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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. \ \

Magnetic Bead‑Based Sandwich Immunoassay for Viral Pathogen Detection by Employing Gold Nanoparticle as Carrier

Enzyme-linked immunosorbent assay(ELISA)provides a convenient way for the detection of viral pathogens.However,conventional ELISA performed on mirowell plates suffers from poor sensitivity,laborious coating and complicated blocking procedures.Herein,we designed a sensitive colorimetric immunoassay by taking advantages of the enrichment and isolation ability of magnetic beads(MBs)and the high loading capacity of gold nanoparticles(AuNPs)for detecting respiratory syncytial virus(RSV)as a pathogen model.RSV was selectively captured and preconcentrated from samples with antibodies functionalized MBs,followed by binding with antibodies labeled AuNPs,which carrying a large amount of alkaline phosphatase(ALP)molecules for colorimetric signal amplification by catalyzing the dephosphorylation of non-colored pNPP to generate colored product pNP.After optimizing the experimental conditions based on the principle of low nonspecific signal,low cost,and high sensitivity,the analytical sensitivity of the developed immunoassay can be improved to 0.27 pg/mL,which is over sevenfold higher than that of commercially available RSV ELISA kits(2 pg/mL).In addition,the total assay time was less than 2.5 h without any pretreatment,which is much more rapid than other reported assays.Therefore,the proposed immunoassay holds great promise for the fabrication of rapid,sensitive,and economic method for the viral pathogen detection.

High HIV incidence epidemic among men who have sex with men in china: results from a multi-site cross-sectional study

Background:Recent upsurge of new HIV infections among men who have sex with men(MSM)is a major concern in China.Paucity of national-level information regarding the burden and predictors of this progressive epidemic of new infections called for a multi-centric,timely and comprehensive investigation.Methods:Mixed methods were used to recruit MSM from seven cities in China between 2012 and 2013.Recent and established HIV infections were estimated by Western Blot and BED HIV-1 capture enzyme immunoassay.Syphilis and herpes simplex virus-2(HSV-2)were also tested.Results:A total of 4496 eligible MSM were recruited.The majority was aged≤35 years(77.5%),migrants(60.3%),never married(69.8%),and played receptive role in anal sex(70.5%).The HIV prevalence was 9.9%,and 41.9%were recently infected,with sensitivity/specificity adjusted HIV incidence of 8.9(95%CI:7.6-10.2)/100 Person-Years.The prevalence of history HSV-2 and syphilis were 12.5%and 8.5%,respectively.Recent HIV infection was associated with having multiple male partners(aOR=1.4,95%CI 1.1-1.9),recreational drug use(aOR=2.2,95%CI 1.6-3.0),anal bleeding(aOR=2.1,95%CI 1.4-3.0),syphilis infection(aOR=2.8,95%CI 1.9-4.3)and history HSV-2 infection(aOR=2.3,95%CI 1.5-3.3).Conclusion:High rate of recent HIV infection is potentially resulting in progressive deterioration of the overall HIV epidemic among MSM in China.Targeted interventions to address high-risk MSM including those having multiple partners,history of recreational drug use and syphilis or HSV-2 infection seemed to be the need of the hour.

The M Protein of SARS-CoV: Basic Structural and Immunological Properties

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.

Hormonal control of seasonal color change in female spiny-footed lizards:an observational and experimental approach

Breeding coloration of females often signals aspects of their reproductive status,suggesting a link between color and sex steroid hormones.In this study,we examined the relationships between 2 sex steroid hormones(progesterone and p-estradiol)and reproductive coloration in female spinyfooted lizards Acanthodactylus erythrurus.We first explored natural variation in female plasma hormone levels and coloration during their reproductive cycle,p-estradiol was negatively related to brightness and positively related to red saturation,whereas progesterone was not significantly related to coloration.After identifying key relationships,plasma hormone concentrations were manipulated by creating 3 experimental female groups(p-estradiol-treated,progesterone-treated,and control),and the effects on coloration were monitored,p-estradiol-treated females,in which there was a rise in both p-estradiol and progesterone levels,lost their red coloration earlier than females in the other 2 experimental groups,whereas progesterone treatment had no significant effect on female coloration.Our results suggest that high levels of either p-estradiol alone or(3-estradiol together with progesterone trigger the loss of red coloration in female spiny-footed lizards,and that progesterone alone does not affect coloration.We hypothesize that changes in female breeding color might be regulated by(3-estradiol in species in which conspicuous coloration is displayed before ovulation,and by progesterone in species in which this color is displayed during gravidity.