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

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Antigens, Bacterial/*immunology ; Argentina ; Bulgaria ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leptospira/isolation & purification/metabolism ; Leptospira interrogans/isolation & purification/metabolism ; Leptospirosis/*diagnosis/microbiology ; Male ; Polysaccharides/*immunology ; Reagent Kits, Diagnostic/*standards ; Republic of Korea ; Sensitivity and Specificity

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.
Antigens, Bacterial/*immunology ; Argentina ; Bulgaria ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leptospira/isolation & purification/metabolism ; Leptospira interrogans/isolation & purification/metabolism ; Leptospirosis/*diagnosis/microbiology ; Male ; Polysaccharides/*immunology ; Reagent Kits, Diagnostic/*standards ; Republic of Korea ; Sensitivity and Specificity

Due to the high variation in test results of indirect enzyme-linked immuno sorbent assay (ELISA) and complicated steps involved in the process of standardization, a platform used for standardizing the test results from indirect ELISA was developed. The platform was designed based on 'Improved Standardization Method for Optical Density' (I-STOD). Gauss-Newton iteration was applied to estimate parameters in a standard formula. Programming Language VB was used for developing interface of platform. The results indicated that the validity of experiment could be verified through platform. A well determined scope of standardization could be generated. The sample with concentration within the scope was standardized and the degree of dilution was calculated for those outside the scope. The platform was successfully developed which normalized the process of standardization. The function provides the researchers with an effective and convenient tool for quickly achieving standardization of ELISA test results.
Antibodies ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; standards ; Humans ; Optical Phenomena

OBJECTIVETo study the pharmacokinetics of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro.

METHODSThe pharmacokinetics of rhG-CSFa and conventional (wild type, WT) granulocyte colony-stimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20, 50, or 100 microg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro, the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat's whole blood or serum.

RESULTSPharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that, at each dose tested, for either route of drug administration, the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF, and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa, respectively.

CONCLUSIONrhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.

Animals ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; methods ; standards ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; genetics ; pharmacokinetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; Sensitivity and Specificity

BACKGROUND: Current status of external quality assessment (EQA) of laboratory tests for syphilis in Korea was analyzed to find out the problems that should be improved in the future. METHODS: Based on the data from the external quality assessment program performed twice a year by the Immunoserology Subcommittee of the Korean Association of Quality Assurance for Clinical Laboratory from the year 2004 to 2006, discordance rates were analyzed according to the test method and commercial kit used. RESULTS: Among the laboratories participating in the EQA program for syphilis test, about 90% of them used non-treponemal tests and about 55% treponemal tests. The non-treponemal tests included RPR (rapid plasma reagin) and VDRL tests used in 88% (363/412) and 11% (45/412), respectively, of the laboratories. The discordance rates were 2.2% for RPR test and 3.6% for VDRL. For the treponemal tests, Treponema pallidum hemagglutination assay (TPHA) was used in 60-76% and Immunochromatography assay (ICA) in about 30% of the laboratories in 2006. A high discordance rate of over 10% was reported in both TPHA and in ICA methods, possibly due to a low titer (1:1 in VDRL) of EQA samples in 2005. Analysis of the accumulated data from year 2004 to 2006 showed that the discordance rates of TPHA, ICA, and FTA-ABS were 4.6%, 3.7%, and 2.7%, respectively. CONCLUSIONS: For syphilis tests, RPR test, TPHA, and ICA are mainly used in Korea. A high discordance rate is still reported in TPHA and ICA, especially when testing samples with a low titer. Further analysis of data and education of laboratory personnel are needed for the improvement of the EQA program.
Enzyme-Linked Immunosorbent Assay ; False Positive Reactions ; Fluorescent Treponemal Antibody-Absorption Test ; Humans ; Korea ; Quality Control ; Reagent Kits, Diagnostic ; Syphilis/*diagnosis ; Syphilis Serodiagnosis/methods/*standards ; Treponema Immobilization Test

OBJECTIVETo evaluate the quality of the ELISA diagnostic kits for detecting hepatitis E virus (HEV) specific IgG antibody.

