Effects of tetrandrine derivative HL-49 on biological property of Bloom DNA helicase / 中国药理学通报

Aim To study the effect of tetrandrine derivative HL-49 on the conformation and biological ac-tivity of Bloom helicase ( BLM ) , and to explore its antitumor mechanism.Methods The effect of HL-49 on the conformation of BLM helicase was studied by ultra- violet spectroscopy.The effects of HL-49 on DNA binding activity, DNA chain dissociation activity and ATPase activity of HL-49 on BLM DNA helicase were analyzed by fluorescence polarization and malachite green-ammonium phosphomolybdate colorimetric method.Results HL-49, a tetrandrine derivative, indirectly inhibited the ATPase activity of BLM DNA heli- case and DNA unwinding activity by reversible binding with DNA.The results of fluorescence polarization experiments showed that HL-49 could not affect the bind ing activity of BLM DNA helicase to DNA (dsDNA/ss- DNA) , but could bind to DNA in a concentration-de- pendent manner (P < 0.01).With the increase of HL- 49 concentration, the DNA unwinding ability of BLM DNA helicase decreased, and the Kobs value decreased gradually.The results of malachite green-ammonium phosphomolybdate colorimetry showed that HL-49 could significantly inhibit the ATPase activity of BLM DNA helicase.Conclusions HL49 can inhibit the ATPase activity and DNA unwinding activity of BLM DNA helicase by the reversible binding with DNA.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53

Development of a fluorescence polarization-based high-throughput screening assay to identify antagonists targeting β-catenin/TCF4 interaction / 药学学报

To develop a fluorescence polarization (FP)-based high-throughput screening (HTS) assay to identify novel small-molecule antagonists targeting β-catenin/TCF4 (T-cell factor 4) interaction, recombinant human β-catenin was expressed in Escherichia coli Rosetta (DE3) cells and purified by HisTrapTM column. The bioactivity of purified β-catenin was further analyzed by enzyme-linked immunosorbent assay (ELISA). According to FP principle, the β-catenin/TCF4 binding model was performed, and fluorescence isothiocyanate (FITC) labeled TCF4 peptide (FITC-TCF4) served as the molecular probe of adaptor for binding to β-catenin. The FITC-TCF4 and β-catenin working concentration were optimized, and the binding conditions (complex stability and dimethylsulfoxide (DMSO) tolerance) have been investigated yet for further hits screening. The results showed that recombinant human β-catenin was successfully expressed and purified β-catenin exhibited favorable bioactivity in ELISA binding assay. Subsequently, the FP-based HTS assay was performed using 20 nmol·L-1 FITC-TCF4 and 100 nmol·L-1 β-catenin. Under these optimized conditions, a high Z´factor of 0.88 was achieved in a 384-well format and this FP-based HTS assay was very stable with regard to DMSO. Through screening of a natural-based product library (NBPL) using the established FP-based HTS assay, three hits (sanguinarine, chelerythrine, and compound S720) were identified as potential β-catenin/TCF4 interaction antagonists. Taken together, we have successfully developed a simple, robust and reliable FP-based HTS assay for screening of novel antagonists targeting β-catenin/TCF4 interaction.

Design, synthesis and biological application of affinity-based small molecular probe for Hsp90 endoplasmic reticulum paralogue of Grp94 / 中国药科大学学报

@#Glucose-regulated protein 94(Grp94), an endoplasmic reticulum resident Hsp90 paralog, has a limited set of client proteins. Selective inhibition of Grp94 has emerged as a new direction for the development of drugs targeting the Hsp90 chaperone system. Now Grp94-Probe, an affinity-based probe of Grp94, was designed and synthesized based on DDO-5813, a most potent Grp94-selective inhibitor we found previously. Using fluorescence polarization(FP)assay and double staining assay with ER-Red in cells, we confirmed the binding of Grp94-Probe with ER Grp94. The FR results showed that the probe exhibited high affinity for Grp94N(EC50=117. 9 nmol/L)without exhibiting obvious Hsp90α inhibition, Moreover, as a fluorescence probe molecule, Grp94-Probe could better distinguish the inhibitory activity of compounds for Grp94N. The results of fluorescence analysis in cells showed that Grp94-Probe could co-stain with ER-Red in the endoplasmic reticulum, and the fluorescence did not decay rapidly with time after 4 h of staining, which further indicated the binding of Grp94-Probe with Grp94 in cells. This Grp94 selective probe can be further used for biology evaluation of Grp94 inhibitor and exploration of Grp94 biological functions.

