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1.
Rev. cuba. hematol. inmunol. hemoter ; 36(4): e1283, oct.-dic. 2020. tab
Article in English | LILACS | ID: biblio-1289419

ABSTRACT

Introduction: Autoimmune hemolytic anemia is a rare disorder characterized by hemolysis mediated by autoantibodies directed against red blood cells. The demonstration of antibody specificity is a very difficult procedure since autoantibodies in general are nonspecific of antigens and react with all erythrocytes analyzed. Occasionally, specificity is observed against the Rh system antigens. Objective: To determinate the specificity of erythrocytes autoantibodies in DAT positive autoimmune hemolytic anemia by MAIEA technique. Methods: The specificity and isotype of erythrocyte autoantibodies were determined in the eluate of 109 blood samples from patients with warm autoimmune hemolytic anemia, by means of the MAIEA technique and the use of monoclonal antibodies that recognized 11 blood group systems and the protein CD47. Results: In 100 percent of cases autoantibodies against Rh system antigens were detected; in 24 cases we detected autoantibodies of IgA and IgM isotypes that recognized different antigens that were recognized by IgG isotype autoantibodies. For idiopathic and secondary warm autoimmune hemolytic anemias, predominance was observed against three or more specificities. IgG was detected in 99.09 percent of the eluates, IgA in 35.77 percent and IgM in 16.51 percent. The high degree of hemolysis was related to the presence of several isotype autoantibodies against four or more blood group specificities. Conclusions: The MAIEA technique is a sensitive method that can be used to determine the specificities and isotypes of autoantibodies in patients with warm autoimmune hemolytic anemia.


Introducción: La anemia hemolítica autoinmune es un trastorno poco común, caracterizado por hemólisis mediada por autoanticuerpos dirigidos contra los glóbulos rojos. La demostración de la especificidad de los anticuerpos es un procedimiento muy difícil, ya que los autoanticuerpos en general no son específicos de los antígenos y reaccionan con todos los eritrocitos analizados. Ocasionalmente, se observa especificidad contra los antígenos del sistema Rh. Objetivo: Determinar la especificidad de los autoanticuerpos eritrocitarios en pacientes con anemias hemolíticas autoinmunes PAD positivas con el empleo de la técnica MAIEA Métodos: Se determinó la especificidad e isotipo de los autoanticuerpos eritrocitarios en el eluido de 109 muestras de sangre de pacientes con anemia hemolítica autoinmune caliente, mediante la técnica de MAIEA y el uso de anticuerpos monoclonales que reconocieron 11 sistemas de grupos sanguíneos y la proteína CD47. Resultados: En el ciento por ciento de los casos se detectaron autoanticuerpos contra los antígenos del sistema Rh. En 24 casos se descubrió autoanticuerpos de isotipos IgA e IgM que reconocieron diferentes antígenos que fueron a su vez reconocidos por autoanticuerpos de isotipo IgG. Se observó para las anemias hemolíticas autoinmunes calientes idiopáticas y secundarias; predominio frente a tres o más especificidades. Se detectó IgG en el 99,09 por ciento de los eluidos, IgA en 35,77 por ciento e IgM en 16,51 por ciento. El alto grado de hemólisis se relacionó con la presencia de varios isotipos de autoanticuerpos contra cuatro o más especificidades de grupos sanguíneos. Conclusiones: La técnica MAIEA es un método sensible que puede usarse para determinar las especificidades e isotipos de autoanticuerpos en pacientes con anemia hemolítica autoinmune caliente.


