Two-site Enzyme-linked Immunosorbent Assay Research Articles

Total protein concentration and stability of Amb a 1 in glycerinated ragweed sublingual immunotherapy stored at room temperature and refrigerated cold temperature.

Few studies have investigated optimal storage conditions or expiration dates for sublingual immunotherapy (SLIT) formulations prepared from glycerinated allergen extracts. The objective of this study was to compare concentrations of short ragweed major allergen (Amb a 1) and total protein in SLIT formulations stored at two different temperatures. It was hypothesised that protein concentrations would show greater decline over time in a formulation stored at room temperature (RT) than in one stored under refrigeration. Two SLIT samples containing equal volumes of 20,000 PNU Amb a 1 extract were prepared and stored at refrigerated cold (CT) (2-8°C) or RT (20-24°C) for 140 days. Changes in total protein and major allergen concentration and composition were measured by Bradford assay, two-site enzyme-linked immunosorbent assay and SDS-PAGE. Presence of Amb a 1 was confirmed with Western immunoblot. Data were analysed using an analysis of covariance, with p < 0.05 considered significant. SDS-PAGE showed compositional changes in a ~26-30 kDa protein band under RT and not CT storage. The Amb a 1 concentration of the RT SLIT sample declined significantly over time, compared to that of the CT SLIT sample (F(1,8) = 47.69, p < 0.0001). There was no significant difference in total protein concentration over time between groups (F(1,8) = 1.79, p = 0.22). These results demonstrate that storage of glycerinated SLIT formulations in refrigerated CT preserved the highest concentration of the specific allergen Amb a 1, suggesting that SLIT formulations containing short ragweed should be stored under refrigeration.

Early monitoring of anti-infliximab antibodies by drug-tolerant assay predicts later immunogenicity and drug survival in rheumatic diseases.

To investigate the appearance of anti-drug antibodies (ADA) against infliximab (IFX) determined by drug-sensitive and drug-tolerant assays and their relationship with drug levels and drug survival. This longitudinal observational study included 45 patients with rheumatoid arthritis (RA) and 61 with spondyloarthritis (SpA). Serum samples were obtained at weeks 2, 6, 12, 24, and 52. Serum IFX levels were measured by a capture enzyme-linked immunosorbent assay (ELISA) and ADA by an in-house drug-sensitive two-site (bridging) enzyme-linked immunosorbent assay (bELISA) and a commercially available drug-tolerant ELISA (IDK, Immundiagnostik, Germany). Anti-drug antibodies were detected earlier by IDK than by bELISA. Once ADA appeared, positivity persisted throughout the study period. Patients who were bELISA ADA+ had higher IDK ADA levels (than bELISA ADA- patients). Circulating IFX levels were detected in all patients except those found to be bELISA ADA+. Serum IFX levels were lower in IDK ADA+ than in IDK ADA-patients.Most patients (64%) discontinued due to inefficacy. The early onset of immunogenicity was related to IFX survival. Both in RA and SpA, the median survival (years) was shorter in patients with earlier development of ADA (IDK+ before or at week 24) than those who became IDK+ later (after week 24) or never developed ADA. A drug-tolerant assay detects ADA during IFX therapy earlier and more frequently than a drug-sensitive assay. The onset of immunogenicity detected by drug-tolerant assays is related to the subsequent detection of ADA by drug-sensitive assays and drug survival.

A-003 Point of Care BNP Values Obtained by the Abbott i-STAT are not Interchangeable With Laboratory Determined Values Using Beckman DXI 800 Analyzer

