Peripheral blood eosinophils from patients with allergic asthma contain increased intracellular eosinophil-derived neurotoxin

BACKGROUND: One mechanism of the eosinophil's contribution to airway inflammation in asthma is through release of cationic granule proteins to cause airway injury. Differences in either the intracellular concentration of granule proteins or the extent of activated degranulation between eosinophils from healthy patients and those with allergy and asthma could, therefore, relate to fundamental differences in this cell's function. OBJECTIVE: To identify phenotypic differences in eosinophil-derived neurotoxin (EDN) content and release in eosinophils from healthy patients, those with allergy, and those with allergy and asthma. METHODS: Peripheral blood eosinophils were isolated by negative anti-CD16 selection. Total intracellular and cytokine-activated release of EDN protein was measured by radioimmunoassay. EDN mRNA was assessed by real-time PCR. RESULTS: Eosinophils from patients with asthma contained significantly more EDN per cell than comparable cells from healthy patients, those with allergy but without asthma, or those with asthma treated with inhaled corticosteroids, but they had concentrations similar to airway eosinophils isolated from bronchoalveolar lavage fluid 48 hours after segmental bronchoprovocation with allergen. Furthermore, this increased granule protein was reflected in more EDN degranulation by IL-5- or GM-CSF-activated eosinophils when calculated as nanograms of protein secreted but not when calculated as a percentage of total EDN release. Levels of EDN mRNA were similar in all subject groups. CONCLUSIONS: These data suggest that peripheral blood eosinophils from subjects with untreated asthma have increased inflammatory capacity, as reflected by greater intracellular concentrations of EDN.     

Detection of transforming growth factor-β in sputum from patients with bronchial asthma by eosinophil survival assay and enzyme-linked immunosorbent assay

Background We have shown that interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are present in sputum from patients experiencing acute asthma attacks, by eosinophil survival assay. The viability of guinea-pig eosinophils was significantly increased in the presence of such sputum extracts after 3 days' culture, and it was inhibited by the addition of anti-IL-5 and anti-GM-CSF antibodies. However, the contribution of IL-5 to the increase in eosinophil viabihty was less than expected from the values of IL-5 measured by enzyme-linked immunosorbent assay (ELISA). Therefore, we speculated that something in sputum inhibited the function of IL-5. Objective Tratnsforming growth factor-β (TGF-β) was the only cytokine we tested that inhibited the prolongation of survival of guinea-pig eosinophils induced by IL-5. The objective of this study is to detect TGF-β in the same sputum. Methods Guinea-pig eosinophils were cultured with or without anti-TGF-β antibody in the presence of sputum extracts, and the eosinophil viability was counted after 3 days. Measurement of TGF-β_l in sputum was performed by ELISA. Results Eosinophil viabilities with and without anti-TGF-β antibody were 79.7 ± 2.9% and 69.0 ± 2.7%, respectively, and the difference between them was statistically significant (P < 0.05, n = 9). The concentration of TGF-β₁ in the sputum was 21.7 ± 3.3 ng/mL (n = 9). Conclusion These observations suggest that TGF-β is present in sputum from patients with bronchial asthma.     

Development of an enzyme-linked immunosorbent assay for octylphenol

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative analysis of octylphenol. A linear carboxylated analogue of 4-nonylphenol was synthesised and characterised as hapten. The hapten was coupled to the carrier protein to produce a specific antibody. An indirect competitive ELISA method was established based on the polyclonal antibody, which exhibited an IC₅₀ value of 51 ng/mL for octylphenol. A monoclonal antibody was produced with an IC₅₀ value of 76 ng/mL for octylphenol. The immunoassay used with the monoclonal antibodies was applied to analyse water samples from Tai Lake in China.     

Development of an enzyme-linked immunosorbent assay for caffeine

A competitive enzyme-linked immunosorbent assay suitable for the measurement of caffeine in plasma and serum has been developed. Sheep immunised with an immunogen prepared by coupling 7-(5-carboxypentyl)1,3-dimethylxanthine to egg albumin produced antibodies with little crossreactivity with the metabolites of caffeine. The enzyme label was prepared by coupling 7-(5-carboxypentyl)-1,3-dimethylxanthine to peroxidase using the mixed anhydride method. The assay, which has a sensitivity of 0.01 mumol/l, permits direct measurement of caffeine in plasma and serum samples. 50 plasma samples measured by ELISA and by an established radioimmunoassay showed a correlation of r = 0.97 (P less than 0.001).     

