Development and verification of double antibody sandwich ELISA method for determination of D antigen content in s IPV vaccine / 中国生物制品学杂志

@#Objective To develop and verify a sandwich ELISA method with bovine polyclonal antibody against rabbit polyclonal antibody for the determination of D antigen content of Sabin strain inactivated poliovirus vaccine(sIPV).Methods The rabbit polyclonal antibodies were prepared with sIPV vaccine bulks of type Ⅰ,Ⅱ and Ⅲ as antigens and detected for the titer and specificity by indirect ELISA.A double antibody sandwich ELISA with bovine polyclonal antibody as coating antibody and rabbit polyclonal antibody as detection antibody was developed to determine D antigen content,and the accuracy,precision and specificity to D antigen of the method were verified.sIPV vaccine samples from five domestic enterprises were detected by the developed method.Results The rabbit polyclonal antibodies for type Ⅰ,Ⅱ and Ⅲ sIPV with good specificity and high titer were prepared,and the double antibody sandwich ELISA method was successfully developed.Using four-parameter fitting,all three standard curves showed good linear relationship,and R~2 values were more than 0.99.The spike recoveries of type Ⅰ,Ⅱ and Ⅲ D antigens were all within 80%～120%,with average values of98.11%,97.41% and 98.66%,respectively.The CVs of repeatability and intermediate precision were all below 10%.The method also distinguished D antigens from C antigens.The developed method determined the D antigen contents of sIPV vaccines from five enterprises.Conclusion A sandwich ELISA method for determination of D antigen content in sIPV vaccine was successfully developed with satisfying accuracy,precision and certain D antigen specificity,which can be used to detect vaccines produced by different manufacturers.

Prevalencia del antígeno D débil y su clasificación fenotípica en donantes voluntarios de sangre / Prevalence of "weak" D antigen and its phenotypic classification in voluntary blood donours / Prevalência do antígeno D "fraco" e sua classificação fenotípica em doadores voluntários de sangue

En el campo de la medicina transfusional la correcta identificación de los fenotipos del sistema Rh y en especial del antígeno D debe ser de manera inequívoca por su relevancia clínica. El antígeno D tiene variantes denominadas D parcial, D débil y DEL, las que se producen por mutaciones de los alelos RHD/RHCE o por una supresión en la expresión fenotípica. Se trató de un estudio descriptivo, retrospectivo de corte transversal en el que se realizó una revisión de registros primarios durante el período 2011-2014 validados de acuerdo con el protocolo de Hernández-Sampieri R. Se utilizó estadística descriptiva mediante la aplicación del software informático SPSS versión 22.0 y se estableció la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado. Se determinó una prevalencia de donantes RhD negativos de 1,8 a 2,5% y RhD débil de 1,79 a 2,28%. La fenotipificación serológica permitió identificar que los tipos 2 y 5 eran los más frecuentes. También se estableció la existencia de aloinmunización por anti-D, anti-C y anti-E. Se estableció de esta manera la existencia de D débil y una importante aloinmunización en la población de donantes de sangre tipificados como D negativo y D débil, por lo que se recomienda implementar un algoritmo de identificación del antígeno D en servicios de medicina transfusional.

In the field of transfusion medicine, the correct identification of the phenotypes of the Rh system and especially of the D antigen must be unequivocal for clinical relevance. The D antigen has variants called partial D, weak D and DEL. These are produced by mutations of the RHD/RHCE alleles or a suppression in phenotypic expression. The objective of this study was to establish the frequency of weak D antigen in the population of blood donours from 17 Ecuadorian states and their phenotypic combinations. It was a descriptive, retrospective cross-sectional study performed during the 2011-2014 period and validated with primary records in accordance with the Hernández-Sampieri R protocol. A descriptive statistics through the application of SPSS computer software version 22.0 was used and the relationship between independent variables through the Chi-square statistic method was established. A prevalence of RhD negative donours from 1.8 to 2.5% and weak D 1.79 to 2.28% was observed The serological phenotyping made it possible to identify that type 2 and 5 were the most frequent. The presence of alloimmunization by anti-D, anti-C and anti-E was also established. Besides, the presence of weak D types and significant alloimmunization in the donour population of blood typed as D negative and weak was established, so it is recommended to implement an algorithm for the identification of D antigen in transfusional medicine services.

