Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).

Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk

Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay （CLIEA） was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer.

Electrochemical Determination of Cortisol by Capillary Electrophoretic Enzyme Immunoassay

An electrochemical method for detection of cortisol based on capillary electrophoretic enzyme immunoassay has been developed. A limit of detection of 1.7?0-9 mol/L was obtained.

Determination of Thyroxine with Capillary Electrophoretic Enzyme Immunoassay

A Capillary electrophortic enzyme linked immunoassay with electrochemical detection (CE-EIA-ED) has been developed. The method can be used to determine thyroxine with a limit of 3.8×10-9 mol/L.

Study on Shigella Detection by ATP Bioluminescence Magnetic Enzyme Immunoassay

[Objective] The research aimed to establish a rapid detection method for Shigella. [Method] Combining immunomagnetic separation technology with ATP bioluminescence technology, a new kind of fast and accurate ATP bioluminescence magnetic enzyme immunoassay technique for Shigella was established. [Result] Using ATP bioluminescence magnetic enzyme immunoassay technique to detect standard solution for Shigella （ATCC 25931 ）, result showed that correlation coefficient between relative light intensity detected by instrument and bacteria concentration detec- ted by culture counting method was 0.981 1. Moreover, relation curve between relative light intensity and Shigella concentration was drawn. [ Conclusion] The method had a high detection speed and accuracy, and could be used for the rapid detection of pathogen in food and environment.

A New Voltammetric Enzyme Immunoassay System for the Detection of Alkaline Phosphatase

A new voltammetric enzyme immunoassay system was investigated based on p-nitrophenyl phosphate (PNPP) as the substrate for alkaline phosphatase (ALP). PNPP is enzymatically hydrolyzed and the product p-nitrophenol (PNP) is detected by differential pulse voltammetry (DPV), which can be oxidized at +1.02 V (vs. Ag/AgCl) on bare glass carbon electrode (GCE). The conditions for enzymatic reaction and electrochemical detection were studied. According to this method, ALP can be detected with a detection limit of 2.8102 mU/L and a linear range of 4.0102 ~ 1.0106 mU/L.

HOMOGENEOUS ENZYME IMMUNOASSAY OF SERUM AFP BY FLUORIMETRIC KINETIC METHOD

The activity of the Ab-bound enzyme(HRP)changed after immunochemical reaction,and can be indicated by a sensitive fluorimetric kinetic method.The finding set the stage for developing sensitive homogeneous immunoassay.AFP was measured as an example.

Electrochemical Enzyme Immunoassay of Tumor Marker CA15-3 with Capillary Electrophoresis

Tumor marker CA15-3 was determined by using capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED). The method can be used to detect CA15-3 with a limit of 0.024 U/mL.

DETECTION OF HELICOBACTER PYLORI STOOL ANTIGEN BY NON-INVASIVE ENZYME IMMUNOASSAY

Objective.To evaluate the clinical utility of a new non-invasive enzyme immunoassay(EIA)for the diagnosis of Helicobacter pylori(H.pylori)infection.Methods.Stool specimens of63patients were collected and tested by using a commercial kit for detecting Helicobacter pylori stool antigen(HpSA),of which61patients also underwent 13 C-Urea breath test( 13 C-UBT).The tissue samples of31patients were obtained endoscopically and were examined with histologic technique(Warthin-Starry silver stain).Regarded 13 C-UBT as a golden standard,HpSA test and histologic techniques were evaluated.Using this method,we also investigated the positive rate of H.pylori infection in children in Beijing.Results.The sensitivity and specificity of HpSA test were94.7%and95.1%respectively;the posi-tive and negative predictive values were97.3%and91.7%respectively;and the accuracy was95.1%.The results showed the prevalence of H.pylori infection was26.0%in children(3～18years)of district of Xicheng in Beijing.After treatment ,HpSA seems to disappear rapidly(3～5days)from the feces.Conclusion.The detection of HpSA in stool samples by HpSA test is a rapid noninvasive test for detecting H.pylori infection,and has both high sensitivity and high specificity.It is suitable for screening and diagnosis of H.pylori infection,monitoring the treatment efficacy in routine in all hospitals.