重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用

目的：制备人自身抗原细胞色素P4502D6（CYP2D6）257-351位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗LKM-1抗体的敏感性和特异性。方法：以肝脏的cDNA混合文库为模板作PCR,将PCR产物与真核表达载体pEGH共同转化酿酒酵母Y258,碱裂解法进行质粒制备,PCR扩增鉴定。表达载体构建成功后,在半乳糖的诱导下表达产生重组融合蛋白,经GST亲和层析法进行纯化后,免疫印迹法鉴定抗原性,ELISA检测抗LKM-1抗体阳性血清及部分其他结缔组织病患者血清中的抗LKM-1抗体。结果：重组融合蛋白在宿主菌中获得表达,免疫印迹法鉴定表明其能与标准抗LKM-1抗体阳性血清反应,而与正常血清、其他抗血清无反应。在26份抗LKM-1抗体阳性血清中,6份抗HCV抗体阳性血清用重组多肽ELISA检测有5份呈阳性,其余20份血清用重组多肽ELISA检测均呈阳性；20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论：重组的257-351位氨基酸片段是CYP2D6抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗LKM-1抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。     

Development of a competitive enzyme-linked immunosorbent assay for detecting cytomegalovirus antibody

A competitive enzyme-linked immunosorbent assay (ELISA) for detecting cytomegalovirus (CMV) antibody was developed. The competitive ELISA was five times more sensitive than the complement fixation test (CFT) and twice as sensitive as indirect ELISA. Testing of paired sera from cardiac transplant patients taken before and after transplantation showed good correlation between results of competitive and indirect ELISA and CFT. The competitive ELISA was more successful than CFT or indirect ELISA in detecting passively acquired antibody, but detection of CMV antibody by competitive ELISA immediately after primary CMV infection was unreliable, possibly because of the high affinity of the monoclonal antibody chosen for the horseradish peroxidase conjugate. However, competitive ELISA may well prove to be more suitable than indirect ELISA for detecting CMV antibody in blood donations.     

Development of colorimetric enzyme-linked immunosorbent assay for human chorionic gonadotropin

The present study demonstrated the development of a solid phase competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of human chorionic gonadotropin (hCG) in serum and urine. Polyclonal antisera raised against the beta- subunit of peak-I hCG was used in the assay. The Peak-IA hCG-penicillinase was used as tracer. The performance of this antiserum and tracer was compared against hCG-beta antisera of NIH, USA and penicillinase conjugated to hCG-beta obtained from NIH, respectively. Almost parallel standard curves were obtained in both cases, suggesting that these antisera and enzyme label have much potential for developing ELISA system. To the anti-rabbit gamma globulin (ARGG) coated polystyrene tubes, standard or serum or urine samples (50 microL), 100 microL of hCG-beta antiserum, 100 microL of peak-I(A) hCG-penicillinase conjugate and 350 microL of assay buffer were incubated at 37 degrees C for 2 hours. Bound enzyme activity was measured using Penicilline V as substrate. In this new strategy, locally available polystyrene tubes were ground from inside and coated with ARGG. The sensitivity of the assay was 17 mIU/mL in urine and 18 mIU/mL in serum. The intra-assay and inter-assay coefficients of variation (CVs) appeared to be within acceptable limits of 10%. The serum and urinary hCG values, obtained by this method, correlated well with those obtained by radioimmunoassay (RIA) r = 0.98 (n = 100 for serum samples; n = 250 for urinary samples).     

