New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA / 药物分析学报

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0 × 10-18 to 1.0 × 10-15 mol/assay,with a detection limit of 1.0 × 10-18 tmol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8％.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Diagnóstico de infecção por vírus linfotrópicos de células T humanas dos tipos 1 (HTLV-1) e -2 (HTLV-2) em população de risco: passado, presente e futuro / Diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) and type 2(HTLV-2) infection in at-risk populations: past, present and future

O Brasil é o país com o maior número de pessoas infectadas pelos vírus linfotrópicos de células T humanas dos tipos 1e -2 (HTLV-1 e HTLV–2) com mais de 2,5 milhões de indivíduos infectados. Em 1993, a realização de testes sorológicos específicos tornou-se obrigatória em Bancos de Sangue. O HTLV-1 causa leucemia/linfoma de células T do adulto e mielopatia associada ao HTLV-1/paraparesia espástica tropical além de outras doenças, enquanto o HTLV-2 pode causar alguns quadros neurológicos e alterar a evolução de HIV/Aids. Os testes sorológicos que identificam anticorpos específicos disponíveis no mercado têm falhado no diagnóstico, principalmente de infecção por HTLV-2. Vários algoritmos de testes de triagem e confirmatórios têm sido propostos, mas nenhum deles se mostrou 100% eficiente com casuística de alto risco. Muitos soros resultam em padrão indeterminado no Western blot, e os isolados virais utilizados na composição dos kits podem ser a causa desses resultados. As técnicas de biologia molecular têm sido descritas como testes confirmatórios, mas não têm sido empregadas na rotina. Desde 1991, a Seção de Imunologia do Instituto Adolfo Lutz tem estudado a infecção por HTLV-1/2, contribuindo para o diagnóstico sorológico e molecular, e tem como desafio implantar um teste laboratorial capaz de detectar infecção causada por cepas brasileiras de HTLV-2.

Usefulness of Short-term Follow-up Enzyme Immunoassay in Mycoplasma pneumoniae Infection / 소아알레르기및호흡기학회지

PURPOSE: The diagnosis of Mycoplasma pneumoniae (M. pneumoniae) infection is usually based on serology using complement fixation assay (CFA), particle agglutination test (PA), enzyme immunoassay (EIA) and polymerase chain reaction (PCR). The objective of this study is to compare the performance of EIA and PCR in diagnosis of M. pneumoniae infection. We also evaluated the usefulness of EIA which were checked on short-term follow-up (3-5 days). METHODS: We included 234 pneumonia children. We used serum specimens for EIA test, which were obtained on admission and 3-5 days after admission. We collected throat swabs or sputums for PCR test, which were obtained on admission or next morning after admission. RESULTS: Of 234 patients, 124 (53.0%) met the diagnostic criteria. The median age was 6 years (from 10 months to 12 years). On admission, the sensitivity and specificity of EIA-specific IgM were 46.1% and 72.8%, respectively. The rate of agreement between PCR and EIA was 64.1%.(kappa=0.187, P=0.004) On 3-5 days after admission, the sensitivity and specificity rates of EIA specific IgM were 85.5%, 69.6%, respectively. The rate of agreement between PCR and EIA was 74.8%.(kappa=0.490, P=0.000) Days after onset had no relation with sensitivity of EIA.(P>0.05) The sensitivity and specificity rates of PCR were 57.5% and 90.0%, respectively. CONCLUSION: This study suggests that PCR and EIA may be the useful diagnostic methods for detecting early phase of M. pneumoniae infection. And EIAs which checked on short-term follow up is also useful. PCR has shown a higher specificity but lower sensitivity. Therefore, PCR must be performed with serologic tests.

Performances of HTLV serological tests in diagnosing HTLV infection in high-risk population of São Paulo, Brazil

Testing problems in diagnosing human T-lymphotropic virus (HTLV) infection, mostly HTLV-II, have been documented in HIV/AIDS patients. Since December 1998, the Immunology Department of Instituto Adolfo Lutz (IAL) offers HTLV-I/II serology to Public Health Units that attend HTLV high-risk individuals. Two thousand, three hundred and twelve serum samples: 1,393 from AIDS Reference Centers (Group I), and 919 from HTLV out-patient clinics (Group II) were sent to IAL for HTLV-I/II antibodies detection. The majority of them were screened by two enzyme immunoassays (EIAs), and confirmed by Western Blot (WB 2.4, Genelabs). Seven different EIA kits were employed during the period, and according to WB results, the best performance was obtained by EIAs that contain HTLV-I and HTLV-II viral lysates and rgp21 as antigens. Neither 1st and 2nd, nor 3rd generation EIA kits were 100 percent sensitive in detecting truly HTLV-I/II reactive samples. HTLV-I and HTLV-II prevalence rates of 3.3 percent and 2.5 percent were detected in Group I, and of 9.6 percent and 3.6 percent in Group II, respectively. High percentages of HTLV-seroindeterminate WB sera were detected in both Groups. The algorithm testing to be employed in HTLV high-risk population from São Paulo, Brazil, needs the use of two EIA kits of different formats and compounds as screening, and because of high seroindeterminate WB, may be another confirmatory assay.

