Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada / Prevalence of low avidity of anti-Toxoplasma gondii immunoglobulin G and risk behavior for toxoplasmosis in pregnant women

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Prevalencia de baja avidez de Inmunoglobulina G anti Toxoplasma gondii y comportamiento de riesgo para toxoplasmosis en embarazada

Introducción: la toxoplasmosis es una infección zoonótica producida por Toxoplasma gondii, protozoo intracelular que puede afectar al hijo de la mujer embarazada y causar severas secuelas por lo que el monitoreo serológico debe ser realizado. Objetivo: determinar la prevalencia de baja avidez IgG anti Toxoplasma gondii y el comportamiento de riesgo para la enfermedad de toxoplasmosis en mujeres que estuvieron embarazadas durante el período 2017-2019 que acudieron al Instituto de Investigaciones en Ciencias de la Salud de la Universidad Nacional de Asunción-Paraguay. Metodología: fueron analizadas 371 fichas de pacientes con serología IgG positiva para toxoplasmosis cuyas muestras fueron procesadas en el Departamento de Producción del Instituto de Investigaciones en Ciencias de la Salud entre los años 2017-2019. Posteriormente, en el año 2020, se realizó 149/371 encuestas digitales de en estas mismas mujeres sobre conocimiento y comportamiento de riesgos para Toxoplasmosis. Resultados: se observó una prevalencia de 18 % de baja avidez para toxoplasmosis. A partir de la encuesta se encontró el 98 % conoce la enfermedad, el 73 % adquirió información durante el embarazo y el 50,3 % recibió orientación de prevención, además, el 65 % refirió como formas de transmisión comer carnes mal cocidas y verduras crudas. En cuanto al comportamiento de riesgo 46 % consume de aguatería, 20 % consume carne a punto medio y 78 % vegetales crudos. El 54 % realiza actividad de cultivo, tienen mascotas como gatos 4,3 %, perros 82 %, además el 9 % refirió dormir con sus mascotas. Conclusión: la prevalencia de baja Avidez en la población estudiada fue del 18 %. Se evidenció algunos comportamientos de riesgo para la toxoplasmosis en las mujeres encuestadas, por lo que se demuestra la necesidad de aplicar programas de prevención primaria en nuestro país.

Introduction: toxoplasmosis is a zoonotic infection caused by Toxoplasma gondii, an intracellular protozoan that can affect children of pregnant women and cause severe sequelae; therefore, serological monitoring should be performed. Objective: to determine the prevalence of low avidity IgG anti-Toxoplasma gondii and the risk behavior for toxoplasmosis disease in pregnant women during the 2017-2019 time period, who attended the Health Sciences Research Institute of the Universidad Nacional de Asuncion - Paraguay. Methodology: a total of 371 patient records with positive IgG serology for toxoplasmosis, whose samples were processed in the Production Department of the Instituto de Investigaciones en Ciencias de la Salud between the years 2017-2019 were analyzed. Subsequently, in 2020, 149/371 digital surveys of the same women were conducted on their knowledge and risk behavior for toxoplasmosis. Results: a low avidity prevalence of 18 % for toxoplasmosis was observed. 98 % knew about the disease, 73 % acquired information during pregnancy, and 50.3 % received preventive orientation. 65 % reported that eating undercooked meat and raw vegetables is a form of disease transmission. Regarding risk behavior, 46 % of the participants consumed poultry, 20 % consumed medium-rare-cooked meat, and 78 % consumed raw vegetables. Fifty-four percent of the patients performed farming activities, 44.3 % had cats as pets, 82 % had dogs, and 9 % slept with their pets. Conclusion: some risk behaviors for toxoplasmosis were evident in the women surveyed, demonstrating the need to implement primary prevention programs in our country.