Subject(s)
Humans , Blood Group Antigens , Immunoglobulin G , Immunoglobulin M , Sensitivity and Specificity , Anemia, Hemolytic, Autoimmune , Antibodies, Monoclonal , Antibody Specificity
2.
Chinese Journal of Biotechnology ; (12): 871-879, 2019.
Article in Chinese | WPRIM | ID: wpr-771323

ABSTRACT

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Subject(s)
Antibodies, Monoclonal , Chemistry , Metabolism , Antibody Specificity , DNA-Binding Proteins , Genetics , Metabolism , Escherichia coli , Genetics , Escherichia coli Proteins , Metabolism , Humans , Peptides , Recombinant Fusion Proteins , Genetics , Metabolism
3.
Article in Korean | WPRIM | ID: wpr-759595

ABSTRACT

Antibodies to high-incidence red blood cell antigens should be considered if panagglutination reactions are noted in all panel cells, and negative reactions to autologous red blood cells are detected on antibody screening and identification tests. In Korea, most of those antibodies are identified through international reference laboratories. To prevent a hemolytic transfusion reaction, antigen-negative red cells should be provided for those patients who have antibodies to red cell antigens. However, this is nearly impossible when the antibody has specificity to high-incidence red cell antigen. In those cases, transfusion of autologous blood, cryopreserved rare blood and the least incompatible blood components can be considered. In the case of surgery, acute normovolemic hemodilution or intraoperative blood salvage can also be considered. For the patients who have antibodies to high-incidence red cell antigens, it should be discussed to set up a national reference laboratory to quickly identify antibody specificities, and to consider establishing rare blood donor registry and frozen rare blood storage/supply system. This article reviews characteristics of antibodies to high-incidence antigens found in Koreans and also the transfusion experiences of those patients based on literature.


Subject(s)
Antibodies , Antibody Specificity , Blood Donors , Erythrocytes , Hemodilution , Humans , Isoantibodies , Korea , Mass Screening , Operative Blood Salvage , Sensitivity and Specificity , Transfusion Reaction
4.
Chinese Journal of Biotechnology ; (12): 1117-1125, 2019.
Article in Chinese | WPRIM | ID: wpr-771816

ABSTRACT

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.


Subject(s)
Animals , Antibodies , Antibody Specificity , Bacterial Proteins , Blotting, Western , Cytoskeletal Proteins , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Plasmids , Rats
5.
Chinese Journal of Biotechnology ; (12): 1723-1735, 2019.
Article in Chinese | WPRIM | ID: wpr-771758

ABSTRACT

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12-200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%-5.6% and 5.6%-7.2%, respectively, and the fortified recovery was 89%-103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.


Subject(s)
Animals , Antibodies, Monoclonal , Antibody Specificity , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas , Interleukins , Mice , Mice, Inbred BALB C
6.
Article in English | WPRIM | ID: wpr-690626

ABSTRACT

<p><b>OBJECTIVE</b>To identify potential serum biomarkers for distinguishing between latent tuberculosis infection (LTBI) and active tuberculosis (TB).</p><p><b>METHODS</b>A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays (ELISA) were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve (ROC) analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability.</p><p><b>RESULTS</b>Microarray results showed that levels of 152 Mycobacterium tuberculosis (Mtb)-antigen- specific IgG were significantly higher in active TB patients than in LTBI patients (P < 0.05), and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031c, Rv1408, and Rv2421c had higher areas under the curve (AUCs) of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability.</p><p><b>CONCLUSION</b>Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.</p>


Subject(s)
Adolescent , Adult , Aged , Antibodies, Bacterial , Antibody Specificity , Antigens, Bacterial , Biomarkers , Blood , Female , Humans , Latent Tuberculosis , Blood , Diagnosis , Logistic Models , Male , Middle Aged , Mycobacterium tuberculosis , Protein Array Analysis , Methods , Proteome , Genetics , Proteomics , Methods , ROC Curve , Young Adult
7.
Article in Korean | WPRIM | ID: wpr-718427