Abstract Background B-type natriuretic peptide (BNP) is used as a biomarker of heart failure. In our hospital, point of care (POCT) BNP test is performed on whole blood using the i-STAT (Abbott, Abbott Park, IL) while in clinical laboratory, the DXI 800 analyzer is used (Beckman, Brea, CA) for determination of BNP in EDTA plasma. Sometimes clinicians interchangeably use POCT BNP with laboratory BNP test for monitoring heart failure patients, but values are sometimes significantly different causing diagnostic confusion. We compared POCT BNP values in 88 patients with laboratory-based BNP values analyzed between less than one hour to less than 12 h. In addition, 12 specimens were simultaneously analyzed for POCT BNP and lab-based BNP. No additional blood was drawn from patients and medical record was not accessed. The study had exempt status: by the IRB of University of Kansas Medical Center. Methods POCT BNP values were obtained using the i-STAT analyzer following manufacturer’s protocol. It is a two-site enzyme-linked immunosorbent assay (ELISA) with an analytical measurement range of 15–5000 pg/mL. In the laboratory BNP concentrations were obtained using the DXI 800 analyzer. It is also a sandwich immunoassay with the analytical measurement range of 5–5000 pg/mL. Results Overall comparison using lab-based BNP as x-axis and POCT BNP as y-axis produced the following regression equation; y = 1.48 x − 23.45 (n = 88, r = 0.96) indicating that although POCT BNP values correlated well with lab-based BNP values, POCT values showed an average 48% positive bias. When specimens analyzed within 4 h were compared, the regression equation was: y = 1.40 x + 74.12 (n = 20, r = 91) and when specimens analyzed within 1 h were compared, the regression equation was: y = 1.56 + 59.0 (n = 23, r = 0.97). In contrast, when 12 specimens were analyzed simultaneously using POCT and lab-based BNP, using x-axis as the lab-based BNP and y-axis as the POCT BNP, the following regression equation was observed: y = 1.83 x − 102.8 (n = 12, r = 0.99). Conclusion Higher positive bias in specimens analyzed simultaneously indicates true positive bias with POCT BNP compared to lab-based BNP because lab-based BNP in real patients were ordered later after POCT BNP. To our knowledge i-STAT BNP values were never compared with DXI 800 BNP values. We communicated with our clinicians not to use lab-based BNP test and POCT BNP in monitoring patients being treated for heart failure interchangeably. POCT BNP can be used for initial screening to identify potential patients with heart failure.

Relationship between Residual Urine Output and Type of Dialysis with FGF23 Levels.

Several studies investigated the role of fibroblast growth factor 23 (FGF23) in the regulation of renal phosphate excretion in chronic kidney disease (CKD). However, patients with residual urine output (UO) seem to control their serum phosphorus levels better. Our aim was to determine whether FGF23 levels are influenced by dialysis modality and UO. We performed a cross-sectional study in hemodialysis (HD) and peritoneal dialysis (PD) patients. The C-terminal FGF23 (cFGF23) levels were determined in plasma with a two-site enzyme-linked immunosorbent assay. The UO collection referred to an mL/day measurement. All p values were two-sided, and the statistical significance was set at p < 0.05. We enrolled 133 patients (58 HD, 75 PD, UO 70%). The median cFGF23 was significantly higher in HD vs. PD patients (p = 0.0017) and not significantly higher in patients without UO (p = 0.12). We found a negative correlation between cFGF23 and the UO volume (p = 0.0250), but the correlation was not significant when considering the type of dialysis treatment. Phosphorus (ß = 0.21677; p = 0.0007), type of dialysis (ß = −0.68392; p = 0.0003), and creatinine (ß = 0.08130; p = 0.0133) were significant and independent predictors of cFGF23 levels. In conclusion, cFGF23 was significantly higher in HD than in PD patients. We found a significant negative correlation between cFGF23 and the residual UO volume, but the correlation was not significant considering the type of dialysis. Our study reveals that dialysis modality is an independent predictor of FGF23 levels. In particular, PD is associated with lower FGF23 levels than HD.

Effects of mechanical washing and drying on the removal of pet allergens.