No campo da medicina transfusional, a correta identificação dos fenótipos do sistema Rh e especialmente do antígeno D deve ser inequívoca devido a sua relevância clínica. O antígeno D tem variantes chamadas de D parcial, D fraca e DEL, as quais são produzidos por mutações dos alelos RHD/RHCE ou por uma supressão na expressão fenotípica. O objetivo deste estudo foi estabelecer a frequência do antígeno D fraco em uma população de doadores de sangue de 17 províncias equatorianas e suas combinações fenotípicas. Foi uma estudo descritivo, retrospectivo de corte transversal em que se realizou uma revisão dos registros primários validados de acordo com o Protocolo Hernández-Sampieri R durante o período 2011-2014. Utilizou-se estatísticas descritivas através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes através da análise estatística de qui-quadrado. Foi determinada uma prevalência de doadores RhD negativos de 1,8 a 2,5% e RhD fraco de 1,79 a 2,28%. A genotipagem serológica permitiu identificar que os tipos 2 e 5 são os mais frequente. A existência de alo imunização por anti-D, anti-C e anti-E também foi estabelecida. A existência de D fraco e uma alo imunização significativa na população de doadores de sangue tipificados como D negativo e fraco, por isso é recomendado implementar um algoritmo de identificação do antígeno D em serviços de medicina transfusional.

Frecuencia de fenotipos del sistema Rh en donantes voluntarios de sangre / Frequency of Rh system phenotypes in voluntary blood donors / Frequência de fenótipos do sistema Rh em doadores de sangue voluntários

El sistema Rh es altamente polimórfico y está relacionado con la producción de aloanticuerpos y la enfermedad hemolítica del recién nacido. Los antígenos codificados por los genes RHD y RHCE forman el fenotipo Rh que es característico en cada población. Las variantes RHCE no han sido identificadas en la población ecuatoriana y así constituyen un riesgo de aloinmunización durante el embarazo o en transfusiones de componentes sanguíneos incompatibles. Prueba de ello es el estudio realizado en Ecuador que determinó una aloinmunización del 0,27%. Los anticuerpos con mayor frecuencia pertenecían al sistema Rh, resultados que motivaron la realización del presente estudio. Se analizaron un total de 1.298 muestras de donantes de sangre provenientes de 22 provincias ecuatorianas. Para la fenotipificación se utilizaron antisueros comerciales de la casa BIORAD y células de fenotipo conocido para el control de calidad interno, y se identificaron 20 fenotipos del sistema Rh distribuidos de forma heterogénea en las 22 provincias; el más frecuente fue Rz/R0. En donantes con fenotipo D débil el más común fue el R2/r, mientras que en los donantes Rh(D) negativo fue el fenotipo r/r. Estos datos demuestran la variedad de fenotipos en la población ecuatoriana y por ende la necesidad de su detección oportuna.

The Rh system is highly polymorphic. It is related to the production of alloantibodies and the hemolytic disease of the newborn. The antigens encoded by the RHD and RHCE genes form the Rh that is characteristic for each population. The RHCE variants have not been identified in the Ecuadorian population, constituting a risk of alloimmunization during pregnancy or in transfusions of incompatible blood components. Proof of this is the study carried out in Ecuador that determined an alloimmunization of 0.27% and the antibodies, more frequently belonged to the Rh system, results that motivated the realization of the present study. A total of 1298 samples from blood donors from 22 Ecuadorian provinces were analyzed. For the phenotyping, commercial antisera from the BIORAD house were used and cells of known phenotype for internal quality control. Identifying 20 phenotypes of the Rh system distributed heterogeneously in the 22 provinces, the most frequent was Rz/R0. In donors with weak D phenotype the most common was R2/r; whereas in Rh(D) negative donors was the r/r phenotype, these data demonstrate the variety of phenotypes in the Ecuadorian population and therefore the need for their timely detection.

O sistema Rh é altamente polimórfico e está relacionado com a produção de aloanticorpos e a doença hemolítica do recém-nascido. Os antígenos codificados pelos genes RHD e RHCE formam o fenótipo de Rh, que é característico para cada população. As variantes de RHCE não foram identificados na população equatoriana constituindo um risco de aloimunização durante a gravidez ou em transfusões de componentes sanguíneos incompatíveis. Prova disso é o estudo realizado no Equador que determinou aloimunização de 0,27%. Os anticorpos com maior frequência pertenciam ao sistema Rh, resultados que motivaram a realização do presente estudo. Um total de 1298 amostras de doadores de sangue de 22 estados equatorianos foram analisadas. Utilizou-se para a fenotipificação anti-soros comerciais BIORAD e células de fenótipo conhecido para controle de qualidade interno, identificando-se 20 fenótipos do sistema Rh heterogeneamente distribuídos nos 22 estados. O mais frequente foi Rz/R0. Em doadores com fenótipo D fraco, o mais comum foi o R2/r; ao passo que nos doadores Rh (D) negativo foi o fenótipo r/r. Esses dados demonstram a variedade de fenótipos na população do Equador, e portanto a necessidade da detecção precoce dos mesmos.