ABSTRACT

BACKGROUND: Anti-E or paired anti-E/-c antibodies can develop in patients with the Rh CDe phenotype. This study examined the differences in transfusion in patients with the CDe phenotype according to formation of anti-E or anti-E/-c antibodies. METHODS: Retrospective reviews were carried out on the results of antibody identification tests performed in 2014. The Rh phenotype and antibody specificity were investigated. The transfusion and medical records of patients with the CDe phenotype were examined. RESULTS: In total, 76 patients were included in the review. Of these 76 patients, 38 (50.0%) were of the CDe phenotype. Anti-E antibodies were the most frequent (60.5%), followed by anti-E/-c antibodies (23.7%). The total transfusion units and platelet transfusion units were significantly higher in patients with anti-E/-c antibodies (P=0.028 and P=0.01, respectively). The distribution of categorized diseases was similar in the patients with the anti-E and anti-E/-c antibodies. A frequency of transfusion episodes greater than or equal to four was higher in patients with hepatobiliary diseases (85.7%). CONCLUSION: In CDe phenotype patients, platelet transfusion was significantly higher in the anti-E/-c positive group than the anti-E positive group, indicating that platelets play a role in red blood cell alloimmunization. Because E is the most immunogenic antigen in Korea, it is important to define the disease group, in which patients with CDe phenotype require a transfusion of E and c-negative blood.


Subject(s)
Antibodies , Antibody Specificity , Blood Platelets , Erythrocytes , Humans , Korea , Medical Records , Phenotype , Platelet Transfusion , Retrospective Studies
8.
Arq. neuropsiquiatr ; 75(8): 580-588, Aug. 2017. tab, graf
Article in English | LILACS | ID: biblio-888309

ABSTRACT

ABSTRACT The polyspecific antibody synthesis in multiple sclerosis (MS) gained diagnostic relevance with the frequent combination of measles-, rubella- and varicella zoster antibodies (MRZ-antibody reaction) but their pathophysiological role remains unknown. This review connects the data for intrathecal polyspecific antibody synthesis in MS and neurolupus with observations in the blood of patients with Guillain-Barré syndrome (GBS). Simultaneously increased antibody and autoantibody titers in GBS blood samples indicate that the polyspecific antibodies are based on a general property of an immune network, supported by the deterministic day-to-day concentration variation of antibodies in normal blood. Strongly correlated measles- and rubella- antibody variations point to a particular connectivity between the MRZ antibodies. The immune network, which provides serological memory in the absence of an antigen, implements the continuous change of the MRZ pattern in blood, not followed by the earlier immigrated B cells without corresponding connectivity in the brain. This may explain the different antibody patterns in cerebrospinal fluid, aqueous humor and blood of the individual MS patient. A complexity approach must implement a different view on causation in chronic diseases and causal therapies.


RESUMO A síntese de anticorpos poliespecíficos em esclerose múltipla (EM) ganhou relevância diagnóstica com a combinação frequente de anticorpos contra sarampo, rubéola e varicela-zoster (reação de anticorpos MRZ), mas seu papel fisiopatológico permanece desconhecido. Esta revisão relaciona os dados da síntese intratecal de anticorpos poliespecíficos em EM e Neurolupus com observações no sangue de pacientes com síndrome de Guillain Barré (SGB). Simultaneamente, os títulos aumentados de anticorpos e autoanticorpos em amostras de sangue de SGB indicam que os anticorpos poliespecíficos se baseiam numa propriedade geral de uma rede imunitária, suportada pela variação determinística da concentração diária de anticorpos no sangue normal. As variações fortemente correlacionadas de anticorpos contra sarampo e rubéola apontam para uma conectividade particular entre os anticorpos MRZ. A rede imunitária, que fornece memória sorológica na ausência de um antígeno, implementa a mudança contínua do padrão MRZ no sangue, não seguida pelas células B que imigraram anteriormente sem conectividade no cérebro. Isto pode explicar os diferentes padrões de anticorpos no LCR, humor aquoso e sangue do paciente individual de EM. Uma abordagem complexa deve implementar uma visão diferente sobre a causalidade em doenças crônicas e terapias causais.


