 29.2%). The histologically confirmed GI tract site in AL amyloidosis was the stomach in 11 patients (55.0%), the colon in nine (45.0%), the rectum in seven (35.0%), and the small bowel in one (5.0%). Patients with GI involvement had a greater frequency of organ involvement (p = 0.014). Median overall survival (OS) in patients with GI involvement was shorter (7.95 months; range, 0.3 to 40.54) than in those without GI involvement (15.84 months; range, 0.0 to 114.53; p = 0.069) in a univariate analysis. A multivariate analysis of prognostic factors for AL amyloidosis revealed that GI involvement was not a significant predictor of OS (p = 0.447). CONCLUSIONS: The prognosis of patients with AL amyloidosis and GI involvement was poorer than those without GI involvement, and they presented with more organ involvement and more advanced disease than those without organ involvement.


Subject(s)
Adult , Aged , Amyloid Neuropathies, Familial/diagnosis , Biomarkers/analysis , Biopsy , Female , Gastrointestinal Diseases/diagnosis , Gastrointestinal Tract/immunology , Humans , Immunoglobulin Heavy Chains/analysis , Immunoglobulin Light Chains/analysis , Kaplan-Meier Estimate , Male , Middle Aged , Multivariate Analysis , Predictive Value of Tests , Prognosis , Proportional Hazards Models , Republic of Korea , Retrospective Studies , Risk Factors , Serum Amyloid A Protein/analysis , Time Factors
11.
Annals of Dermatology ; : 496-500, 2014.
Article in English | WPRIM | ID: wpr-124796

ABSTRACT

Intravascular lymphoma (IVL) is a rare disorder characterized by the presence of large neoplastic lymphoid cells restricted to the lumens of small vessels with a predilection for the skin and the central nervous system. While the vast majority of cases involving IVL are of B-cell lineage, the disease rarely affects the T-cell, the histiocytes, and the natural killer cells. We report a case of intravascular T-cell lymphoma (IVTL) associated with Epstein-Barr virus (EBV). A 23-year-old healthy woman presented with tender indurated erythematous patches with overlying telangiectasia on her right breast, abdomen, both the upper and the lower extremities and the back for 3 months. The pathology revealed an infiltration of dermal and subcutaneous vessels by large and atypical lymphoid cells with immunohistochemical features of the T-cell lineage with a cytotoxic phenotype (CD3+, CD8+, granzyme B+, TIA-1+, CD4-, CD5-, CD20-, CD56-). Interestingly, the DNA extracted from the skin biopsies demonstrated evidence of a monoclonal immunoglobulin heavy chain gene rearrangement, but no T-cell receptor gene rearrangement was found. In situ hybridization study for EBV-encoded RNA was positive. She was diagnosed with an EBV-associated IVTL. The patient's skin lesions were refractory to the combination of chemotherapy and autologous stem cell transplant, and she expired. The findings in the present case may highlight the unique clinicopathologic aspects of EBV-associated cytotoxic IVTL that occurred in a young, immunocompetent woman.


Subject(s)
Abdomen , B-Lymphocytes , Biopsy , Breast , Central Nervous System , DNA , Drug Therapy , Female , Gene Rearrangement , Genes, T-Cell Receptor , Granzymes , Herpesvirus 4, Human , Histiocytes , Humans , Immunoglobulin Heavy Chains , In Situ Hybridization , Killer Cells, Natural , Lower Extremity , Lymphocytes , Lymphoma , Lymphoma, T-Cell , Pathology , Phenotype , RNA , Skin , Stem Cells , T-Lymphocytes , Telangiectasis , Young Adult
12.
Article in English | WPRIM | ID: wpr-247076

ABSTRACT

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')2 fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-Id) responses. Antigen-specific elution method was used for panning private anti-Id VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.


Subject(s)
Aflatoxin B1 , Allergy and Immunology , Amino Acid Sequence , Animals , Antibodies, Anti-Idiotypic , Chemistry , Camelids, New World , Allergy and Immunology , Immunoglobulin Heavy Chains , Chemistry , Molecular Sequence Data
13.
Chinese Journal of Biotechnology ; (12): 1351-1361, 2014.
Article in Chinese | WPRIM | ID: wpr-345589

ABSTRACT

Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.