Background: In Korea, the number of households with indoor pets is rapidly increasing in parallel with changes in cultural lifestyles. The sensitization rate of pet allergens is also increasing in Korea. Objective: We evaluated the effectiveness of washing machines to remove dog and cat hair and their allergens. In addition, this study aimed to investigate whether only a mechanical dryer without mechanical washing could be used for pet allergen removal. Method: We brushed cats and dogs, and thereafter collected their hair and used a residential vacuum cleaner to obtain dust and other particulate matter from a household. The contents of the vacuum bag were sifted through a 300-μm sieve filter. Some of the contents were placed in phosphate-buffered saline solution with 0.5% Tween 20 to make a liquid extract. Hair, dust, and liquid extract-contaminated fabric samples after mechanical washing or after drying without mechanical washing were analyzed for pet allergens (Fel d l [cat], Can f 1 [dog]) by using a two-site enzyme-linked immunosorbent assay. We assessed the remaining allergens in the contaminated fabrics after mechanical drying and washing. Results: The mean Fel d l and mean Can f 1 removal ratios after mechanical washing with detergent were > 99.99% for the dust, hair, and liquid extract. The removal ratios after mechanical washing without a detergent were lower for both Fel d 1 and Can f 1, for hair, dust, and their respective liquid extracts (p < 0.05). Mechanical drying was just as effective as mechanical washing with detergent for removing Can f 1 but was less effective for Fel d 1 (p < 0.05). Conclusion: Mechanical washing with detergent is important to remove pet allergens from contaminated fabrics. If washing is difficult, then using just a dryer without washing can be an alternative method to remove allergens from contaminated bedding or clothing.

Development of active affibody aggregates induced by a self-assembling peptide for high sensitive detection of alpha-fetoprotein

Alpha-fetoprotein (AFP) is one of the most important biomarkers associated with primary liver cancer and the development of accurate and sensitive methods for detecting the serum level of AFP is of great importance in cancer diagnosis. In this study, three self-assembling peptides (SAPs) tagged AFP specific affibody dimer (ZAFP D2)2 recombinant protein, (ZAFP D2)2-ELK16, (ZAFP D2)2-L6KD and (ZAFP D2)2-R18A were successfully produced and characterized. The (ZAFP D2)2-ELK16 affibody aggregates showed higher and more specific binding affinity to AFP compared to (ZAFP D2)2 and other two SAP-tagged (ZAFP D2)2 and in addition displayed enhanced AFP-detection sensitivity and improved stability. A sensitive two-site enzyme-linked immunosorbent assay for AFP using (ZAFP D2)2-ELK16 as the detection reagent was also established. The AFP detection limit and linear range using (ZAFP D2)2-ELK16 based two-site ELISA were 9.7 ng mL−1 and 20–600 ng mL−1, which were significantly improved compared to those of the (ZAFP D2)2 based two-site ELISA for AFP. To detect AFP in serum samples, the average recovery ranged from 95.02% to 110.41% and the relative standard deviation was in the range of 2.45%-3.97%, which meet the standard clinical testing requirements. This proposed strategy provides a high yield, easy purification and green biosynthesis method to prepare SAP-based affibody aggregates for enhanced detection sensitivity by improving the immunoassay signal amplification activity.

γ' Fibrinogen as a Predictor of Survival in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disorder related to neuroinflammation that is associated with increased risk of thrombosis. We aimed to evaluate γ' fibrinogen plasma level (an in vivo variant of fibrinogen) as a biomarker in ALS, and to test its role as a predictor of disease progression and survival. Sixty-seven consecutive patients with ALS were followed and the results were compared with those from 82 healthy blood donors. Patients were clinically evaluated at the time of blood sampling and on follow-up (every 3 months for the beginning of the follow-up until death) by applying the revised ALS Functional Rating Scale. Human plasma γ' fibrinogen concentration was quantified using a specific two-site sandwich kit enzyme-linked immunosorbent assay. We found, for the first time, a positive association between γ' fibrinogen concentration and survival in ALS patients: patients with higher γ' fibrinogen plasma levels survived longer, and this finding was not influenced by confounders such as age, gender, respiratory impairment, or functionality (ALSFRS-R score). Since increased levels have a positive impact on outcome, this novel biomarker should be further investigated in ALS.