Subject(s)
Humans , Guillain-Barre Syndrome/immunology , Antibodies, Viral/blood , Multiple Sclerosis/immunology , Antibody Specificity/immunology , Rubella/immunology , Immunoglobulin G/blood , Cerebrospinal Fluid/chemistry , Herpes Zoster/immunology , Measles/immunology , Antibodies, Bacterial , Multiple Sclerosis/cerebrospinal fluid , Mumps/immunology , Antigens, Viral/immunology
9.
Article in English | WPRIM | ID: wpr-100903

ABSTRACT

BACKGROUND: Basic National Institute of Health (NIH) and sensitive antihuman globulin (AHG) methods are widely used for T-cell complement-dependent cytotoxicity crossmatch (XM) tests. Whereas NIH-negative, AHG-positive (NIH⁻/AHG⁺) results are caused by weak antibodies, NIH⁺/AHG⁻ results are usually due to autoantibodies. We found that solid organ transplantation candidates with NIH⁺/AHG⁻ XM results are repeatedly excluded from allocation of deceased donor organs by the Korean Network for Organ Sharing (KONOS) allocation system. Here, we attempted to demonstrate that these patients do not have donor-specific HLA antibodies (DSAs). METHODS: Sera showing NIH⁺/AHG⁻ results in the analysis of 1,668 KONOS T-cell XM tests were screened for panel reactive antibody (PRA) using a Luminex test. For screen-positive samples, antibody identification was conducted using a Luminex single antigen assay and the presence or absence of class I DSAs was determined. For positive controls, 42 KONOS XM tests showing probable true-positive (NIH⁻/AHG⁺ or NIH⁺/AHG⁺) results were reviewed for PRA results based on electronic medical records and the presence or absence of DSAs was determined. RESULTS: NIH⁺/AHG⁻ results were observed in 1.3% (21/1,668) of KONOS XM tests analyzed. Most of these (18/21, 85.7%) were negative for PRA or DSAs. All probable true-positive cases were either positive for DSAs (24/42, 57.1%) or had high PRA (mean, 92% [range; 42%~100%]), complicating accurate identification of antibody specificities. CONCLUSIONS: NIH⁺/AHG⁻ results are not rare (1.3%) in KONOS XM tests. Most of these results are not due to DSAs, and these patients should not be excluded from organ allocation.


Subject(s)
Antibodies , Antibody Specificity , Autoantibodies , Electronic Health Records , Humans , Organ Transplantation , T-Lymphocytes , Tissue Donors , Transplants
10.
Yonsei Medical Journal ; : 399-406, 2016.
Article in English | WPRIM | ID: wpr-21016

ABSTRACT

PURPOSE: Japanese hop (Humulus spp.) and mugwort (Artemisia spp.) are notable causes of autumn pollinosis in East Asia. However, Japanese hop and mugwort pollen extracts, which are widely used for the diagnosis, have not been standardized. This study was performed to standardize Japanese hop and mugwort pollen extracts. MATERIALS AND METHODS: Allergen extracts were prepared in a standardized way using locally collected Humulus japonicus and purchased Artemisia vulgaris pollens. The immunoglobulin E (IgE) reactivities of prepared extracts were compared with commercial extracts via IgE immunoblotting and inhibition analyses. Intradermal skin tests were performed to determine the bioequivalent allergy unit (BAU). RESULTS: The IgE reactive components of the extracts via IgE immunoblotting were similar to those of commercial extracts. A 11-kDa allergen showed the strongest IgE reactivity in Japanese hop, as did a 28-kDa allergen in mugwort pollen extracts. Allergenic potencies of the investigatory Japanese hop and mugwort extracts were essentially indistinguishable from the commercial ones. Sums of erythema of 50 mm by the intradermal skin test (SigmaED50) were calculated to be 14.4th and 13.6th three-fold dilutions for Japanese hop and mugwort extracts, respectively. Therefore, the allergenic activity of the prepared extracts was 90827.4 BAU/mg for Japanese hop and 34412 BAU/mg for mugwort. CONCLUSION: We produced Japanese hop and mugwort pollen extracts using a standardized method. Standardized Japanese hop and mugwort pollen extracts will facilitate the production of improved diagnostic and immunotherapeutic reagents.