METHODSFive diagnostic kits (WT, GD, HM, GeneLabs and KH) for anti-HEV IgG were assayed by detecting HEV IgG in the HEV diagnostic reference sera from 24 positive cases and 30 negative cases. 42 cases clinical suspect patients with hepatitis E from Ditan hospital in Beijing in 1994 to 1995 and 230 normal persons' sera from Chengdu city, Sichuan province in September in 2005.

RESULTSThe conformity rates of positive and negative sera was 95.83% (23) and 96.67% (29) for WT kit, 87.50% (21) and 100% (30) for GD kit, 87.50% (21) and 96.67% (29) for HM kit, 66.67% (16) and 100% (30) for GeneLabs kit, 45.83% (11) and 100% (30) for KH kit for detecting anti-HEV positive and negative reference sera, respectively. The five kits were used to detect anti-HEV IgG among 42 clinical suspect patients with hepatitis E and 230 normal persons' sera, The positive rate was 97.62% (41) and 31.74% (73) by WT, 100% (42) and 17.39% (40) by GD kit, 97.62% (41) and 23.91% (55) by HM kit, 90.48% (38) and 7.83% (18) by GeneLabs kit, 90.48% (38) and 3.48% (8) by KH, respectively.

CONCLUSIONThe positive rates of all the 5 kits were over 90% and had a very high conformity in detecting anti-HEV IgG in clinical patient with hepatitis E, positive rates of 3.48% to 31.74% were found in detecting the normal persons' sera. The research showed insufficiency of the ELISA kits for epidemiological survey of HEV infection in population.

Adolescent ; Adult ; China ; Enzyme-Linked Immunosorbent Assay ; instrumentation ; methods ; Hepatitis Antibodies ; blood ; Hepatitis E ; blood ; diagnosis ; virology ; Humans ; Immunoglobulin G ; blood ; Reagent Kits, Diagnostic ; standards ; Reproducibility of Results ; Sensitivity and Specificity ; Young Adult

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen- free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/immunology/prevention & control/*veterinary/virology ; Chickens ; Enzyme-Linked Immunosorbent Assay/veterinary ; Escherichia coli/genetics ; Immunization/standards/*veterinary ; Infectious bursal disease virus/genetics/*immunology/pathogenicity ; Poultry Diseases/*immunology/prevention&control/virology ; Recombinant Proteins/genetics/*immunology ; Specific Pathogen-Free Organisms ; Vaccines, Attenuated/immunology/pharmacology ; Vaccines, Synthetic/immunology/pharmacology ; Viral Structural Proteins/biosynthesis/genetics/*immunology ; Viral Vaccines/*immunology/pharmacology

BACKGROUNDTo evaluate homemade and imported HbsAg ELISA kits on screening blood donors.

METHODSSamples for evaluation included 120 HbsAg serum plates for the golden criteria and 400 sets of serum from blood donors in Dongguan. The samples underwent blind screening with homemade and imported ELISA kits respectively.

RESULTSThe sensitivity of homemade (Xinchuang) and imported (Diasorin) HbsAg ELISA kit were 85.71% (72/84) and 100% (84/84), respectively. Their specificity was 100% (436/436) and 96.55% (421/436) respectively. The consistency of two ELISA kits was 100%.

CONCLUSIONThe imported ELISA kit had the highest sensitivity, but its specificity was not as good as that of homemade ELISA kit. The two kinds of ELISA kits had good repetition. The combination of the two reagents may ensure the safety of blood transfusion.

Blood Donors ; China ; Enzyme-Linked Immunosorbent Assay ; instrumentation ; methods ; standards ; Hepatitis B ; blood ; diagnosis ; prevention & control ; Hepatitis B Surface Antigens ; blood ; Humans ; Mass Screening ; methods ; standards ; Reagent Kits, Diagnostic ; standards ; Reference Standards ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice).

METHODSBALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants.

RESULTSFour hybridomas, named F1, D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 1x10(-4) to 1x10(-5). Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay.

CONCLUSIONAnti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Biomarkers ; analysis ; Blotting, Western ; Consumer Product Safety ; Enzyme-Linked Immunosorbent Assay ; Female ; Food, Genetically Modified ; standards ; Hybridomas ; Mice ; Mice, Inbred BALB C ; Oryza ; genetics ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; analysis ; immunology ; metabolism ; Plants, Genetically Modified ; metabolism ; Rabbits