Problemas nos testes diagnósticos de infecção pelos vírus linfotrópicos de células T humanas (HTLV), principalmente HTLV-II, têm sido observados em pacientes com HIV/Aids. Desde Dezembro de 1998, a Seção de Imunologia do Instituto Adolfo Lutz (IAL) oferece a sorologia para HTLV-I/II para Serviços de Saúde Pública que atendem populações consideradas de risco para esta infecção. Duas mil trezentas e doze amostras de soro: 1.393 de Centros de Referência em Aids (Grupo I) e 919 de Clínicas de Especialidade em HTLV (Grupo II) foram encaminhadas para o IAL para a pesquisa de anticorpos anti-HTLV-I/II. A maioria delas foram testadas por dois ensaios imunoenzimáticos (EIAs) e confirmadas por Western Blot (WB 2.4, Genelabs). Sete kits diferentes de EIAs foram empregados durante o período e de acordo com os resultados do WB a melhor performance foi obtida com os EIAs que continham lisado viral dos HTLV-I e -II e a rgp21 como antígenos. Nenhum kit de EIA de 1ª, 2ª ou 3ª geração foi 100 por cento sensível para detectar todas as amostras verdadeiramente HTLV-I/II reagentes. A prevalência de HTLV-I e HTLV-II, respectivamente, foi de 3,3 por cento e 2,5 por cento no Grupo I e de 9,6 por cento e 3,6 por cento no Grupo II. Em ambos os Grupos, foram detectadas altas percentagens de soros com padrão indeterminado no WB. O algoritmo de testes sorológicos para ser usado em população de alto risco para HTLV de São Paulo, Brasil, necessita de dois kits EIAs de princípios e composição diferentes para a triagem sorológica e, pelo elevado número de WB indeterminado, talvez de um outro teste confirmatório.

Clinicial Outcome of Pregnancies with Positive or Equivocal Anti-rubella IgM Antibody and Comparison of the Results Performed by 4 Different Enzyme Immunoassays / 대한임상병리학회지

BACKGROUND: The test for the anti-rubella IgM Ab (R-IgM) is important in early pregnancies because therapeutic termination may be considered depending on the results. METHODS: The subjects consisted of 52 pregnant women with positive or equivocal R-IgM by Cobas Core Anti-Rubella IgM EIA test (Roche, Basel, Swiss) between January 1997 and July 2000. Three different EIA methods such as the Enzygnost Anti-Rubella-virus/IgM test (DADE Behring, Marburg, Germany), the AxSYM Anti-Rubella IgM test (Abbott, USA), and the IMx Anti-Rubella IgM test (Abbott, USA) were simultaneously performed on 44 specimens as well as the Cobas Core Anti-Rubella IgM EIA test. RESULTS: Among 52 pregnancies, 9 (17%) experienced an artificial abortion due to positive or equivocal R-IgM result. The clinical symptoms associated with rubella infection were observed in 3 cases and the persistent R-IgM positivity was noted for more than 1 year in 4 cases. The concordance rate between 4 different EIAs was 41%. When performed with serial diluted pool and 3 sera, the results of Cobas Core were similar to those of AxSYM, IMx and Enzygnost. The R-IgM were detected one titre higher in diluted sera performed by IMx and Enzygnost than those of Cobas Core and AxSYM. CONCLUSIONS: For pregnancies with positive or equivocal R-IgM, it is recommended that results should be interpreted with caution, considering the possibilities, such as a persistent R-IgM response and discrepant R-IgM depending on the different EIA methods.