Determination of the Mycelium and Antigens of a Number of Micromycetes in Soil Extracts via Enzyme-Linked Immunosorbent Assay

Sixteen variants of test systems (two-site sandwich and competitive enzyme-linked immunosorbent assay (ELISA)) were studied to determine the concentration of microfungi, some of which are known opportunistic pathogens for humans, in soil extracts. The correlation of the amount of fungal biomass (the length of the growing mycelium) with the concentrations of antigens detected by the two-site sandwich ELISA method and the competitive ELISA method was evaluated. It has been shown that the methods are highly effective in the case of long incubation (more than 5 days) of soil samples and are ineffective for short-term incubation, because there was no correlation between hypha length and antigen concentration during the first day of cultivation. The perspectivity of ELISA methods for the detection of some soil microfungi from genera Aspergillus (Asp. flavus, Asp. fumigatus, Asp. niger), Fusarium (F. solani, F. poae), and others (Alternaria alternata, Phoma lingham, Mucor hiemalis) was shown. The competitive ELISA method was more efficient than two-site sandwich ELISA.

Роль тканевого эритропоэтина и механизмов обратной связи в локальной регуляции эритропоэза

Erythroblastic islets (EI) of rat bone marrow were cultured for 24, 48, or 72 h together with various erythrocytes, simulating the state of erythron hyper- or hyporegeneration in the in vitro system. The concentration of erythropoietin was determined in the control and experimental cultures of EI by two-site enzyme-linked immunosorbent assay. With the addition of reticulocytes/young red blood cells (erythron hyperregeneration model) at a dose of 30 or 60 cells / 1 EI, after 24 h of cultivation, the concentration of erythropoietin in the culture medium increased by 37% compared to control cultures. In cultures with 120/1 EI reticulocytes / young red blood cells, the concentration of erythropoietin decreased by 25% by 48 h. The addition of mature/old red blood cells (an erythron hyporegeneration model) caused inhibition of erythropoietin synthesis in EI culture. By 48 h, the content of this hormone in the cultivation medium significantly decreased in all series of the experiment: in cultures with 30 cells / 1 EI - by 18%, and in cultures with a large erythrocyte load - by 28%. In 72-h cultures containing 30, 60, or 120 red blood cells / 1 EI, the concentration of erythropoietin decreased by 26%, 40%, and 53%, respectively, compared with the control. We found that local regulation of erythropoiesis is directly related to the synthesis of erythropoietin in EI and depends on the number and maturity of cells produced by red bone marrow. The data obtained suggest that it is tissue erythropoietin that is the most important link in the implementation of feedback mechanisms that provide an adequate response of the erythron to an oxygen demand in physiological conditions and support erythropoiesis in pathological conditions accompanied by small production of erythropoietin in the kidneys.

Stability of extracts from pollens of allergenic importance in Korea.

Background/AimsAccurate diagnosis and the effects of allergen-specific immunotherapy for pollinosis are greatly dependent on the potency and stability of the extract. This study aimed to examine factors, such as temperature and storage buffer composition, that affect the stability of allergen extracts from pollens of allergenic importance in Korea.MethodsWe prepared four pollen allergen extracts from ragweed, mugwort, Japanese hop, and sawtooth oak, which are the most important causes of seasonal rhinitis in Korea. Changes of protein and major allergen concentration were measured over 1 year by Bradford assay, two-site enzyme-linked immunosorbent assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reconstitution of the lyophilized allergen extract in various buffers and stored at room temperature (RT, 18°C to 26°C) or refrigerated (4°C).ResultsMore than 90% of the original protein concentration in all four extracts examined was detected over 1 year when 50% glycerol was added and refrigerated, whereas 57.9% to 94.5% remained in the extracts at RT. The addition of 50% glycerol to the storage buffer was found to prevent protein degradation at RT. Amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at RT when reconstituted in a buffer without 50% glycerol. However, 55.6% to 92.8% of Amb a 1 content was detected after 1 year of incubation at 4°C in all buffer conditions except 0.3% phenol.ConclusionsAddition of 50% glycerol as well as refrigeration was found to be important in increasing the shelf-life of allergen extracts from pollens of allergenic importance.