Effects of dihydromyricetin on biological properties of Bloom syndrome helicase / 中国药理学通报

; Aim To explore the anti-tumor mechanism of dihydromyricetin (DMY), a kind of flavonoid compound with anti-inflammatory and anti-tumor effects, via studying the effect of DMY on biological activities of Bloom helicase. Methods The effect of DMY on the biological activities of BLM helicase was studied by ultraviolet spectrum (UV), circular dichroism (CD), fluorescence polarization and free phosphorus detection. Results The results of CD and UV showed that DMY could bind to a site of the BLM helicase. In the concentration of DMY in 0 ~ 25 μmol . L 1 range, DMY showed a positive correlation with the interference ability of BLM helicase secondary structure with the increase of concentration, while in the concentration of DMY in 25 ~ 75 μmol . L 1 range, DMY showed a negative correlation. Fluorescence polarization and free phosphorus detection experiments showed that DMY could bind to BLM helicase, thus inhibiting the helicase activity of BLM helicase. Conclusions DMY can competitively bind to the DNA binding site of BLM helicase and change the spatial structure of BLM helicase, inhibiting the binding of BLM helicase to DNA and the biological activity of BLM helicase accordingly.

Effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on biological properties of BLM helicase / 中国药理学通报

Aim To investigate the effects of bisbenzyl-isoquinoline alkaloid tetrandrine derivative HL-27 on the biological properties of the BLM642-1290 helicase. Methods Fluorescence polarization technique was used to investigate the effects of bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27 on the DNA bind-ing activity and unwinding activity of the BLM642-1290 helicase. Malachite green-phosphate ammonium molyb-date colorimetry was used to investigate the effects of HL-27 on the ATPase activity of the BLM642-1290 heli-case. Ultraviolet spectral scanning was used to investi-gate the effects of HL-27 on the conformation of the BLM642-1290 helicase. Results When the concentra-tion of HL-27 reached 33.34 μmol·L-1, the inhibi-tion ratio of dsDNA and ssDNA binding activity of the BLM642-1290 helicase was 41.35% and 59.54% , re-spectively. When the concentration of HL-27 reached 50 μmol·L-1, the inhibition ratio of DNA unwinding activity of the BLM642-1290 helicase was 78.68% . When the concentration of HL-27 reached 100 μmol· L-1, the inhibition ratio of ATPase activity of the BLM642-1290 helicase was 43.8% . Conclusion The DNA binding activity, ATPase activity and unwinding activity of the BLM642-1290 helicase can be inhibited by bisbenzylisoquinoline alkaloid tetrandrine derivative HL-27.

A fluorescence polarization immunoassay for the detection of aflatoxins in herbal teas / 药学学报

A rapid fluorescence polarization immunoassay (FPIA) has been developed for the determi-nation of aflatoxins in samples of naturally-contaminated herbal teas. The tracers were synthesized by chemical method and determined by thin layer chromatography (TLC) and mass spectroscopy (MS). Fluorescence polarization was evaluated by the detection of polarized light. The results showed that the limit of detection (LOD) of FPIA for aflatoxins was 20 ng·mL-1, the IC50 was 371.80 ng·mL-1, and the linear range of the developed FPIA was 92.76-252.32 ng·mL-1. Compared with conventional HPLC methods, the FPIA developed in this study has the advantages of short analysis time and low cost. This method may be suitable for high- throughput screening of aflatoxins in herbal teas.