Laboratory Evaluation of the LG HCD 3.0TMB (LG HCD3.0 plus) for the Detection of HCV Antibodies / 대한임상병리학회지

BACKGROUND: The purpose of this study was to evaluate the newly improved third generation enzyme immunoassay kit, LG HCD 3.0TMB (LG Chemical Ltd., Korea) for the detection of antibodies for the hepatitis C virus. METHODS: The 1,068 clinical samples and 3 seroconversion panels were subjected to compare LG HCD 3.0TMB with Ortho HCV 3.0. The discordant clinical samples were confirmed by RT-PCR (Roche Amplicor HCV test) and RIBA (LG HCD confirm, Ortho RIBA HCV 3.0). Reproducibility was estimated using six samples with different anti-HCV levels for each assay. RESULTS: In clinical samples, concordance between LG HCD 3.0TMB and Ortho HCV 3.0 was 98.8% (1,055 of 1,068 tests) in the screening test. After a repeat test of 13 discordant samples, overall concordance was 99.3% (1,061 of 1,068 tests). In the seroconversion panel testing, the LG HCD 3.0TMB detected HCV antibodies earlier than the Ortho HCV 3.0 in one of the three panels. Results of both EIA assays were constant on the reproducibility test. CONCLUSIONS: Based on the good agreement with Ortho HCV 3.0 and superior seroconversion sensitivity, the LG HCD 3.0TMB assay appears to be suitable for detecting HCV antibodies in clinical laboratories.

Impact of Anti-HCV(+) on Renal Transplantation

BACKGROUND: A high incidence of chronic liver disease is reported in end-stage renal failure patients due to hemodialysis and blood transfusion. An average of 20% of the patients who received renal hemodialysis are infected with hepatitis C virus, but the incidence of infection in these patients varies widely according to geographic location and the diagnostic methods used. Controversy exists regarding the impact of pretransplantation HCV infection on the outcome of renal transplantation. We measured the seroprevalence of the antibody to hepatitis C (anti-HCV) in renal transplant candidates and compared the prevalence of posttransplantation liver disease, graft, and patient survival among renal transplant recipients with and without anti-HCV at the time of the transplantation, and we attempted to define the possible factors affecting the clinical course following renal transplant in positive HCV patients. METHODS: Between June 1990 and December 1997, 634 patients underwent renal transplants at our institute. Viral infection with hepatitis were analyzed in these patients by using anti-HCV positivity using first, second, and third generation EIA, and RT-PCR. RESULTS: Twelve (12) of the 634 (1.9%) had positive anti-HCV before renal transplantation. During a mean follow-up of 29.4 months, viral mRNA was detected in the pretransplantation serum in 3 out of 8 (37.5%) positive anti-HCV patients. Among the 12 patients with positive anti-HCV, 2 (16.6%) showed early liver dysfunction, and 1 (8.3%) showed histologic progression to chronic hepatitis leading to hepatic failure and death. Graft loss occurred in 1 of the 12 (8.3%) patients with positive anti-HCV and in 62 of the 622 (9.8%) patients with negative anti-HCV. Three (3) out of the 12 (25%) patients with positive anti-HCV, and 121 of the 622 (19.6%) patients with negative anti-HCV had episodes of rejection. One (1) of the 12 (8.3%) patients with positive anti-HCV and 26 of the 622 (4.2%) patients with negative anti-HCV died after kidney transplantation. There were no statistical differences in patients or graft survival between the positive anti-HCV (+) and the negative anti-HCV patients. CONCLUSION: From these results, we can assume that the presence of anti-HCV without advance liver disease should not be a contraindication for kidney transplantation.

HCV Serotypes and HCV RNA Quantitation / 대한임상병리학회지

BACKGROUND: There are several serological subtypes (serotypes) of hepatitis C virus (HCV) which can be detected by a serological enzyme immunoassay (EIA) method which detects the HCV subtype-specific antibodies against the NS4 region of the virus. We determined the HCV serotypes chronic HCV infected patients in terms of clinical applications. METHODS: Subtypes based on Simmonds' classification were detected in chronic HCV patients serologically and viral loads quantitated with branched DNA (bDNA) assay. The EIA was compared with multiplex PCR method. RESULTS: The serotype 1 (Simmonds' classification, 1a, 1b, 1c), the most prevalent form in Korea followed by serotype 2 (Simmonds' classification, 2a, 2b, 2c). The distribution of serotypes and HCV viral loads were not different according to disease severity. The patients infected with serotype 1 had higher viremic level (serotype 1, 7.4+/-15.5 MEq/mL; serotype 2, 1.1+/-2.2 MEq/mL, P=0.017). The serotype matched with the genotype in 85.7% (18/21) of cases. CONCLUSIONS: HCV serotype 1 is the most prevalent form in Korea and seemed to be able to more actively replicate than serotype 2. Both multiplex PCR and EIA can be used for detecting HCV subtypes and mutually interpretable.