Effects of Obesity on Serum Calcium and Parathyroid Hormone in Zucker Rat Model (P08-032-19)

ObjectivesObesity has been epidemic in the United States and world for more than two decades. Obesity is associated with type 2 diabetes, cardiovascular disease and certain types of cancer. Primary Hyperparathyroidism (pHPT) is also more commonly seen in western societies and is also increasing. Previously, we have reported that obesity caused a significant reduction in serum Vitamin D using obese rat model. The role of obesity on serum Ca and PTH is less known. The main objective of this study was to investigate the effect of obesity on serum calcium (Ca) and parathyroid hormone (PTH) using obese Zucker rat model. Perhaps this could serve as an animal model for pHPT. MethodsFive-week-old female lean and obese Zucker rats were used. After one week of acclimation, lean and obese rats were maintained on AIN-93 G diet for 155 days. Rats were weighed twice per week. At the end of the experiment, rats were sacrificed and serum were collected to measure serum protein, Ca and PTH levels. Serum protein was measured by atomic absorbance spectrophotometry, serum calcium was measured by using with Arsenazo III method and serum PTH by a two-site enzyme-linked immunosorbent assay using a commercially available rat intact PTH ELISA kit. ResultsOur results shows that 1) obese rats gained significantly (P < 0.001) more weight than lean rats; 2) obesity caused a significant increase in serum protein (P < 0.05); 3) obesity increased significantly serum Ca (P < 0.05) and 4) increased a significant decrease PTH levels (P < 0.05). ConclusionsOur results show that obesity increased serum Ca and decreased PTH levels. This is a physiologic negative feedback relationship expected in a normal parathyroid system. If this had been a model for pHPT, the calcium and PTH levels would have been simultaneously elevated. It seems this animal model may not be model for pHPT. Funding SourcesArkansas Bioscience Institute.

Monoclonal Antibodies to Recombinant Fag e 3 Buckwheat Allergen and Development of a Two-site ELISA for Its Quantification.

PurposeBuckwheat is a major cause of anaphylaxis, and Fag e 3 is the key major allergen in buckwheat. However, an immunoassay system for the quantification of Fag e 3 has yet to be developed.MethodsWe developed a 2-site enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (mAbs) produced against recombinant Fag e 3. We applied this ELISA to quantify native Fag e 3 in total buckwheat extract.ResultsFour clones of mAbs were produced, and all recognized vicilin allergens not only from buckwheat, but also from peanut and walnut. However, the ELISA using these antibodies was only able to quantify Fag e 3 in the total extract after addition of 1% sodium dodecyl sulphate (SDS) and heating, which facilitated dissociation of the allergen. The detection limit of the developed 2-site ELISA was 0.8 µg/mL. The measurement of Fag e 3 in the total extract of buckwheat showed that approximately 12% of protein in total buckwheat extract was Fag e 3.ConclusionsWe have developed an ELISA system for the quantification of the group 3 buckwheat allergen, Fag e 3, specifically. This assay will be useful for standardization of buckwheat allergens and monitoring of buckwheat contamination in foods.

Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines

ObjectiveThe present study investigated the effect of the Iodoform-containing root canal filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion. DesignThe effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha (TNFα) from macrophages was examined. Cell vitality was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-linked immunosorbent assay (ELISA). ResultsDirect exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by ∼70% compared to control media (P<0.05). Media dilution from 1/16 to 1/1024 demonstrated a proliferative effect, increasing cell viability by about 60% compared to media without Iodoform-containing root canal filling material. ConclusionsDirect and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation.