Screening of small molecule inhibitors for PLK1 PBD and evaluation of antitumor activities / 药学学报

With the method of fluorescence polarization (FP), we screened small molecule inhibitors for PLK1 PBD to identify the lead compounds for antitumor drugs. FP led to the identification of a potent hit, F083-0063, whose inhibition rate was (99.7±0.4)% at 10 μg·mL-1. The IC50 was calculated to be 1.9±0.1 μmol·L-1 using Graphpad Prism 5. The effect of the compound on cells' multiplication was measured by MTT assay which showed that F083-0063 inhibited the proliferation of many tumor cell lines. Flow cytometry analysis indicated that the F083-0063 promoted cell apoptosis and induced cell G2/M arrest. Migration abilities of cells, evaluated using scratch test, increased significantly in the presence of F083-0063 with the mi-gration rate as low as (37.6±0.7)% at 20 μmol·L-1. Molecular linkage technique found F083-0063 had good affinity with PLK1 PBD. The results of Western blotting showed that the expression of cyclin-dependent proteins was increased after treatment with F083-0063. In summary, F083-0063 has an antitumor activity and is expected to be an antitumor lead compound targeting PLK1 PBD.

Development of the high-throughput screening fluorescence polarization assay for HSP90 inhibitor / 中国生化药物杂志

Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21 ( DE3) competent cells were transformed with pET24α( +)-HSP90αplasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile, the binding activity of GA and NVP-AUY922 for HSP90αwas measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01μg/mL HSP90α, with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90, with IC50 of 55 nM and 13 nM, respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.

A comparative study of Sodium valproate concentration in peripheral blood monitoring by FPIA and HPLC in epilepsy children / 中国生化药物杂志

Objective To investigate the comparation of sodium valproate concentration in peripheral blood monitoring by fluorescence polarization immunoassay method(FPIA)and high performance liquid chromatography method(HPLC)in epilepsy children.Methods 87 cases of epilepsy children received Sodium valproate treatment in our hospital from February 2014 to June 2016 were selected,fasting venous blood of elbow vein were collected the next morning after last medication, blood concentrations of Sodium valproate in serum samples were detected by FPIA method and HPLC method respectively,the correlation and consistency of results of the two methods were observed and compared.Results The intra day and inter day RSD of Sodium valproate concentration in peripheral blood in epilepsy children detected by FPIA were <5%,the recovery rate was 90%-110%,the precision and accuracy were high;The intra day and inter day RSD of Sodium valproate concentration in peripheral blood in epilepsy children detected by HPLC were <5%,the recovery rate was 90%-110%,the precision and accuracy were high; the linear regression equation between determination value of HPLC method (X) and determination value of FPIA method (Y) was:Y=0.8355X+1.8231,correlation coefficient r=0.914,the detection results were positively related;the Sodium valproate blood concentration detected by FPIA was significantly lower than that detected by HPLC method, the difference was statistically significant (P<0.05);Bland-Altman deviation chart results show that determination of blood drug concentration by HPLC method was higher than that of FPIA method by 7.2μg/mL.Conclusion The precision and accuracy of sodium valproate concentration in peripheral blood monitoring by FPIA method and HPLC method were all high, and the correlation was good, but the detection results of the two methods were significantly different,the detection result of HPLC method was higher than that of FPIA method,need to choose and judge according to the clinical situation.

Comparison of Reversed Phase High Performance Liquid Chromatography and Fluorescence Polarization Immunoassay for the Determination of Plasma Concentration of Carbamazepine / 医药导报

Objective To evaluate the correlation and difference of reversed phase high performance liquid chromatography (RP-HPLC) and fluorescence polarization immunoassay (FPIA) on determining serum concentration of carbamazepine.Methods Fifty serum samples were collected,both RP-HPLC and FPIA methods were employed to determine the concentration of carbamazepine.The results were analyzed by paired t test,Bland-Altman and Deming regression methods,respectively.Results The results of measuring 50 samples by the two methods showed that FPIA datas were significantly higher than RP-HPLC datas,and there was statistically significant difference(P<0.05) and poorer consistency between two methods;There was good correlation between carbamazepine concentrations determined by the two methods.Deming regression equation was CFPIA=1.195 3 CRP-HPLC-0.144 0,and Pearson correlation coefficient was 0.968 5.Conclusion Clinicians should pay more attention to the difference of carbamazepine concentration determination by different methods when carbamazepine individualized dosage regimen was adjusted according to therapeutic drug monitoring.