Development of an enzyme-linked immunosorbent assay for the pyrethroid fenpropathrin

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of fenpropathrin was developed. Two haptens, FPa (α-carboxy-3-phenoxyphenyl-2,2,3,3-tetramethyl-cyclopropane-carboxylate) and FPb (α-(N-butyrical)-3-phenoxybenzyl-2,2,3,3-tetramethyl-cyclopropane-carboxylate), were synthesised and conjugated with ovalbumin (OVA) by the mixed anhydride method as coating antigens (FPa–OVA and FPb–OVA), and the hapten FPb was conjugated to bovine serum albumin (BSA) by the carbodimide method to produce an immunogen (FPb–BSA). Polyclonal antibody against fenpropathrin was raised for screening the more sensitive coating antigen. Under optimised assay conditions, the 50% inhibitory concentration (IC₅₀) was 0.34±0.090 mg/L and the limit of detection (LOD) was 0.0093±0.00065 mg/L. The cross-reactivities with other pyrethroids, such as deltamethrin, cypermethrin, fenvalerate and cyhalothrin, were all lower than 0.1%. Water samples spiked with different concentrations of fenpropathrin (0.01–1.0 mg/L) were analysed according to this method. The ic-ELISA developed could successfully be applied to residue analysis of fenpropathrin in aquatic.     

Development of an enzyme-linked immunosorbent assay for fecal bile acid

It has been suggested that the bile acids in the feces act as a promoter of colon cancer. Among the bile acids, deoxycholic acid (DCA), which is one kind of the secondary bile acid, is said to have strong influence. DCA/cholic acid (CA) ratio in feces is also said to have a diagnostic significance in colon cancer. With this in mind, we created a CA and DCA's monoclonal antibody (MoAb) to measure them through the enzyme linked immunosorbent assay (ELISA) method. Using these MoAb, we were able to measure CA and DCA concentrations with low cross-reaction to other bile acids compared with the method with polyclonal antibody (PoAb). We measured CA and DCA concentrations and calculated the DCA/CA ratios in healthy subjects and patients with colon cancer. All subjects had been screened for colon cancer. We then compared the healthy subjects, the cancer patients before surgery and the same cancer patients after surgery. Cancer patients after surgery had significantly low DCA/CA ratios compared to before surgery, whereas there was no significant difference between healthy subjects and the pre-operative colon cancer patients.     

Development of an enzyme-linked immunosorbent assay for caprine arthritis-encephalitis virus.

Because relatively few caprine arthritis-encephalitis virus (CAEV)-infected animals exhibit clinical signs of illness, efforts to control and eradicate this virus will depend heavily on a sensitive diagnostic test that can be easily carried out. The currently utilized tests are of limited usefulness because of relatively low sensitivity or because of incomplete cross-reactivity of goat sera with heterologous test antigens. An enzyme-linked immunosorbent assay (ELISA) with purified CAEV antigen and biotin-avidin amplification steps was therefore developed and compared with a radioimmunoassay (RIA) against CAEV p28. Of over 500 sera tested, there was 99% concordance between the two tests. On the other hand, 23 of 24 sera obtained from animals with clinical signs of disease that were negative by agar gel immunodiffusion test (with ovine progressive pneumonia virus antigen) were positive by ELISA and RIA. These results suggest that an ELISA with CAEV antigen is superior to the agar gel immunodiffusion test and is easier and faster than an RIA, and therefore may be the method of choice for diagnosing CAEV infection.     

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for mitozantrone

A mouse monoclonal antibody (NO-1) with specificity for the anti-cancer drug mitozantrone (MZ) (Novantrone) was produced by immunization of a BALB/c mouse with mitozantrone-keyhole limpet haemocyanin (MZ-KLH) conjugate. When used in an indirect competitive enzyme-linked immunosorbent assay (ELISA), NO-1 permitted the accurate and reproducible detection of between 0.25-50 ng/ml of MZ in pooled human serum, the standard curve obtained within this range being virtually linear. The assay demonstrated good reproducibility with intra-assay coefficients of variation (CV) of between 1.41% and 7.02% and an inter-assay CV of 3.45%. Regression analysis of levels of MZ detected by ELISA vs. the actual amounts added to pooled human serum gave a very good correlation coefficient of r = 0.995. NO-1 showed no cross-reactivity with either bisantrene or daunorubicin. A simple pharmacokinetic study was undertaken in rabbits given MZ intravenously at a dose of 0.5 mg/kg of body weight. Levels of MZ in rabbit serum measured with the assay ranged between 82 and 170 ng/ml for rabbits 1 and 2, respectively at 15 min falling to 1.25 ng/ml by 48 h for rabbit 1 and falling to undetectable levels by 120 h for rabbit 2.     