Subject(s)
Allergens/analysis , Antibody Specificity , Artemisia , Bronchial Hyperreactivity/blood , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , Immunoglobulin E/blood , Pollen/chemistry , Reference Standards , Republic of Korea , Rhinitis, Allergic, Seasonal
11.
Article in Chinese | WPRIM | ID: wpr-815005

ABSTRACT

OBJECTIVE@#To explore the effect of pre-transplant donor specific antibody (DSA) on antibody-mediated rejection (AMR) and function of transplanted kidney.
@*METHODS@#A total of 88 cases of renal transplant recipients were selected. Before surgery, DSA was examined by Luminex liquid phase chip in renal transplant recipients. The recipients were divided into a DSA positive group (n=20) and a DSA negative group (n=68). The follow-up time was 2 years. After the operation, the pathologic morphology of the transplanted kidney was evaluated and classified according to the Banff 2005 standard. The situation for the transplanted kidney was evaluated.
@*RESULTS@#The incidence of AMR in the DSA positive group and negative group was 20% and 1.5%, respectively, with significant difference between them (P0.05).
@*CONCLUSION@#The detection of DSA level before renal transplantation can predict the risk of AMR and the function of transplanted kidney. The MFI intercept point of the highest DSA (MFI>
7909.5) can be used to predict the risk of AMR.


Subject(s)
Antibody Specificity , Graft Rejection , Humans , Incidence , Isoantibodies , Blood , Kidney , Pathology , Kidney Transplantation , ROC Curve
13.
Acta Pharmaceutica Sinica ; (12): 1225-1231, 2015.
Article in Chinese | WPRIM | ID: wpr-320097

ABSTRACT

Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over the traditional chemotherapy. However, it is difficult to control the site and stoichiometry of conjugation in mAb, typically resulting in heterogeneous mixtures of ADCs that are difficult to optimize. New methods for site-specific drug attachment allow development of more homogeneous conjugates and control of the site of drug attachment. In this article, the new literature on development of ADCs and site-specific ADCs is reviewed. In addition, we summarized the various strategies in production of site-specific ADCs.


Subject(s)
Antibodies, Monoclonal , Chemistry , Antibody Specificity , Binding Sites, Antibody , Immunoconjugates , Chemistry
14.
Chinese Journal of Virology ; (6): 379-387, 2015.
Article in Chinese | WPRIM | ID: wpr-296273

ABSTRACT

The severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative pathogen of an emerging infectious disease severe fever with thrombocytopenia syndrome and a new member in the genus Phlebovirus of family Bunyaviridae. Immune responses and pathological lesions in SFTSV-infected Balb/C mice and hamsters were evaluated by inoculation of SFTSV at 105 TCID50 or 103 TCID50 per animal through four different routes of infection, including intravenous, intramuscular, intraperitoneal, and intracerebral injections. The vehicle control groups were also included. At different time points after the inoculation blood and plasma samples were collected. Blood cell counts, blood viral RNA copies, and plasma antibodies were detected by automatic blood cell counters, real-time PCR, and luminex assays, respectively. At two weeks post inoculation, the animals were sacrificed. Tissues including heart, liver, spleen, lung, kidney, intestine, muscle, and brain, were collected for pathological analyses. Results showed that the SFTSV could infect Balb/C mice and hamsters with SFTSV-specific immunoglobulin (Ig) M and IgG antibodies detected in plasma samples on day 7 post inoculation. The SFTSV-specific IgM levels peaked on day 7 post inoculation and then decreased, whereas the SFTSV-specific IgG levels started to increase on day 7 and then peaked on day 14 post inoculation. Pathological analyses indicated significant pathological lesions in liver and kidney tissues. In conclusion, SFTSV could can infect different strains of rodent animals and cause similar immunological and pathological responses.