Evaluation of three commercially-available chikungunya virus immunoglobulin G immunoassays / Evaluación de tres inmunoensayos comercializados de inmunoglobulina G para el diagnóstico del virus del chikungunya

ABSTRACT The emergence of chikungunya virus in the Americas means the affected population is at risk of developing severe, chronic, rheumatologic disease, even months after acute infection. Accurate diagnostic methods for past infections are essential for differential diagnosis and consequence management. This study evaluated three commercially-available chikungunya Immunoglobulin G immunoassays by comparing them to an in-house Enzyme-Linked ImmunoSorbent Assay conducted by the Centers for Disease Control and Prevention (Atlanta, Georgia, United States). Results showed sensitivity and specificity values ranging from 92.8% - 100% and 81.8% - 90.9%, respectively, with a significant number of false-positives ranging from 12.5% - 22%. These findings demonstrate the importance of evaluating commercial kits, especially regarding emerging infectious diseases whose medium and long-term impact on the population is unclear.(AU)

RESUMEN Como consecuencia de la aparición del virus del chikungunya en las Américas, la población afectada corre el riesgo de padecer reumatismos crónicos graves, aun meses después de la infección aguda. Es fundamental contar con métodos precisos para diagnosticar los antecedentes de la infección a fin de elaborar un diagnóstico diferencial y abordar las manifestaciones de la fase crónica. Se han estudiado tres inmunoensayos comercializados de detección de inmunoglobulinas G para el diagnóstico del chikungunya, comparándolos con el enzimoinmunoanálisis de adsorción (ELISA) propio. Los resultados señalan valores de sensibilidad del 92,8% al 100% y de especificidad del 81,8% al 90,9%, así como un número significativo de falsos positivos, de entre el 12,5% y el 22%.(AU)

Brucella spp. in equines slaughtered in the south region of Brazil / Anticorpos contra Brucella spp. em equídeos da região Sul do Brasil abatidos em matadouro-frigorífico

Bacteria of the genus Brucella are widespread in many countries. These microorganisms can infect humans and many wild and domestic animal species. These bacteria have zoonotic potential, and can cause economic and public health problems since they can be transmitted by direct contact with sick animals, through consumption of contaminated milk, raw meat and its derivatives (Soares et al., 2015). Brucellosis is considered a chronic evolving disease, unusual in horses, predominantly caused by Brucella abortus. However, it is not characterized by reproductive disorders in horses, but primarily by abscess in the cervical region, bursa, tendons, and joints. Transmission is likely to occur via ingestion of contaminated water and pastures, especially in areas endemic for bovine brucellosis (Ribeiro et al., 2008). The slaughterhouse is a strategic point for obtaining information about the animal and animal products, edible or not. This study investigated the presence of anti-Brucella spp. immunoglobulins in the serum samples from horses slaughtered in a slaughterhouse in southern Brazil, to estimate the frequency of Brucella spp. antibodies and determine the spatial distribution of the cases.(AU)

Objetivou-se investigar a presença de imunoglobulinas anti-Brucella spp. em amostras de soros sanguíneos de equídeos abatidos em matadouro-frigorífico, sob Serviço de Inspeção Federal, localizado na região Sul do Brasil. Utilizaram-se 767 amostras de sangue de equídeos adultos abatidos no período de abril a maio de 2013. Os animais foram provenientes de 45 municípios dos estados do Rio Grande do Sul, Santa Catarina e Paraná. Para diagnóstico, foram utilizados os testes do antígeno acidificado tamponado (AAT), sendo os resultados positivos confirmados pelos testes de polarização fluorescente (TPF), reação de fixação de complemento (RFC) e 2-mercaptoetanol (2-ME). Foram sororreagentes no AAT 65 (8,47%) animais. Destes, apenas dois (3,07%) foram positivos também na RFC e três (4,62%) animais foram positivos no TPF. Apesar da baixa frequência de animais positivos para Brucella spp., pode-se afirmar que a infecção em equinos está presente na área estudada, o que é demonstrado pela presença de animais sororreatores. No âmbito da saúde animal, pública e ocupacional, sugere-se a atenção a essa doença, visando diminuir o risco de infecção.(AU)