Subject(s)
Humans , Immunoglobulin Heavy Chains , Chemistry , Single-Domain Antibodies , Chemistry
14.
Chinese Medical Journal ; (24): 1872-1877, 2013.
Article in English | WPRIM | ID: wpr-273079

ABSTRACT

<p><b>BACKGROUND</b>Much is known about the cytogenetic lesions that characterize multiple myeloma (MM) patients from the USA, Europe, and East Asia. However, little has been published about the disease among Southeast Asians. The aim of this study was to determine the chromosomal abnormalities of MM patients in our Singapore population.</p><p><b>METHODS</b>Forty-five newly-diagnosed, morphologically confirmed patients comprising 18 males and 27 females, aged 46 - 84 years (median 65 years) were investigated by karyotyping and fluorescence in situ hybridization (FISH). FISH employing standard panel probes and 1p36/1q21 and 6q21/15q22 probes was performed on diagnostic bone marrow samples.</p><p><b>RESULTS</b>Thirty-four cases (75.6%) had karyotypic abnormalities. Including FISH, a total detection rate of 91.1% was attained. Numerical and complex structural aberrations were common to both hyperdiploid and non-hyperdiploid patients. Numerical gains of several recurring chromosomes were frequent among hyperdiploid patients while structural rearrangements of several chromosomes including 8q24.1 and 14q32 characterized non-hyperdiploid patients. With FISH, immunoglobulin heavy chain (IGH) gene rearrangements, especially fibroblast growth factor receptor 3 (FGFR3)/IGH and RB1 deletion/monosomy 13 were the most common abnormalities (43.4%). Amplification 1q21 was 10 times more frequent (42.5%) than del(1p36) and del(6q21).</p><p><b>CONCLUSIONS</b>We have successfully reported the comprehensive cytogenetic profiling of a cohort of newly-diagnosed myeloma patients in our population. This study indicates that the genetic and cytogenetic abnormalities, and their frequencies, in our study group are generally similar to other populations.</p>


Subject(s)
Aged , Aged, 80 and over , Chromosome Aberrations , Cytogenetics , Female , Humans , Immunoglobulin Heavy Chains , In Situ Hybridization, Fluorescence , Karyotyping , Male , Middle Aged , Monosomy , Genetics , Multiple Myeloma , Genetics , Pathology , Receptor, Fibroblast Growth Factor, Type 3 , Genetics , Retinoblastoma Protein , Genetics , Singapore
15.
Article in Chinese | WPRIM | ID: wpr-306487

ABSTRACT

<p><b>OBJECTIVE</b>To construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.</p><p><b>METHODS</b>The total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.</p><p><b>RESULTS</b>The libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).</p><p><b>CONCLUSION</b>We have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.</p>


Subject(s)
Amino Acid Sequence , Animals , Antibodies , Genetics , Cell Surface Display Techniques , Gene Library , Humans , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin kappa-Chains , Genetics , Peptide Library , Urinary Bladder Neoplasms , Genetics , Allergy and Immunology
16.
Article in Chinese | WPRIM | ID: wpr-306456

ABSTRACT

<p><b>OBJECTIVE</b>To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.</p><p><b>METHODS</b>Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.</p><p><b>RESULTS</b>Using 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].</p><p><b>CONCLUSION</b>Using 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).</p>


Subject(s)
Animals , Antibodies, Viral , CHO Cells , Cell Surface Display Techniques , Cricetinae , Cricetulus , Dengue Virus , Allergy and Immunology , Gene Library , Genetic Vectors , Humans , Immunoglobulin Heavy Chains , Allergy and Immunology , Transfection
17.
Acta Pharmaceutica Sinica ; (12): 66-70, 2013.
Article in Chinese | WPRIM | ID: wpr-274589