A novel validated enzyme-linked immunosorbent assay to quantify soluble hemojuvelin in mouse serum

Hemojuvelin is a critical regulator of hepcidin expression and can be cleaved by proteases to form soluble hemojuvelin. Soluble hemojuvelin has been recently identified in human serum but the presence and quantity of soluble hemojuvelin in mouse serum is unknown. We developed a two-site enzyme-linked immunosorbent assay using a monoclonal anti-hemojuvelin as the capture antibody and a biotinylated polyclonal anti-hemojuvelin as the detection antibody to quantify the levels of soluble hemojuvelin in mouse serum. We validated this assay using cell-conditioned media and serum from Hemojuvelin-null and Bone morphogenetic protein 6-null mice. We also used this validated assay to measure serum soluble hemojuvelin concentrations in mice receiving an acute low iron or high iron treatment. This two-site enzyme-linked immunosorbent assay was highly specific for mouse hemojuvelin, with a lower limit of detection at 13.2-26.8 ng/mL of soluble hemojuvelin in mouse serum. The median serum soluble hemojuvelin concentration in wild-type C57BL/6J mice was 57.9 ± 22 ng/mL, which is 4- to 20-fold less than that reported in healthy human volunteers. After acute low iron diet treatment in these mice, serum soluble hemojuvelin levels were increased and correlated with lowered serum iron levels and decreased hepatic hepcidin expression. An acute high iron diet in wild-type mice or chronically iron-overloaded Bone morphogenetic protein 6-null mice did not significantly lower serum soluble hemojuvelin concentrations. Here we report reliable quantitation of mouse serum soluble hemojuvelin using a novel and validated enzyme-linked immunosorbent assay. This assay may provide a useful tool to elucidate the source and physiological role of serum soluble hemojuvelin in hepcidin regulation and iron metabolism using well-established mouse models of iron-related disorders.

P-0264 Carboxy-Terminal Telopeptide of Type I Collagene and Tartrate-Resistant Acid Posphatase Measurement in Patients with Colorectal Cancer and Bone Metastases

ABSTRACT Introduction Despite significant advances in detection and treatment, colorectal cancer (CRC) still remains one of the most prevalent cancers, and one of the leading causes of mortality due to malignancy. In patients with stage I-II CRC surgical resection usually represents the sole treatment, while in those with metastasized tumor (stage IV) any therapy is rarely successful. Unfortunately, up to 60% of patients with CRC undergoing primary surgery with curative intention die from metastatic disease. Thus, occult tumor cells, not detected preoperatively, likely colonize and remain vital in different tissues of these patients, such as lymph nodes, blood, and bone marrow. Usually, CRC metastasizes to the liver and lungs more frequently than to bone. Isolated bone metastasis (BM) is considered truly rare, but it has been observed that the improved survival for patients with metastatic CRC following expanded treatment options is associate with an increased incidence of BMs. Because metastases in uncommon sites often indicates the terminal phase of CRC, clinicians should be more vigilant about possible BMs. For this purpose, several serum biomarkers have been tested for early detection of BMs, such as carboxy-terminal telopeptide of type I collagen (ICTP), a cross-link product of collagen I degradation, and tartrate-resistant acid phosphatase 5b (TRACP), specifically derived from osteoclasts. Recent studies showed that TRACP activity and ICTP were increased in up to 90% of patients with breast cancer and BM. The aim of this study was to evaluate the usefulness of TRACP, ICTP, and bone alkaline phosphatase (BAP) measurements in patients with CRC and BM. Methods Fourteen patients (9 men, 5 women, mean age 56.1±4.8, range 49-63 years) with CRC and confirmed BMs (cases), and a group of 15 age- and gender matched (10 men, 4 women, age 57.1±4.9 years, p=0.08) patients (controls) without BM (negative F-18 FDG PET/CT) underwent serum TRACP, ICTP, and BAP measurements. Written informed consent was obtained from all the participants. TRACP and BAP were measured by two-site enzyme-linked immunosorbent assay (ELISA), while ICTP was measured by commercially available radioimmunoassay. The sensitivity and specificity of serum TRACP, ICTP, and BAP as a marker for BM were estimated by receiver operator characteristic (ROC) curves. Results The mean levels of TRACP, ICTP, and BAP (cases vs. controls) were: 5.9±1.6 vs. 4.8±1.3 U/L (95% CI 0.11-2.11, p=0.08), 6.9±1.4 vs. 5.9±1.3 U/L (95% CI 0.94-3.04, p=0.0003), and 82.6±18.2 vs. 79.3±16.2 (95% CI 9.81-16.39, p=0.59). ROC analysis established a cutoff value for ICTP of 4.51 U/mL to identify patients with extensive BM with a specificity of 97% and a sensitivity of 92% (area under the curve=0.97; 95% CI=0.95-0.98). A strong relationship was found only between TRACP and ICTP (R=0.95, p Conclusion Serum ICTP is a useful diagnostic marker in the detection of BMs in patients with CRC, more accurate than TRACP and BAP.