A comparison of fluorescence polarization assay with four classical serological methods in diagnosis of human brucellosis / 中华地方病学杂志

Objective To analyze the diagnostic value of fluorescence polarization assay (FPA) for human brucellosis.Methods From April 2013 to August 2014,240 positive sera of brucella patients were collected as case group and 287 normal sera as control group in Heilongjiang Agricultural Reclamation Bureau General Hospital.Four milliliters of venous blood were withdrawn for separation of serum.Fluorescence polarization assay was used and the consistency,sensitivity and specificity were compared with those of four classical serological methods,including rose bengal plate agglutination test (RBPT),standard tube agglutination test (SAT),Coomb's test and cysteine test.Results The results of FPA were complied with those of the four conventional serological methods,Kappa ≥ 0.75.The sensitivity of FPA (89.17％) was higher than that of RBPT (87.08％),SAT (85.00％),Coomb's (74.17％) and cysteine test (75.83％).The specificity of FPA (94.08％) was higher than that of RBPT (89.90％) and Coomb's (81.53％),but lower than that of SAT (98.95％) and cysteine test (99.30％).Conclusion FPA can be used as a laboratory diagnostic method for human brucellosis,which is worthy to be widely used.

Binding mechanism between bovine serum albumin and echinacoside / 中草药

Objective: To explore the binding mechanism between echinacoside (ECH) and serum albumin (SA). Methods: The binding parameters were detected by spectrum experiment under physiological conditions, and the molecular modeling techniques had been used to investigate the binding mechanism between ECH and bovine serum albumin (BSA). Results: Molecular docking revealed that ECH binded to BSA mainly by hydrogen bonds and van der Waals forces, and there was a hydrophobic interaction. The results from spectroscopy indicated that the drug could bind with BSA to form static complex with significantly strong bond. The value of binding distances (r) was low, which indicated the occurrence of energy transfer. ECH affected the conformation of micro-domain and changed the hydrophobicity of the binding domain. The fluorescence phase diagram revealed that the changes on the conformational pattern of proteins had been affected by drug conformed to the "all-or-none" pattern. According to the obtained thermodynamic parameters, it also showed that the main interactional force of ECH binding with BSA was hydrogen bonds and van der Waals forces. The fluorescence polarization proved quantitatively that ECH-BSA generated a non-covalent complex. Conclusion: The experimental results agree with computer molecular modeling, which provides helpful reference for the interaction mechanism of ECH binding with BSA.

Fluorescence polarisation assay for detection of brucellosis / 中国人兽共患病学报

Brucellosis is a zoonotic disease of global importance for infecting humans .The early diagnosis of brucellosis infection plays a significant role in the treatment and rehabilitation .This paper reviews the different methods used to diagnose brucellosis ,particularly introduces the basic principles and applications of fluorescence polarisation assay as a diagnostic tool for brucellosis ,which could provide the reference for clinical diagnosis and epidemiological investigation on brucellosis .

A review on biological matrices and analytical methods used for determination of drug of abuse.

Drug addiction is a chronic disease with a potential for fatality if not treated. The drugs with potential for abuse are mostly psychoactive drugs. Serious widespread medical and health consequences associated with drug abuse involve neurotoxicity, cardiovascular complications, impairment of the immune system function, and many other physiological effects. Illicit drug use remains the second most common mode of HIV infection. Various analytical techniques and number of biological matrices has been used for the detection of drug of abuse in cases such as drug addiction, driving under influence of drugs, neonatal drug exposure in case of drug abuse by pregnant women etc. Urine and blood sample remain the most widely used conventional biosample for the detection of drug of abuse. Various other alternative biological matrices such as saliva, hair, nails, tears and meconium have also been used for the same purpose. Number of analytical techniques such as liquid chromatography with mass spectrometry (LC-MS) and LC with tandem MS (LC-MS2), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrospray ionization Time-of- Flight mass spectrometry (ESI-TOF), combination of ultra-performance liquid chromatography (UPLC) and TOF, fluorescence polarization immunoassay (FPIA) and enzyme multiplied immunoassay technique (EMIT) have been used for the detection of drugs of abuse in above mentioned biosamples. This review summarizes the conventional as well as alternative biological matrices and various analytical techniques used for the determination of drugs of abuse.