ABSTRACT

To prepare large naive phage antibody library, the host bacteria with high transformation efficiency is used in the Cre-LoxP recombination system. The variable regions of immunoglobulin light and heavy genes were amplified from lymphocytes collected from adult peripheral blood and newborn cord blood. The genes were spliced to form the single-chain variable fragments (scFv) by overlap PCR, cloned into pDAN5a vector and then transformed into XL2-blue MRF' with the Hte gene. Compared with XL1-blue strain, the size of the primary library was increased by 3.9 times. The primary library infected Cre recombinase-expressing bacteria, and the genes between phagemids created many new VH/VL combinations. The library was calculated to have a diversity of 1.7 x 10(11) and validated by the selection of antibodies against six different protein antigens. This library provides the basis for further selection of antibody-based drugs. It is the first time to report that XL2-blue MRF' can be used to improve the diversity of the library in the recombination system.


Subject(s)
Adult , Escherichia coli , Genetics , Allergy and Immunology , Genetic Vectors , Humans , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Light Chains , Genetics , Immunoglobulin Variable Region , Genetics , Infant, Newborn , Integrases , Metabolism , Lymphocytes , Allergy and Immunology , Peptide Library , Recombination, Genetic , Genetics , Single-Chain Antibodies , Genetics , Metabolism , Transformation, Genetic
18.
Acta Pharmaceutica Sinica ; (12): 1651-1656, 2013.
Article in Chinese | WPRIM | ID: wpr-298030

ABSTRACT

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.


Subject(s)
Animals , Complementarity Determining Regions , Genetics , Allergy and Immunology , Cytokines , Allergy and Immunology , Humans , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Immunoglobulin Heavy Chains , Genetics , Allergy and Immunology , Mice , Peptide Library , Protein Binding , Receptors, Immunologic , Allergy and Immunology , Single-Chain Antibodies , Genetics , Allergy and Immunology
19.
Blood Research ; : 287-291, 2013.
Article in English | WPRIM | ID: wpr-25181

ABSTRACT

Nodular lymphoid hyperplasia of the stomach is a rare lymphoproliferative disorder. Here, we report a 38-year-old man who presented with multiple submucosal tumors of the stomach. Histologically, the lesions were characterized by multiple discrete submucosal nodules of lymphoid cells. The infiltrates between the lymphoid follicles were composed mainly of medium-sized lymphoid cells with abundant clear cytoplasm, as well as a few large cells with vesicular nuclei. The gastric mucosa exhibited multifocal lymphoid aggregates and some of the epithelial cells were infiltrated by small lymphocytes mimicking lymphoepithelial lesions. Histopathology was consistent with mucosa-associated lymphoid tissue lymphoma. However, the infiltrating lymphoid cells were positive for CD2, CD3, CD5, and CD7. In addition, polymerase chain reaction analysis of the immunoglobulin heavy chain and T-cell receptor gene rearrangements demonstrated polyclonality. This case was diagnosed as reactive lymphoid hyperplasia of the stomach.


Subject(s)
Adult , Cytoplasm , Epithelial Cells , Gastric Mucosa , Genes, T-Cell Receptor , Humans , Hyperplasia , Immunoglobulin Heavy Chains , Lymphocytes , Lymphoma, B-Cell, Marginal Zone , Lymphoproliferative Disorders , Polymerase Chain Reaction , Pseudolymphoma , Stomach
20.
Indian J Cancer ; 2012 Jan-Mar; 49(1): 137-143
Article in English | IMSEAR | ID: sea-144564

ABSTRACT

Chronic lymphocytic leukemia (CLL) was largely considered to be a disease of slow progression, standard treatment with Chlorambucil and having almost similar prognosis. With the introduction of molecular methods for understanding the disease pathophysiology in CLL there has been a remarkable change in the approach towards the disease. The variation in B-cell receptor response and immunoglobulin heavy chain variable region (IGHV) mutation, genetic aberration and defect in apoptosis and proliferation has had an impact on therapy initiation and prognosis. Early diagnosis of molecular variant is therefore necessary in CLL.


Subject(s)
Chromosome Aberrations , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Lymphocytosis/diagnosis , Mutation , Prognosis , Receptors, Antigen, B-Cell/genetics , Tumor Suppressor Protein p53/genetics , ZAP-70 Protein-Tyrosine Kinase/genetics
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