The effect of expression level of IL-23R mRNA on Th17/IL-17 immune response in mycobacterium tuberculosis infection / 中华疾病控制杂志

Objective To investigate the effect of interleukin 23 receptor (IL-23R) on T helper cell 17 (Th17) call-mediated immune response in mycobacterium tuberculosis (TB) infection,and to explore the role of IL-23R in the pathogenesis of pulmonary tuberculosis.Methods 21 active lung tuberculosis (ATB) patients were enrolled in Beijing chest hospital from July to October in 2015,21 cases of latent tuberculosis infection (LTBI) and 21 healthy Healthy Donors (HD) were selected from Beijing Changping center for tuberculosis control and prevention from May to July in 2015.The peripheral blood mononuclear cells (PBMCs) were isolated and cultured.The expression of IL-23R mRNA in PBMCs was detected,IL-23 and IL-17A levels in the supernatant of PBMCs were measured.The expression of IL-23R mRNA in different groups and the effect of IL-23R expression on IL-17A level were analyzed.Results The expression of IL-23R mRNA in ATB group was lower than that in LTBI group (Z =-2.528,P =0.011),and in ATB group was higher than that in HD group (Z =-3.849,P ＜ 0.001).The expression of IL-17A in ATB group was lower than that in LTB group (t =2.238,P =0.031),and ATB group was higher than that in HD group (t =4.733,P ＜ 0.001).There was no significant difference in IL-23 level between the three groups (F =0.432,P =0.651).IL-23R mRNA expression was positively correlated with IL-17A level (rs =0.438,P =0.047).Conclusions The expression level of IL-23R in mycobacterium tuberculosis infection can regulate the immune response mediated by Th17 cells,which may affect the susceptibility and infection outcome of pulmonary tuberculosis.

Rh Blood Grouping of the Individuals Born of Rh Negative Mother and Rh Positive Biological Father.

Unlike ABO antigens, Rh antigens are present only on Red Blood Cells (RBC), RhO(D) antigen is clinically the most important in the Rh system because it is highly antigenic. In India, as reported, 95% population is Rh positive whereas 05% is Rh Negative(approximately). Rh antibodies are the major cause3 of haemolytic disease of newborn (HDN) and lead to destruction of transfused Rh Positive red cells. A total number of 799 pregnant mothers having RhD Negative Blood Group (and also their husband possessing RhD Positive Blood Group) were surveyed. Out of the 799 babies born, 662 possessed RhD Positive and 137 possessed RhD Negative Blood Group. Besides this, twin babies born of RhD Negative mother and RhD Positive Biological Father were computed separately (Total No.of such mothers being 12). In case of twin delivery, the twins babies all possessed RhD Positive Blood Group. Calculation shows that about 17% of babies were RhD Negative and 83% were RhD Positive.

Weak D antigen - Revisited.

D antigen is the most immunogenic antigen in the complex Rh blood group system discovered in the year 1939. There is a lot of polymorphism in its phenotype due to genetic heterogeneity. Certain mutations and /or deletions lead to a weak phenotype defined by decreased density of antigen sites which require the use of anti human globulin for detection. The need for detection of the weak D antigen was to prevent alloimmunization by this blood if transfused to a D negative patient especially to women in child bearing age group. This contention is however, controversial and not proven beyond doubt. Moreover, the use of potent monoclonal D typing antisera detects low density of weak D antigens thus obviating the use of anti human globulin. We have assessed the incidence of Rh negative and weak D blood groups in the Garhwal region of Uttarakhand and reviewed the literature regarding the controversies in the clinical significance of weak D antigen.