Standardization of House Dust Mite Extracts in Korea

PurposeHouse dust mites are the most important cause of respiratory allergy in Korea. Standardization of allergen extracts is essential for improving diagnostics and immunotherapeutics. This study was undertaken to evaluate the allergenicity of standardized house dust mite allergen extracts from Korean house dust mite isolates.MethodsAllergen extracts were prepared from cultured Korean house dust mites (Dermatophagoides farinae and D. pteronyssinus). Allergenic activities of Korean house dust mite extracts were compared to standardized extracts from a company in the United States whose allergen concentrations were expressed as Allergy Units (AUs). Specifically, we compared group 1 and 2 major allergens using two-site enzyme-linked immunosorbent assay (ELISA) kits and an in vivo intradermal test.ResultsMajor allergen concentrations were 17.0 µg/mg (5.0 µg/mg of Der f 1 and 12.0 µg/mg of Der f 2) for a D. farinae extract and 24.0 µg/mg (11.6 µg/mg of Der p 1 and 12.4 µg/mg of Der p 2) for a D. pteronyssinus extract. Using chloramphenicol (CAP) inhibition assays, AUs were 12.5 AU/µg for a D. farinae extract and 12.8 AU/µg for a D. pteronyssinus extract. Allergenic activities were 3- to 4-fold stronger when assessed by intradermal skin tests for in vivo standardization.ConclusionsAllergen extracts were prepared from Korean house dust mites and the allergenicities of the extracts were estimated using AU measurements. House dust mite extracts prepared in this study could be utilized as a reference material, which will be useful for the development of diagnostic and immunotherapeutic reagents in Korea.

Protective effect of epicardial adiponectin on atrial fibrillation following cardiac surgery

Inflammation has been implicated in the pathogenesis of postoperative atrial fibrillation (AF). Adipose tissue secretes both pro-inflammatory cytokines such as interleukin-6 (IL-6) and anti-inflammatory mediators such as adiponectin. We set out to examine the association of adiponectin and IL-6, both circulating and locally produced by the epicardial adipose tissue, with AF development after cardiac surgery. A total of 90 consecutive patients undergoing cardiac surgery were evaluated. Blood samples were collected before induction of anaesthesia. Epicardial fat was obtained upon commencement of cardiopulmonary bypass. IL-6 and adiponectin levels were determined in serum and supernatant of epicardial adipose tissue organ cultures with two-site enzyme-linked immunosorbent assay (ELISA). Heart rhythm was assessed with continuous tele-monitoring for 72 h postoperatively, and with 6-hourly clinical examinations and daily electrocardiograms (ECGs) thereafter. A total of 36 patients developed postoperative AF (40%). Baseline-serum IL-6 and adiponectin were not associated with AF (p = 0.86 and 0.95, respectively). Epicardial adipose tissue IL-6 levels did not correlate with the development of the arrhythmia either (p = 0.37). However, epicardial adiponectin release was lower in patients who developed AF than in those who remained in sinus rhythm (76 (interquartile range (IQR) 35-98) vs 53 ((IQR) 35-69) ng h(-1)g(-1) of tissue cultured, p = 0.066). Following linear regression, the association of epicardial adiponectin with AF almost reached statistical significance (p = 0.066). Multivariate logistic regression analysis of identified risk factors for AF, with the inclusion of epicardial adiponectin as an independent variable, revealed increased age (odds ratio (OR) 1.09, 95% confidence interval (CI) 1.02-1.17, p = 0.013) and epicardial adiponectin levels (OR 0.98, 95% CI 0.97-1.00, p = 0.054) as independent predictors of postoperative AF. Increased epicardial adiponectin is associated with maintenance of sinus rhythm following cardiac surgery. This reinforces the inflammatory hypothesis in the pathogenesis of postoperative AF and may represent a novel therapeutic target for its effective prevention.