Expression and purification of a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein as well as preparation and identification of its polyclonal antibody / 中华皮肤科杂志

Objective:To prokaryotically express a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein, and to prepare and identify its polyclonal antibody. Methods:The pCold TF-NGO2105 660-1468 aa recombinant plasmid was transformed into the bacterium Escherichia coli BL21 (DE3) for protein expression. After the inclusion body protein was denatured and renatured, the target protein was purified. Then, BALB/c mice were immunized with the target protein to prepare a polyclonal antiserum; the antibody potency was evaluated by enzyme-linked immunosorbent assay, the specificity of the antibody against NGO2105 protein in Neisseria gonorrhoeae was analyzed by Western blot analysis, the affinity of the antiserum with Neisseria gonorrhoeae was analyzed by flow cytometry, and adhesion inhibition assay was performed to evaluate the inhibitory effect of anti-NGO2105 660-1468 aa antibody on the adhesion of Neisseria gonorrhoeae to human cervical epithelial ME-180 cells. Comparisons between different groups were performed by using t test. Results:The NGO2105 660-1468 aa protein was expressed as the inclusion body, and the soluble target protein was obtained by denaturation, renaturation, and purification. After immunization of mice with the target protein, the antiserum titer was 5.12 × 10 6, and flow cytometry showed that the antibody bound well to the Neisseria gonorrhoeae NGO2105 660-1468 aa. Adhesion inhibition assay showed that the anti-NGO2105 660-1468 aa antibody significantly inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells, and the inhibitory effect was concentration-dependent to some extent, with the adhesion rates of Neisseria gonorrhoeae treated with 20- and 40-fold dilutions of the anti-NGO2105 660-1468 aa antibody being 52.9% and 79.2% respectively, significantly lower than the adhesion rate in the untreated group (100%, t = 8.40, 5.29, P < 0.001, = 0.006, respectively) . Conclusion:The NGO2105 660-1468 aa protein was successfully expressed and purified, and a highly potent polyclonal antibody was prepared, which had a good affinity with Neisseria gonorrhoeae and an adhesion inhibition ability.

Anticuerpos neutralizantes, nuevas pruebas de laboratorio contra el SARS-CoV-2 / Neutralizing antibodies, new laboratory tests against SARS-CoV-2

RESUMEN Introducción: En la presente revisión conoceremos los detalles de esta nueva prueba de laboratorio, utilizada para cuantificar los anticuerpos neutralizantes contra el SARS-CoV-2. Esta prueba diagnóstica comienza a tener un mayor protagonismo, a razón del proceso de infección y vacunación en el mundo, para comprender los misterios del correlato de protección inmunológica. Contenido: Los anticuerpos neutralizantes pueden bloquear la capacidad del virus, para unirse al receptor ACE2 en las células humanas y estos anticuerpos permiten eliminar el efecto de microorganismos invasores. Su actividad se genera por las proteínas situadas en la superficie de los virus, a las que se unen para «bloquear¼ la infección. Los anticuerpos neutralizantes se definen in vitro por su capacidad para bloquear la entrada, fusión o salida del coronavirus, es decir son anticuerpos funcionales. En la actualidad existen diferentes pruebas de laboratorio (pruebas de inmunoensayo de alto rendimiento), que pueden detectar anticuerpos inmunoglobulinas G anti proteína S del SARS-CoV-2 y que se correlacionan con las pruebas de laboratorio idóneas para la determinación de estos anticuerpos. Es crucial que estas pruebas de inmunoensayo de alto rendimiento, sean validadas en su fabricación comparándolas con los métodos gold standard para determinar la presencia de anticuerpos neutralizantes. Perspectiva: Se pretende ampliar el conocimiento de esta nueva prueba, que en un futuro permitirán definir los valores de correlato inmunológico generados por las vacunas o por una infección previa.