Cloning and expression of human anti-Rh(D) single-chain Fv fragment / 医学研究生学报

Objective: To construct a phage display library of human single-chain Fv antibodies against blood group Rh(D) substance. Methods: Combining phage display library techniques, isolated total RNA from B lymphoblastoid cell lines secreting anti-Rh(D) antibodies was used for the synthesis of the first strand of cDNA, V_ H and V_ L genes were amplified by 2nd PCR and linked together by splicing overlap extension (SOE) with the use of a (Gly_ 4Ser)_ 3 linker. The resulted scFv genes were then cloned into pCANTAB5E vectors and displayed on the phage. Phage clones were selected using intact red cells as a source of antigen. After 4 rounds of "binding-elution-enrichment", each clone was assayed for specificity by Dot ELISA. Results: A phage antibody library, with the sink size being 1.2?107, was obtained. The percentage of full-length scFv gene inserted into phage DNA was 0.80. Rescued by helper phage, a phage scFv library with titer of 3?108 pfu/ml was established. Specific phages with scFv were acquired after 4 rounds of panning, one clone exhibiting specific binding to Rh+ cell was identified by Dot ELISA. Conclusion: A strategy for construction phage antibody library by means of phage display technique was practicable, which would be useful in screening engineered antibodies against human Rh (D) blood group substances.

A Case of Partial-D Category Va / 대한수혈학회지

The patient, a 65-year-old woman, was admitted for chronic subdural hematoma. ABO and Rh blood typing were performed as a pre-operation test. Her red blood cells were not agglutinated with anti-D reagent (Ortho Diagnostic System, USA). But they were positive in subsequently performed weak-D test and also agglutinated with three other anti-D reagents (Baxter Dade, USA; Biotest Diagnostics, Korea; Bioscot Ltd., UK). The patient s Rh phenotype was CcDe. Antibody screening test, direct and indirect antiglobulin tests showed negative results. Different reactivity to various anti-D reagents as shown in this case suggested that her cells have partial-D antigen which lack one or more components of the Rh D antigen. We considered that this case was category Va according to the reactivity patterns of monoclonal anti-D antibodies with various partial- D cells.

Chemical Modification of RBC Surface Antigen with Methoxy Polyethylene Glycol / 대한임상병리학회지

BACKGROUND: Today, blood group antigens are a strong barrier of safe transfusion. We evaluated the change of agglutinability of antibody to RBC surface antigen before and after activated methoxy polyethylene glycol (mPEG) modification. METHODS: We collected blood from healthy volunteers and the blood were treated by activated mPEG (MW 5,000, Sigma, USA). Agglutinability of RBC was measured using anti-sera (Green Cross, Korea) in ABO and Rh(D) groups, and compared the agglutinability changes before and after mPEG treatment. RESULTS: The agglutinability of Rh(D) surface antigen (n=20) was disappeared after mPEG treatment. However, ABO antigens showed variable agglutinability against antisera, some of which showed no change at all. CONCLUSIONS: In the case of Rh(D) antigen, it would be useful to apply mPEG treated RBCs for clinical use, if the safety problem were solved. But in the case of ABO antigen, the more evaluation of the condition of reaction and the concentration of mPEG should be needed.

The study of RHD gene mutation in weak D / 대한수혈학회지

BACKGROUND: The weak D is characterized serologically by a weak or negative agglutination reaction with polyclonal anti-D in an immediate-spin test and agglutination is enhanced in the indirect antiglobulin test. Weak D has a lower number of D antigen or weaker antigen density than are normal D positive red cells. Here we studied the cause of weak D antigenicity at genetic level and compared to that of normal D RBCs. METHODS: The amplification of RHD gene and RHCcEe gene site was done in normal D(n=20), weak D(n=8), D negative group(n=20) by polymerase chain reaction and by based on D typing in these individuals compared to that of serologic D typing. In addition, to detect RHD gene mutation and nucleotide sequence difference of weak D group compared to normal D RBCs, single stranded conformational polymorphism PCR was simultaneuosly perfomed in two group by RHD amplified product(189 bp). We analysis the correlation RHD genotyping and serological phenotyping, and also analysis the difference of nucleotide sequence between two group in genetic level. RESULTS: The RHD genotyping was completely matched normal D(n=20), D negative group(n=20) but weak D group(n=8) showed same genotype of normal D RBCs. In single stranded conformational polymorphism PCR, weak D phenotypes does not show any abnormalities at the genomic level when compared to the RHD gene in normal D phenotypes. CONCLUSIONS: RHD PCR showed good correlation with conventional serologic test but weak D genotype was same as that of normal D RBCs. The weaker immunogenicity of weak D is not explained by genomic DNA difference itself.