Determination of Progesterone Receptor by Chemiluminescent Enzyme Immunoassay

A new method of chemiluminescent enzyme immunoassay (CLEIA) was developed and the standard curve and regression equation for determination of progesterone receptor (PR) made. The luminosity of tissue samples was tested and PR level was calculated by the regression equation. Correlation analysis revealed that there was a linear relationship between different concentrations of the standard PR samples and the corresponding values of luminosity: Y=3748+463.77X, γ=0 9958. The values of the luminosity in 38 cases of tumor tissues were determined with the highest being 267.32 fmol/mg, the lowest 3.69 fmol/mg and the mean 78.53 fmol/mg. The new method of CLEIA was a stable, creditable,specific and sensitive assay for determination of PR.

A NOVEL POLYMER ENZYME LINKED IMMUNOASSAY METHOD AND ITS APPLICATIONS

During the course of study, we found that both poly (N-isopropylacrylamide ) (PNIP) and PNIP-Ab (enzyme-labelled antibody)could be adhere tightly to a cellulose acetale-nitrate membrane, and that the retention of PNIP-Ab on the membrane increased over 30-fold when compared with the unconjugated Ab.Thus we used this characteristic to develop a novel immunoassay method-polymer enzyme linked immunoassay method: homogeneous antigen-antibody reaction and heterogeneous separation process. When applied for detection of human serum HBsAg, this immunoassay system can detect as little as 1 ng/ml of human serum HBsAg.

Fast Diagnosis of Gonorrhea With Enhanced Luminescence Enzyme Immunoassay

Objective:To evaluate the value of enhanced luminescence enzyme immunoassay in the diagnosis of Neisseria gonorrhea(NG) infection. Methods: Anti-catalase antibody for Neisseria gonorrheae combined with enhanced luminescence enzyme immunoassay were used to test for N. gonorrhea. Results: A minimum of 1×10^4/CFU of GC in genital tract secretions or urine could be detected with the technique of luminescence enzyme immunoassay. Conclusion : The enhanced luminescence enzyme immunoassay has the advantage of high sensitivity and specificity for diagnosing NG from genitourinary tract secretion and urine.

Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients

We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.

Current knowledge on the laboratory diagnosis of Clostridium difficile infection

Clostridium difficile (C. difficile) is a spore-forming, toxin-producing, gram-positive anaerobic bacterium that is the principal etiologic agent of antibiotic-associated diarrhea. Infection with C. difficile (CDI) is characterized by diarrhea in clinical syndromes that vary from selflimited to mild or severe. Since its initial recognition as the causative agent of pseudomembranous colitis, C. difficile has spread around the world. CDI is one of the most common healthcare-associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Due to extensive antibiotic usage, the number of CDIs has increased. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with the disease, mainly with regards to antibiotic management. The diagnosis of CDI is primarily based on the clinical signs and symptoms and is only confirmed by laboratory testing. Despite the high burden of CDI and the increasing interest in the disease, episodes of CDI are often misdiagnosed. The reasons for misdiagnosis are the lack of clinical suspicion or the use of inappropriate tests. The proper diagnosis of CDI reduces transmission, prevents inadequate or unnecessary treatments, and assures best antibiotic treatment. We review the options for the laboratory diagnosis of CDI within the settings of the most accepted guidelines for CDI diagnosis, treatment, and prevention of CDI.

A Putative Protein with No Known Function in Arabidopsis thaliana Harbors a Domain with Adenylyl Cyclase Activity

Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC261-388) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule. From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.

Accuracy of Helicobacter pylori stool antigen for the detection of Helicobacter py/ori infection in dyspeptic patients

AIM: The premier platinum Helicobacter pylon (Hpylori) stool antigen (HpSA) test is an enzyme immunoassay (EIA) that detects an Hpyloriantigen present in human stools. However, at present there is no uniformity about the cut off level required to consider the test as positive or negative. So we need the cut off level for our local population. The aim of this study was to evaluate the HpSA for the detection of H pylori infection in dyspeptic patients and to determine the sensitivity, specificity of the HpSA test in the diagnosis of H pylori infection, as compared to other standardized diagnostic techniques. METHODS: Sixty-three dyspeptic patients were selected from patients who came to the Division of Gastrointestinal Clinic in Cipto Mangunkusumo Hospital, Jakarta, Indonesia. H pylori infection was confirmed in all patients by histology and rapid urease test (CLO test). Positive results for H pylori were based on positive results from both rapid urea test and microscopic detection of H pylori. Stool specimens were analyzed for H pylori antigen using HpSA immunoassay. RESULTS: A total 63 patients consisted of 31 (49.2%) males and 32 (50.8%) females ranging in ages between 16 and 73 years with a mean age of 42.4±15 years. The mean age of men was 43.2±15.7 years and women was 41.6±14.4 years. Endoscopic findings in this study included gastric cancer 1.6%, peptic ulcer 4.8%, duodenal ulcer 7.9%, esophagitis 6.3%, gastritis 77.7%, and gastroduodenitis 4.8%. According to the predefined study criteria, 6 (9.5%) of 63 patients were positive for H pylori. In the diagnosis of infection, the area under the receiver operating characteristic (ROC) curve for the HpSA test was 0.722 (95% CI, 0.518-0.927). Using a cut-off value of 0.274 instead of 0.16 (as recommended by the manufacturer) the sensitivity and the specificity were 66.7% and 78.9% respectively. CONCLUSION: The HpSA stool test, using a cut-off value of 0.274, may be useful for the primary diagnosis of H pylori infection, its specificity is similar to other Standard tests but its sensitivity was lower.