ABSTRACT Introduction: In this review we will know the details of this new laboratory test, used to quantify neutralizing antibodies against SARS-CoV-2. This diagnostic test begins to have a greater role, due to the process of infection and vaccination in the world, to understand the mysteries of the correlate of immune protection. Content: Neutralizing antibodies can block the ability of the virus to bind to the ACE2 receptor in human cells and these antibodies allow to eliminate the effect of invading microorganisms. Their activity is generated by proteins on the surface of viruses, which they bind to "block" infection. Neutralizing antibodies are defined in vitro by their ability to block the entry, fusion or exit of the coronavirus, that is, they are functional antibodies. Currently there are different laboratory tests (high-throughput immunoassay tests), which can detect immunoglobulin G anti-protein S antibodies to SARS-CoV-2 and which correlate with the gold standard laboratory tests for the determination of these antibodies. It is crucial that these high-throughput immunoassay tests are manufacturing validated against gold standard methods to determine the presence of neutralizing antibodies. Perspective: This review aims to expand the knowledge of this new test, which in the future will allow defining the immunological correlate values generated by vaccines or by a previous infection.

Analysis of serum anti-EB virus antibody in children with infectious mononucleosis / 中华实用儿科临床杂志

Objective:To understand the profile type of serum Epstein-Barr virus (EBV) antibodies in children with infectious mononucleosis (IM), and to analyze the significance of viral capsid antigen (VCA) IgG antibody affinity in the diagnosis of IM.Methods:Retrospective analysis was performed on the results of the serum anti-EBV antibody profile and plasma EBV nucleic acid test of 150 hospitalized children with IM diagnosed in Beijing Children′s Hospital, Capital Medical University, from May 2016 to May 2019.Anti-EBV antibody profiles, including anti-VCA-IgG, anti-VCA-IgM, anti-early antigen (EA) IgA, anti-EBV nuclear antigen (EBNA) IgG, and anti-VCA-IgG affinity, were detected by enzyme-linked immunosorbent assay (ELISA). Plasma EBV nucleic acids were detected by real-time quantitative PCR.Results:There were mainly two types of anti-EBV antibody profiles in 150 children with IM: (1)130 cases who were positive for anti-VCA-IgM/IgG, negative for anti-EBNA-IgG and positive for anti-VCA-IgG antibodies with low affinity, accounting for 86.7% (130/150 cases), of which 50 cases were positive for anti-early antigen IgA; (2)18 cases who were negative for anti-VCA-IgM, positive for anti-VCA-IgG, negative for anti-EBNA-IgG and positive for anti-VCA-IgG antibody with low affinity, accounting for 12.0% (18/150 cases), of which 5 cases were positive for anti-EA IgA.EBV DNA was measured in 132 children, with a posi-tive rate of 37.9% (50/132 cases).Conclusions:There were several types of serum EBV antibody profiles in children with IM, 12.0% of patients with IM in this study were negative for anti-VCA-IgM, and the diagnosis of IM was confirmed by the affinity of anti-VCA IgG.

Current status of cytomegalovirus infection in pregnant women and its correlation with pregnancy outcomes in Huzhou / 中华实验和临床病毒学杂志

Objective@#To understand the current situation of human cytomegalovirus(HCMV) infection in local pregnant women and its impact on pregnancy outcomes, so as to provide reference for strengthening health care during pregnancy and promoting the policy of "fewer and better children"@*Methods@#A total of 2 000 pregnant women underwent prenatal examination who had delivery in the Maternal and Child Health Hospital of Huzhou from July 2017 to December 2018. HCMV IgM, IgG and IgG- avidity index (AI) were detected by enzyme-linked immunosorbent assay (ELISA), and HCMV DNA was detected by real-time polymerase chain reaction (PCR) -fluorescent probe. We also analyzed the positive status of HCMV and its relationship with pregnancy outcome.@*Results@#Among the 2 000 pregnant women, 1 941 cases (97.05%) were positive for HCMV IgG, 26 cases (1.30%) had active infection with HCMV, of whom 5 cases (0.25%) were serum HCMV IgM/IgG dual positive, 24 cases (1.20%) were urine HCMV DNA positive. HCMV IgG AI test result showed that 3 cases of AI ≤30%, which means that they had primary infections. The detection rates of HCMV DNA positive and HCMV IgM/IgG dual positive were 12.12%, 4.54% in 66 pregnant women with adverse pregnancy and 0.83%, 0.10% in 1 934 pregnant women with normal pregnancy. The difference was significant (χ2=68.663, 50.499, P<0.01). Logistic regression analysis showed that HCMV IgM/IgG dual positive (OR=12.743, 95%CI: 1.202-135.100, P=0.035), HCMV-DNA positive (OR=10.426, 95%CI: 3.635-29.909, P<0.01) were independent risk factors for adverse pregnancy outcomes. In addition, low education level, living in rural areas and having a history of adverse pregnancy also increased the incidence of adverse pregnancy outcomes.@*Conclusions@#HCMV infection is prevalent in pregnant women in this area, but only a few of them had active infection. HCMV active infection is a risk factor for adverse pregnancy outcomes.