Desarrollo y evaluación de un protocolo serológico de fluorescencia polarizada en el estudio de anticuerpos contra Brucella spp en humanos / Development and evaluation of a serological protocol of fluorescence polarization for the preliminary study of Brucella spp antibodies in humans

A objeto de mostrar el desarrollo y alcance de un método de análisis serológico basado en la técnica de fluorescencia polarizada (FPA) a partir de una gota de sangre obtenida mediante punción capilar, se realizó la determinación de anticuerpos antibrucelosis de un conjunto de 321 personas de alto riesgo laboral. Los resultados se compararon con la data proveniente del análisis de sueros sanguíneos mediante FPA e inmunoanálisis enzimático competitivo (ELISA-c). El número de concordantes fue 318 (99,06%), los 3 discordantes (0,93%) resultaron negativos con fluorescencia polarizada en suero (FPAs) y ELISA-c, pero positivos con FPA capilar (FPAc). Los resultados comparativos de FPAc fueron: sensibilidad: 100%; especificidad: 99,05%; valor predictivo positivo: 66,67%; valor predictivo negativo: 100,0%; proporción de falsos positivos: 0,95%; proporción de falsos negativos: 0%; exactitud: 98,0%; razón de probabilidades: 203,00. La J de Youden para ambos métodos de FPA fue de 0,667. La determinación se consideró confiable y la concordancia de ambos procedimientos de FPA y ELISA-c resultó sin diferencias estadísticas (P>0,05%), lo que permite recomendar ampliamente la implementación del estudio de la brucelosis humana con sangre proveniente de punción capilar como método preliminar.

In order to show the development and scope of a serological analysis method based on fluorescence polarization assay (FPA) from a drop of blood obtained by the capillary technique, a Brucella antibody assay was performed on a group of 321 high-risk workers. The results were compared with data from the analysis of blood serum by FPA and a competitive enzyme immunoassay (ELISA-c). The number of concordance was 318 (99.06%), and discordant 3 (0.93%), which were negative in serum by fluorescence polarization (FPAs) and ELISA-c, but positive with capillary FPA (FPAc). The comparative results FPAc were: sensitivity 100%; specificity: 99.05%; positive predictive value 66.67%; negative predictive value 100.0%; false positive rate: 0.95%; false negative rate: 0%; accuracy: 98.0%; odds ratio: 203.00. The youden J for both FPA methods was 0.667. The identification was considered reliable and the correlation of both procedures, FPA and ELISA-c, was no statistically different (P> 0.05%), which allows to highly recommend the study implementation of human brucellosis with capillary blood as a preliminary method.

Validação interlaboratorial do teste de polarização fluorescente para o diagnóstico sorológico da brucelose bovina / Interlaboratorial validation of the fluorescence polarization assay for the serodiagnosis of bovine brucellosis