Toremifene use does not alter serum inhibin A and B levels during mid-luteal phase in women with premenstrual mastalgia

Aims. To find out if there is any link between the therapeutic effect of toremifene on premenstrual mastalgia and luteal phase serum inhibin A and/or B levels.Methods. Forty-eight patients participating in a randomized cross-over trial on toremifene vs. placebo for premenstrual mastalgia gave three blood samples during the luteal phase of the menstrual cycle: the first at baseline, the second during the third toremifene/placebo cycle, and the third during the third placebo/toremifene cycle, respectively. The blood samples were analyzed for inhibin A and B with respective specific two-site enzyme-linked immunosorbent assays. Toremifene (20 mg/d) and placebo were administered during the luteal phase only.Results. When all the toremifene-treated cycles were compared with all the placebo cycles and with the baseline, the median inhibin A levels were 42, 38, and 40 pg/ml, respectively (baseline versus toremifene, p = 0.638; baseline versus placebo, p = 0.468; and toremifene versus placebo, p = 0.365). The median inhibin B levels were at baseline 19 ng/l, during placebo 20 ng/l, and during toremifene 17 ng/l (baseline versus toremifene, p = 0.983; baseline versus placebo, p = 0.519; and toremifene versus placebo, p = 0.880).Conclusion. A luteal administration of toremifene does not seem to result in any changes in mid-luteal concentrations of inhibin A or B in serum.

Correlation of vascular endothelial growth factor with the severity of thalassemia intermedia

Several lines of evidence indicate that thalassemia intermedia is associated with disturbances in vascular endothelial cell proliferation. In addition, autopsy studies of thalassemia intermedia reported obstructive lesions in the pulmonary artery in 44% of cases, especially splenectomized patients, and a recent in-vivo study reported increased level of vascular endothelial growth factor (VEGF) in thalassemia intermedia. The aim of the present study was to determine whether VEGF levels are correlated with the severity of the disease. Blood samples were collected from 21 patients with thalassemia intermedia and assayed for VEGF by a two-site enzyme-linked immunosorbent assay. A significant correlation was noted between VEGF levels and patient age (P = 0.0022, r = 0.82), presence of splenomegaly (P = 0.004, r = 0.79) and hepatomegaly (P = 0.023, r = 0.82). VEGF levels were also significantly correlated with both left ventricular end-diastolic diameter and left ventricular end-systolic diameter (P = 0.02, r = 0.84 and P = 0.01, r = 0.86, respectively). The study indicates that VEGF levels may be related to the clinical severity of thalassemia intermedia, as expressed by the degree of hepatomegaly and splenectomy and cardiac indexes. Further and larger studies are needed to confirm these observations.

Neurotrophin and neuropeptide expression in mouse brain is regulated by knockout of the norepinephrine transporter

Neurotrophins [e.g. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)] and neuropeptides such as corticotropin-releasing factor (CRF) are reported to contribute to the action of antidepressants (ADs). Norepinephrine transporter (NET) knockout (NETKO) mice represent a model of chronic AD treatment. In the present study, we examined brain-region-specific regulations of NT-3, NGF, BDNF and CRF at the mRNA and protein level in NET wild-type (NETWT) and NETKO mice by means of quantitative real-time PCR (qPCR) and two-site enzyme-linked immunosorbent assays (ELISAs), respectively. NETKO-induced changes were detected for NT-3 in olfactory bulb, brainstem and whole brain at the mRNA and for olfactory bulb at the protein level, for NGF mRNA and protein in olfactory bulb, cerebellum and brainstem and for CRF mRNA and protein in the hippocampus. In contrast, BDNF levels remained unaltered. Our results suggest that NETKO mice represent a useful model to examine gene regulation of downstream targets potentially involved in the action of ADs. We could delineate NT-3, NGF and CRF as being regulated in distinct brain regions by KO of the NET.