Avidity of serum immunoglobulin G antibodies to Porphyromonas gingivalis in the elderly with chronic periodontitis

The present study aimed at evaluating serum immunoglobulin G (IgG) avidity to Porphyromonas gingivalis in elderly patients with mild and severe chronic periodontitis. The avidity of antibodies against P. gingivalis present in the sera of 18 patients with mild chronic periodontitis and 18 patients with severe chronic periodontitis was evaluated using an ammonium thiocyanate-dissociated enzyme-linked immunosorbent assay (ELISA). The results showed that the mean absorbance value in serum IgG antibody titers was significantly higher in the severe chronic periodontitis group than in the mild chronic periodontitis group (198 ± 35 ELISA unit [EU] vs. 142 ± 32 EU, p < 0.01). However, there was no significant difference between the two groups in antibody avidity (65 ± 57 EU vs. 54 ± 27 EU). These findings suggest that humoral immune responses to P. gingivalis between mild and severe chronic periodontitis in elderly patients are characterized by the differences in the quantity rather than the quality of the antibodies.

Higher Binding Affinity and In Vitro Potency of Reslizumab for Interleukin-5 Compared With Mepolizumab

Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [K(D)]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (k(on)) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 10⁶ and 1.83 × 10⁵, respectively. The dissociation constant (k(off)) values were 4.29 × 10⁻⁴ and 2.14 × 10⁻⁴, respectively. Calculated K(D) values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the k(on) values for human IL-5 for reslizumab and mepolizumab were 3.17 × 10⁶ and 1.32 × 10⁵, respectively. The k(off) values were 1.36 × 10⁻⁵ and 1.48 × 10⁻⁵, respectively. Measured K(D) values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC₅₀) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC₅₀ values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.

Avaliação da transferência placentária e pelo colostro de anticorpos IgG e IgA anti-Staphylococcus aureus em mães com e sem colonização nasal / Evaluation of placental and colostral transfer of anti-Staphylococcus aureus IgG and IgA antibodies in mothers with and without nasal colonization

A transferência passiva de anticorpos da mãe para o filho auxilia na adaptação ao meio externo. No recém-nascido (RN), a colonização pelo Staphylococcus aureus (S. aureus) é precoce, sendo este um importante agente etiológico em infecções neonatais e no lactente jovem, para o qual ainda não se dispõem de vacina. OBJETIVOS: Avaliar as concentrações, títulos e avidez de anticorpos maternos anti-S. aureus do tipo IgG e IgA e a passagem desses anticorpos para os RN por transferência placentária e pelo colostro. MÉTODOS: Estudo caso-controle de 147 parturientes saudáveis. Foram coletadas amostras de soros maternos, de cordão umbilical e colostro. O grupo caso foi definido pela colonização nasal natural pelo S. aureus, sendo que para cada caso (n=49) foram selecionados 2 controles (n=98). Foram utilizadas as metodologias de imunoturbidimetria para dosagem de IgG total, ensaio imunoenzimático para dosagem IgA total e para a aferição das concentrações e títulos de anticorpos específicos anti-S. aureus (IgG sérica, subclasses séricas IgG1 e IgG2, IgA de colostro e os índices de avidez). Foram aplicados testes não paramétricos de Wilcoxon para amostras pareadas e de Mann-Whitney para amostras não pareadas, com intervalo de confiança de 95%, nível de significância p < 0,05. RESULTADOS: No grupo caso, as concentrações séricas de IgG total materna foram maiores mas com menor taxa de transferência placentária de IgG total, ocorrendo o inverso para o grupo controle. Não foram observadas diferenças nas concentrações séricas de IgG materna anti-S. aureus entre os grupos, mas com taxa de transferência placentária significantemente menor no grupo caso. Observou-se que os títulos específicos de IgG1 anti-S. aureus foram mais baixos no soro materno e no cordão do grupo caso, com taxas de transferência similar para os grupos caso e controle. Para os títulos específicos de IgG2 anti-S. aureus, não foram observadas diferenças entre os grupos caso e controle, com taxas de...