An enzyme-linked immunosorbent assay for thyroxine in dried blood spotted on filter paper

We have developed a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for thyroxine (T4) in dried blood samples spotted on filter paper. The assay is carried out on microtiter plates without extraction or centrifugation steps. The detection limit of the assay is 5 pg/disc/well, equivalent to 1.25 micrograms/1 of whole blood or 2.5 micrograms/1 of serum. Intra- and inter-assay coefficients of variation for various T4 concentrations are 2.4-9.0% and 5.9-17.5% respectively. Correlation between the proposed ELISA method and the RIA is good (r = 0.900, n = 62, y(RIA) = 0.99x(ELISA) + 9.90). The ELISA method is useful for mass-screening of neonatal congenital hypothyroidism using dried blood samples on filter paper, is very simple and one person can assay more than 300 samples per day.     

Development of a blocking enzyme-linked immunosorbent assay for the detection of avian polyomavirus-specific antibodies

Avian polyomavirus, described originally as budgerigar fledgling disease virus, has been associated with devastating contagious disease outbreaks in budgerigar aviaries. At present, this virus affects a wide range of psittacine and non-psittacine birds worldwide, and the serum neutralisation test is used for the serodiagnosis of avian polyomavirus infections. A blocking enzyme-linked immunosorbent assay was developed for the screening of large numbers of sera collected from various avian species. The assay employs a monoclonal antibody directed against the major structural protein VP1 as a blocking antibody in a sandwich blocking procedure. Either purified avian polyomavirus particles or avian polyomavirus VP1 expressed in recombinant baculovirus-infected Sf9 cells were used as antigen. The specificity of the blocking enzyme-linked immunosorbent assay was evaluated by testing sera directed against mammalian polyomaviruses. Using sera obtained from chicken infected experimentally with avian polyomavirus and a collection of psittacine field-origin sera, a good correlation was observed between the results of the blocking enzyme-linked immunosorbent assay and the serum neutralisation test. However, the blocking enzyme-linked immunosorbent assay is more rapid and more economic. Both, avian polyomavirus particles and VP1 produced by recombinant DNA technology proved to be suitable antigens.     

斑蝥素间接竞争性ELISA检测方法的建立

目的通过获得的斑蝥抗血清,建立快速、简便、有效的斑蝥素间接竞争ELISA检测方法。方法以合成的斑蝥素完全抗原免疫家兔,获得高效价的抗血清,通过优化条件,建立斑蝥素间接竞争ELISA检测方法的标准曲线,并设定浓度梯度,测定检测灵敏度、检测稳定性和可靠性。结果建立的标准曲线方程为y=-13.421x+102.45,R2=0.988,检测范围为:0.1～24μg/ml。其最低检测下限为0.01μg/ml,批内和批间变异系数分别为4.27%和4.56%。结论本方法具有良好的灵敏性和可重复性,可为进一步开发斑蝥素检测试剂盒提供基础。     

Development of sandwich enzyme-linked immunosorbent assay for determination of tetanus toxoid concentration

According to the recommendation of the World Health Organization (WHO), the use of an in vivo test for measuring of the potency of tetanus toxoid vaccine (TTdV) is still unavoidable, but the establishment of a convenient in vitro test would significantly improve the work in this field. A sandwich enzyme-linked immunosorbent assay (sELISA) was developed for a rapid and sensitive quantification of tetanus toxoid (TTd). We produced four monoclonal antibodies (MAbs) designated 41, 51, 62, and 71 that reacted with TTd and recognized different antigenic determinants on TTd. We also used two of these antibodies for developing a sELISA, with MoAb 71 as an immobilized and MoAb 51 as a capture antibody. The measurement range of this assay was from 31-1000 ng/mL and the minimum detection limit for TTd was 31 ng/mL. This high sensitivity of this sELISA and its good reproducibility suggest that the developed method could be reliably used to estimate the concentration of TTd, which could be easily extrapolated to the estimation of vaccine potency.     