EXPRESSION OF X-GENE TRANSLATION PRODUCT IN HUMAN PRIMARY CHOLANGIOCARCINOMA AND ITS SURROUNDING NONTUMOROUS TISSUES

Intrahepatic cholangiocarcinoma is an uncommon type of primary hepatoma and its mechanism is still unknown. Twenty samples of the cancer and the tissues surrounding it were all taken from the Xi' an area and examined for the presence of X-gene product (HBxAg) by the avldin-biotin-peroxidase complex method. It was found that 17 cases (85%) showed positive reaction, HBxAg was present in 15 cancer samples and 16 non-tumor tissue samples. This result suggests that there is a close relationship between human choiangiocarcinoma and HBV, and the HBxAg might play an important role in the pathogenesis of cholanglocarcinoma.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

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 mol/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.

Investigation of 4-hydroxynonenal-delved Epitopes on Apolipoprotein B in Human Serum

hydroxynonenal(HNE) is one of the important products generated in lipid peroxidation. An enzyme linked immunoassay for HNE derived epitopes on apolipoprotein B(apo B) was established and 263 human sera were measured. The mean expression of HNE epitopes in men(156.5 mg/L, n=157) was higher than that in women(147.6 mg/L, n=106). In the subjects less than 70 years of age the serum levels of HNE epitopes increased with the age. Expression of HNE epitopes on apo B in serum positively related to serum contents of ferritin, total cholesterol, triglycerides, and low density lipoprotein cholesterol(LDL C). In addition, mean level of HNE epitopes on apo B in patients with coronary heart disease (CHD, 183.5 mg/L, n=103) was higher than that of controls(133.3 mg/L, n=160). This difference was statistically significant. A multiple regression analysis furth showed that serum concentration of LDL C and HNE epitopes were positively related to CHD as well as the age, but high density lipoprotein cholesterol and female were negatively related to CHD. Thus, increased expression of HNE epitopes on apo B might be an independent risk factor of CHD. \ \

Sero-Epidemiological Study of Respiratory Syncytial Virus

Background: Respiratory syncytial virus (RSV) is one of the major viruses that cause respiratory infections in all generations, not only in neonates and infants. There is a limited number of reports on serological epidemiology of RSV subgroups A and B. Neutralizing test (NT) antibody reflects protective immunity but bothersome. Sero-epidemiological study should be performed using practical NT method. Methods: Two wild-type viruses subgroups A and B, isolated in 2013, and the Long strain was used as the challenge viruses. NT antibody with 100% inhibition of cytopathic effect (CPE) was examined. A total of 91 serum samples obtained from 0 to 12 years subjects without RSV infection who visited our hospital with some health problems and 121 sera obtained from healthy subjects in different age groups were used. Serological epidemiology of subgroups A and B was investigated in this study using new NT methods. Results: 1) A simple and practical NT method was developed. 2) The NT antibody titer was lowest in <1 year of age (5 × 21.70 ± 2.03 against subgroup A and 5 × 20.85 ± 1.31 against subgroup B) and increased in 3 years of age or older, and high antibody titers were maintained during school age. 3) A slight difference was observed in the NT antibody titers against subgroups A and Bin young children <3 years, but not after 3 years of age, reflecting the repeated infections. 4) Specific IgG antibody against RSV was measured. The IgG EIA values decreased with age. No association was observed between IgG EIA and NT titers. Conclusions: A simple NT assay method was developed in the present study. By the age of 3 years, high NT antibody titers were observed and maintained until 12 years. The IgG (EIA) values decreased with age. No association was observed between IgG (EIA) and NT titers.