The passive transfer of antibodies from mother to child assists in adjustment to the external environment. In the newborn (NB), colonization by Staphylococcus aureus (S. aureus) occurs early, which is an important etiologic agent in neonatal and young infant infections, for which no vaccine is available. AIMS: To evaluate concentrations, titers and avidity of anti-S. aureus maternal IgG and IgA antibodies and transmission of these antibodies to the newborns via placental transfer and colostrum. METHODS: Case-control study of 147 healthy pregnant women. Samples of maternal serum, cord blood and colostrum were collected. The case group was defined by natural nasal colonization with S. aureus, and for each case (n = 49) were selected 2 controls (n = 98). Immunoturbidimetric assay was used to measure total IgG, and immunoenzymatic assay to measure total IgA in colostrum and anti-S. aureus concentrations and titers (serum IgG, serum IgG1 and IgG2, colostrum IgA and IgG and IgA avidity indexes). Nonparametric Wilcoxon test for paired samples and the Mann-Whitney test for unpaired samples were applied, with a confidence interval of 95%, significance level of p < 0.05. RESULTS: In the study group, maternal total IgG serum concentrations were higher but with lower total IgG placental transfer ratio, while the opposite occurred for the control group. No differences were observed in anti-staphylococcal maternal IgG serum concentrations between groups, but placental transfer ratio was significantly lower in the case group. It was observed that anti-S. aureus IgG1 titers were lower in maternal and cord serum from the case group, with with similar transfer ratios for case and control groups. Regarding antistaphylococcal IgG2 titers, no differences were observed between case and control groups, with similar transfer ratios between groups. It was observed that specific IgG2 titers were higher than those of IgG1 in both maternal and cord serum from both groups. In...

Clinical utility of high avidity anti-dsDNA antibody in the diagnosis of systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the clinical utility of anti-dsDNA antibody in the diagnosis of systemic lupus erythematosus (SLE).Methods Total and high affinity anti-dsDNA antibodies of 431 serum samples were measured by total and high affinity anti-dsDNA antibody EIA kits.Modified SLE disease activity index (M-SLEDAI) was scored for each SLE patients at the time of serum collection.The agreements between total and high affinity anti-dsDNA EIA tests and the correlation of anti-dsDNA antibody levels with disease activity and clinical manifestations were analyzed.Statistical analysis was performed using x2 test,Spearman correlation,Mann Whitney U test & Fisher exact,Student t test.Results ① The overall agreement between total and high affinity anti-dsDNA antibodies was 93.3％,and the total agreement of SLE patients was 89.6％.Twenty-two out of 23 (95.7％) SLE patients with positive total anti-dsDNA antibody but high affinity antidsDNA antibody were inactive with the M-SLEDAI score less than 4.Four patients with other autoimmune diseases had positive total anti-dsDNA antibody but negative on high affinity anti-dsDNA antibodies.② The total and high affinity anti-dsDNA antibody levels were significantly positively correlated with disease activity (M-SLEDAI) and negatively correlated with serum C3 and C4 levels (P＜0.01).③ The ratio of high affinity to total anti-dsDNA antibody was significantly higher in patients with M-SLEDAI ≥4 or with active kidney damage (P＜0.01).Conclusion Both total and high affinity anti-dsDNA antibodies are significantly correlated with SLE disease activity and kidney damage.However,the high affinity anti-dsDNA antibody may be more specific for active SLE and helpful in the differential diagnosis with other autoimmune diseases.High affinity anti-dsDNA antibody is more valuable in monitoring SLE disease activity status,especially on kidney damage,compared to total anti-dsDNA antibodies.