Subject(s)
Animals , Antibody Specificity , Bunyaviridae Infections , Blood , Pathology , Cricetinae , Immunoglobulin G , Blood , Immunoglobulin M , Blood , Leukocyte Count , Mice , Mice, Inbred BALB C , Organ Specificity , Phlebovirus , Allergy and Immunology , Physiology
15.
Article in Chinese | WPRIM | ID: wpr-357238

ABSTRACT

<p><b>OBJECTIVE</b>To prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cells (LSC).</p><p><b>METHODS</b>BALB/c mice were inoculated intraperitoneally with hybridoma cells (3H6E10, 10D8A7) and their ascites were collected. The monoclonal antibody against hu-IL1RAP specifically was purified from ascites, the nondenaturing-PAGE, ELISA and Western blot were used to detect the purity, titer and sensitivity of antibody.</p><p><b>RESULTS</b>Two purified antibodies were obtained and named as 3H6E10 McAb and 10D8A7 McAb, whose purity was 95% and 94% respectively. The titer of two purified monoclonal antibodies was 1 : 81000 and specific conjugation of IL1RAP purified protein and endogenous protein from normal people and leukemia patients with purified antibodies were confirmed.</p><p><b>CONCLUSION</b>The purified monoclonal antibodies which can specifically bind to hu-IL1RAP are successfully prepared, thus providing novel way to effectively clear LSC in the future.</p>


Subject(s)
Animals , Antibodies, Monoclonal , Antibody Specificity , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Humans , Hybridomas , Interleukin-1 Receptor Accessory Protein , Leukemia , Mice , Mice, Inbred BALB C , Neoplastic Stem Cells
16.
Article in Chinese | WPRIM | ID: wpr-345525

ABSTRACT

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.


Subject(s)
Animals , Antibodies, Monoclonal , Antibody Specificity , Blotting, Western , Epitope Mapping , Hybridomas , Immunoglobulin G , Mice , Mice, Inbred BALB C , Reticuloendotheliosis virus , Allergy and Immunology , Viral Envelope Proteins , Allergy and Immunology
17.
Article in Chinese | WPRIM | ID: wpr-255174

ABSTRACT

<p><b>OBJECTIVE</b>To prepare rabbit monoclonal antibody (RabMab) against guanosine 3', 5'-cyclic monophosphate (cGMP) and to develop a competitive ELISA for the detection of cGMP.</p><p><b>METHODS</b>New Zealand white rabbits were immunized with synthesized cGMP-keyhole limpet hemoeyanin (cGMP-KLH) to prepared a RabMAb with monoclonal antibody technique of Epitomics. A competitive ELISA kit was produced with cGMP RabMAb. The specificity, the precision and the recoveries of the method were determined.</p><p><b>RESULTS</b>The RabMAb with high sensitivity towards cGMP were prepared with an antibody timer of 3.1 ng/mL and 50% inhibitive concentration (IC50) of 12.57 ng/mL. The cGMP RabMAb had 33% cross-reactivity to inosine 3', 5'-cyclic monophosphate (cIMP) and little or no cross-reactivity to other compounds. A competitive ELISA was developed for detection of cGMP. The range of detection was 0~120 ng/mL with a minimal limit of 1.95 ng/mL. The recovery of assay was 89%~103%. The inter-assay and intra-assay coefficient variations were below 11.68% and 13.85%, respectively.</p><p><b>CONCLUSION</b>The RabMab against cGMP with high affinity and high specificity has been generated successfully, and a competitive ELISA for detection of cGMP has been developed with the prepared cGMP RabMAb.</p>