Development of an enzyme-linked immunosorbent assay for the detection of lolines in pastures

Polyclonal antibodies were produced in sheep for the development of a competitive enzyme-linked immunoassay for use in quantifying loline alkaloids in pasture samples. Lolines are aminopyrrolizidine secondary metabolites produced by fungal endophytes present in tall fescue and meadow fescue grasses, and confer increased resistance to a range of grass pests. Immunizing and plate coating antigens were prepared with derivatized loline dihydrochloride. Cross-reactivity studies indicated that the assay developed has broad specificity and antibody binding to loline analogues is affected by structural changes in the side chain of the loline molecule. Results obtained using the optimized immunoassay were compared with those determined by gas chromatography. The assay provides a sensitive and rapid analytical method that detects the loline analogues of interest and has been applied in pasture breeding programmes. The assay limit of quantitation for lolines in pasture is 3?µg/g in dried herbage.     

Dot enzyme-linked immunosorbent assay for more reliable staging of patients with Human African trypanosomiasis

Human African trypanosomiasis (HAT) or sleeping sickness is a disease characterized by a hemolymphatic stage 1 followed by a meningoencephalitic stage 2 which is fatal without specific treatment. Furthermore, due to the toxicity of drugs used to treat stage 2 (mainly melarsoprol) accurate staging is required. Actual criteria employed during field surveys are not sensitive enough for precise staging. Antineurofilament (anti-NF) and antigalactocerebrosides (anti-GalC) antibodies have been identified in cerebrospinal fluid (CSF) as potential markers of central nervous system (CNS) involvement. We describe a dot enzyme-linked immunosorbent assay (dot-ELISA) to detect anti-GalC and anti-NF antibodies and its value in staging. NF- and GalC-dotted nitrocellulose strips were first developed in our laboratory. They were then evaluated in Angola and Central African Republic on 140 CSF samples. Compared to our staging criteria (i.e., CSF cell count > or = 20 cells/microl, CSF immunoglobulin M concentration > or = 100 mg/liter, and/or the presence of trypanosomes in the CSF), combined detection of both CSF anti-NF and CSF anti-GalC by dot-ELISA showed 83.2% sensitivity and 100.0% specificity. Dot-ELISA could be a useful test to diagnose CNS involvement in HAT in the less-equipped laboratories or in the field situation and to improve patient treatment.     

Accuracy of a commercial enzyme-linked immunosorbent assay for CagA in patients from Brazil with and without gastric carcinoma

We validated a commercial enzyme-linked immunosorbent assay for the detection of anti-CagA antibodies in Brazilian patients with Helicobacter pylori infection. The test presented high sensitivity (97.4%) and specificity (88.9%) when employed in patients without gastric carcinoma. However, in gastric carcinoma patients, the test was neither sensitive nor specific enough to detect cagA-positive H. pylori infection.     

A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative

An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.     

Efficacy of an enzyme-linked immunosorbent assay for detection of urinary tract immunoglobulins for diagnosis of urinary tract infections.

Results of the Uristat test (Shield Diagnostics Ltd.), a novel enzyme-linked immunosorbent assay (ELISA) for detection of urine antibodies to seven common bacterial pathogens, were compared with results of urine culture, urinalysis, and clinical history to determine the usefulness of Uristat in the diagnosis of urinary tract infections (UTIs). Midstream, catheterized, and indwelling catheter urine specimens sent to the laboratory for culture were included in the study. Quantitative cultures were performed on both 5% sheep blood agar and eosin-methylene blue agar. Uristat ELISAs were performed according to the manufacturer's instructions. By using a Bacillus subtilis bioassay technique, antibacterial activity was detected in the urine of 236 (22.2%) of 1,061 patients. Probable, possible, or asymptomatic UTIs were diagnosed for 258 (24.3%) of the 1,061 patients. Of those infections, 219 (84.9%) were caused by bacterial species whose antibodies were detectable by Uristat. Uristat's sensitivity and specificity were 76.7 and 56.0%, respectively. Uristat's predictive values of positive and negative results were 31.2 and 90.2%, respectively. Further development of the Uristat test is necessary before it can be of assistance in the diagnosis of UTIs.