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (KD) of ~5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.

The effect of the human cytomegalovirus pp65 IgG-avidity index for the human cytomegalovirus primary infection in mice model / 中华检验医学杂志

Objective To investigate the significance of human cyomeg Movirus(HCMV)pp65 IgG antibody avidity index(AI)for the clinical diagnosis of HCMV primary infection through the experimental model of HCMV primaly infection in BALB/c mice.Methods 6～8 weeks,female,specific-pathogen-free BALB/c mice were divided into 5 groups.6 mice in each group. And he mice were injected with 2×106 PFU/m1,2×105 PFU/mi,2×104 PFU/ml,2×103 PFU/ml and 2×102 PFU/ml of HCMV intraperitoneally respectively. Another 6 mice were injected intraperitoneally with the maximum dose of HCMV kept at 56℃ for 30 min as inactivated virus group.And HF negative control group was established at same time. All the mice ere sacrificed to obtain brain and lung tissues for the following experiments after 1 montll.(1)Tissue samples obtained from mice were inoculated in human embryo fibroblasts(HF)monolayers after routine treatment for virus isolation.HCMV specific eytopathie effect(CPE) was observed bv inverted phase-contrast microscopy.HCMV UL83 DNA in the ultures as tested by PCR and pp65 antigen was detected by indirect immunofluorescence.(2)Extracted mRNA from tissue samples and HCMV pp67 mRNA were analyzed by reverse transcriptase PCR(RT-PcR).(3)Immunoglobulin M(IgM)antibody and immunoglobulin G(1gG)antibody avidity Was investigated for their usefulness in distinguishing primary genital HCMV infections rom nonprimary infections with ELISA kit using truncated pp65 protein.ResultsHCMV can be isolated in the tissues from the mice injected with 2×106 PFU/ml and 2×105 PFU/m1.RT- PCR and ELISA showed positive results in the same groups.The infective rates were 100%.The analysis of the low doses groups,inactivated group and HF negative ontrol group all showed negative results.Conclusions BALB/c mice can be infected with HCMV and appeared as primary infection after1 month.Determination of HCMV pp65 IgM and HCMV p065 IgG-AI by ELISA incorporated with virus isolation and RT-PCR are helpful for distinguishing primary infections from nonpfimary infections.The detection of HCMV p65 IgM and HCMV pp65 IgG-AI by ELISA utilizing recombinant protein pp5 as antigens can be used for preliminary screening.

Quantification and affinity assessment of autoantibodies against different epitopes of BP180 from patients with bullous pemphigoid / 中华皮肤科杂志

Objective To purify and quantify the autoantibodies against various epitopes within BP180-NC16A domain from the sera of patients with bullous pemphigoid (BP). Methods Three epitopes within BP180-NC16A domain, NC16A-1, NC16A-2 and NC16A-3, were prepared. Blood samples were obtained from 10 patients who were diagnosed as active BP by clinical, pathological and immunofluorescence examination. Autoantibodies against these epitopes were purified with affinity chromatography column from the sera of these patients with active BP, and quantified separately. The relative binding affinity of autoantibodies to NC 16A-1, NC 16A-2 and NC 16A-3 was measured using thiocyanate elution method. Results The autoantibodies against NC16A-1, NC16A-2 and NC16A-3 were successfully purified from the sera of patients. On average, the amount was 49.0±20.7 μg, 117.7±22.4 μg and 39.5±18.9 μg respectively for autoantibodies to NCI6A-1, NC16A-2 and NC16A-3 purified from a portion of serum containing about 20 mg IgG. Both the amount and binding affinity of anti-NC16A-2 autoantibody were significantly higher than those of anti-NC16A-1 and anti-NC16A-3 autoantibodies. Conclusion BP180 NC16A-2 (aa507-aa520) may be the major epitope recognized by pathogenic autoantibodies in patients with BP.