Esta investigação teve por objetivo validar o teste de polarização fluorescente (TPF) para o diagnóstico sorológico da brucelose bovina, determinando a sensibilidade (SE) e a especificidade relativas (SP) e verificando a reprodutibilidade do teste em quatro laboratórios no Brasil. Foram selecionadas 1.389 amostras de soro sanguíneo, as quais foram inicialmente submetidas aos testes do antígeno acidificado tamponado (AAT) e mercaptoetanol (2-ME). As mesmas amostras foram submetidas à reação de fixação de complemento (RFC) e ao TPF. Para a avaliação do TPF, foi adotada a combinação dos resultados do AAT, da RFC e do 2-ME, utilizados como população de referência (padrão-ouro). Para a determinação do ponto de corte do TPF que proporciona a melhor combinação de sensibilidade e especificidade, foi usada a análise TG-ROC. A concordância entre os resultados dos quatro laboratórios foi determinada com base no indicador kappa e no coeficiente de correlação de Pearson. Os pontos de corte do TPF situaram-se entre 85,2 e 93,6 mP, conforme o laboratório. A sensibilidade variou de 91,7 a 97,3 por cento, e a especificidade situou-se na faixa de 82,6 a 98,3 por cento. Na comparação entre os resultados do TPF dos quatro laboratórios, o indicador kappa ficou entre 0,69 e 0,95, o que indica, na maioria das situações, reprodutibilidade excelente, e o coeficiente de correlação variou entre 0,76 e 0,99. Os resultados indicaram que o TPF apresentou bom desempenho, na maioria das situações, com sensibilidade e especificidade elevadas. Em comparação com os testes convencionais, o TPF apresenta as vantagens de ser de execução mais rápida e mais fácil e não estar sujeito à ocorrência de prozona, como a RFC e o 2-ME, nem de atividade anticomplementar, como a RFC.

The purpose of this research was the interlaboratorial validation of the polarization fluorescence assay (PFA) for the serodiagnosis of bovine brucellosis, verifying the relative sensitivity, the relative specificity and the reproducibility of the test in four Brazilian laboratories. Serum samples from 1,389 bovines were selected and submitted to the rose Bengal (RBT) and 2-mercaptoethanol tests in one of the laboratories. The same samples were tested by the complement fixation (CFT) test and by the PFA in the four laboratories participating of the research. The reference population (golden standard) used to evaluate the PFA was the combination of the results of RBT, CFT and 2-ME. TG-ROC analysis was used to obtain the cut-off that provided the best combination of sensitivity and specificity. The agreement between laboratories was obtained by the kappa statistic and Pearson correlation coefficient (r). The PFA cut-off values were from 85.2 to 93.6. The sensitivity of the PFA assay varied from 91.7 percent to 97.3 percent, and the specificity values varied from 82.6 percent to 98.3 percent. When comparing PFA results from the four laboratories, the kappa values was between 0.69 and 0.95, which indicates, in most situations, excellent reproducibility, and the correlation coefficient varied from 0.76 to 0.99. The results showed that the PFA had a good performance, with high sensitivity and specificity. Compared to the conventional tests, the PFA has the advantages of being easy and quick to perform, and it is not prone to the occurrence of prozone, as the CFT or the 2-ME, nor to the occurrence of anti-complementary effect, as the CFT.

Influence of matrix effect upon cyclosporine A test by fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique methods / 中华检验医学杂志

Objective To explore the matrix effect on cyclosporine A (CsA) test by fluorescence polarization immunoassay (FPIA) and enzyme-multiplied immunoassay technique (EMIT), explain the discrepancy of external quality control results between these two methods and find the corrective action.Methods One hundred whole blood samples with various concentrations were adopted and CsA levels were detected by FPIA and EMIT.The results were compared with each other.Moreover, the influence of residual metal ions upon immunoreactions was assessed by adding Cu2+ and Zn2+.The effect of non-whole blood matrix on extraction efficiency for quality control materials and CsA calibrator was evaluated by adding identical volume of Hb-rich reagents followed with re-extraction.Results There is good correlation between results measured with FPIA(X) and EMIT(Y) methods ( Y=0.926 8X -8.115,R2 =0.996 9).Neither FPIA nor EMIT was affected by residual metal ions ( P ＞ 0.05 ). Non-whole blood matrix decreased the extraction efficiency of two methods, but it could be corrected by supplementation of the Hb-rich reagents (≥30 g/L).Conclusions Non-whole blood matrix may be the main reason for the inconsistent results measured by FPIA and EMIT methods.It could be corrected by using Hb-rich reagents.In addition,we should consider the influence of low lib on CsA test,espocially for organ transplant patients with lower Hb ( ＜30 g/L).