Subject(s)
Animals , Antibodies, Monoclonal , Antibody Specificity , Cross Reactions , Cyclic GMP , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Rabbits
18.
Article in Korean | WPRIM | ID: wpr-73599

ABSTRACT

Development of luminex-based solid phase assays enables advanced measurement of HLA antibody with sensitivity, specificity, and increasing knowledge of unacceptable antigens. In this review, we described the principle of the luminex-based assay and its current applications for organ transplantation including C1q assay, calculated panel reactive antibody, and virtual cross-matching. We also discussed the technical aspects and limitations for clinical utilization. The variables related to measurement of HLA antibody specificities and their clinical relevance remain unclear, therefore the interpretation of results requires comprehensive knowledge and clinical information in critical cases.


Subject(s)
Antibody Specificity , Immunoassay , Organ Transplantation , Sensitivity and Specificity , Transplantation , Transplants
19.
Chinese Journal of Virology ; (6): 18-23, 2015.
Article in Chinese | WPRIM | ID: wpr-280301

ABSTRACT

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.


Subject(s)
Animals , Antibodies, Monoclonal , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Antibody Specificity , Bunyaviridae Infections , Allergy and Immunology , Virology , Female , Humans , Hybridomas , Allergy and Immunology , Mice , Mice, Inbred BALB C , Phlebovirus , Allergy and Immunology , Viral Structural Proteins , Allergy and Immunology
20.
Salud pública Méx ; 56(5): 502-510, sep.-oct. 2014. ilus, tab
Article in English | LILACS | ID: lil-733323

ABSTRACT

Objective. To estimate the annual cost of the National Cervical Cancer Screening Program (CCSP) of the Mexican Institute of Social Security (IMSS). Materials and methods. This cost analysis examined regional coverage rates reported by IMSS. We estimated the number of cytology, colposcopy, biopsy and pathology evaluations, as well as the diagnostic test and treatment costs for cervical intraepithelial neoplasia grade II and III (CIN 2/3) and cervical cancer. Diagnostic test costs were estimated using a micro-costing technique. Sensitivity analyses were performed. Results. The cost to perform 2.7 million cytology tests was nearly 38 million dollars, which represents 26.1% of the total program cost (145.4 million). False negatives account for nearly 43% of the program costs. Conclusion. The low sensitivity of the cytology test generates high rates of false negatives, which results in high institutional costs from the treatment of undetected cervical cancer cases.


Objetivo. Estimar el costo anual del Programa Nacional de Detección Oportuna de Cáncer Cervical en el Instituto Mexicano del Seguro Social (IMSS). Material y métodos. Este análisis de costos examinó las distintas coberturas por región reportadas por el IMSS. Se estimó el número de citologías, colposcopías, biopsias y evaluaciones de patología y los costos de pruebas de diagnóstico y de tratamientos por neoplasia cervical intraepitelial de grado II y III (NIC 2/3) y cáncer cervical. Los costos de las pruebas de diagnóstico se estimaron utilizando una técnica de microcosteo. Se llevó a cabo un análisis de sensibilidad. Resultados. El costo de realizar 2.7 millones de citologías fue de 38 millones de dólares, lo que representa 26.1% del costo total del programa (145.4 millones). Los falsos negativos corresponden a casi 43% de los costos del programa. Conclusiones. La baja sensibilidad de la citología genera un alto número de falsos negativos que resultan en costos elevados para la institución por el tratamiento de estos casos no detectados.


Subject(s)
Animals , Female , Rats , Collagen/metabolism , Fibronectins/metabolism , Laminin/metabolism , Liver Cirrhosis, Experimental/metabolism , Malonates/pharmacology , Antibody Specificity , Dimethylnitrosamine , Evaluation Studies as Topic , Extracellular Matrix/metabolism , Immunoenzyme Techniques , Liver Cirrhosis, Experimental/chemically induced , Rats, Sprague-Dawley
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