Effect of tetracycline on the avidity of serum IgG antibody against porphyromonas gingivalis in patients with aggressive periodontitis / 中国病理生理杂志

AIM: To evaluate the effect of mechanical periodontal treatment combined with tetracycline on per iodontal attachment level (AL) and the avidity of serum antibody against porphyromonas gingivalis( P. gingivalis) in patients with aggressive periodontitis(AgP). METHODS: Twenty- five patients with AgP and twenty periodontally healthy controls were studied (HS). Clinical examination and recordings of AL were performed before and 3,6 and 12 months after the periodontal treatment. The avidity of IgG antibody against P. gingivalis lipopolysaccharide(LPS) was measured by diethylamine dissociation ELISA. RESULTS: A significant improvement in AL was observed following treatment ( P ＜ 0. 01 ). The avidity of serum IgG antibody against P. gingivalis increased compared with controls, and was decreased significantly after mechanical periodontal treatment combined with tetracycline (P ＜ 0. 01 ). CONCLUSION: Ourresults demonstrate that mechanical periodontal treatment combined with tetracycline provides clinically favorable results in patients with AgP.

The Research on Expression and Purification of a Chimeric Anti-p185 Antibody / 中国生物工程杂志

The Her-2 proto-oncogene encodes a 185kDa transmenbrane glycoprotein p185 which has intrinsic tyrosine kinase activity. It is overexpressed in several malignant human tumors like breast cancer. A chimeric antibody by assembling a single-chain Fv antibody and a human IgG1 Fc fragment was constructed. This chimeric antibody reacts with tumor surface antigen p185c-erbB-2 specifically. In order to put the antibody into clinical application, two steps purification method was used to attain the antibody’s purity more than 95%. Both the lyophilized pharmaceutical formulations of the antibody were found. The formulations can keep the stability and activity of the antibody for at least one year. These results were the foundation of the chimeric antibody for cancer therapy.

Genetical Engineering of Humanized Anti-keratin Antibodies with High Affinity / 中华皮肤科杂志

Objective To produce humanized anti-keratin antibodies with high affinity. Methods Panning of phage antibody libraries against human epidermal keratin was conducted to select specific antibodies. Random mutation method and chain-shuffling technique for the gene of the antibodies were employed to improve antibody affinity. The binding activity, specificity and affinity of the obtained antibodies were de-termined and analyzed. Results Four clones of either IgG or IgM anti-keratin antibodies were seleeted from semisynthetic or natural antibody libraries. One of them was seleeted to undergo affinity maturation. Random mutation method for the gene did not result in any improvement in antibody affinity. By chain-shuffling technique, however, affinity level was markedly increased and an antibody with affinity of 8?1010/M was isolated. Conclusion It is feasible to generate humanized antikeratin antibodies using genetical engineering technique.

Determination of functional affinity of rhEndoglin conjugated eptide / 第三军医大学学报

Objective To assess functional affinity of rhEndoglin conjugated eptide in order to identify the affinity.Methods We measured the functional affinity of rhEndoglin conjugated eptide with non-competitive ELISA method.After coating the peptide by BSA binding with glutaral couple,affirming the best concentration,best time of peptide to coat the plate and the coating coefficient,and the proper binding time of peptide to Endoglin to reach an equilibrium,we plotted the standard curve of the binding reaction of peptide and Endoglin.Results The affinity constant of peptide and anti-hEndoglin IgG is respectively(2.956?0.749)?106mol/L and(7.403?10.76) ?108mol/L.Conclusion The peptide we got from the peptide liberary can strongly bind to Endoglin,providing a theoretical basis for its clinical use.