Biotin-streptavidin Enzyme-linked Immunosorbent Assay Research Articles

Determination of Peach Gum in Ancient Chinese Paintings by Biotin–Streptavidin-Enzyme-Linked Immunosorbent Assay

ABSTRACT Peach gum was the most common and widely used exudate gum in ancient China. In this study, polyclonal antibodies to peach gum were prepared using the protein of peach gum as the antigen. The polyclonal antibodies were used to identify peach gum with high sensitivity and specificity by biotin–streptavidin-enzyme-linked immunosorbent assay (BA-ELISA). The binders used in the ancient paintings of the Forbidden City and the Dazu Rock Carvings were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the binders of the Dazu Rock Carvings were diverse, including collagen, ovalbumin, casein, peach gum, and lacquer, and that collagen and peach gum coexisted in most samples of the Forbidden City. This study demonstrates ELISA's high efficiency in analyzing binders in ancient paintings.

Development of a highly sensitive biotin–streptavidin enzyme-linked immunosorbent assay for detecting diethyl phthalate based on a specific polyclonal antibody

An indirect competitive biotin–streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established for the determination of diethyl phthalate (DEP) in wine samples. A highly sensitive and specific polyclonal antibody (pAb-DEP) targeting DEP was prepared. Under optimized conditions, good linearity was achieved within a range of 0.021–9.512 μg·L−1. The limit of detection (IC10) was 0.0079 μg·L−1 and the median inhibitory concentration (IC50) was 0.443 μg·L−1. Besides, the BA-ELISA was highly selective, with low cross-reactivity values with DEP analogs (below 5%). Finally, the concentrations of DEP in wine samples ranged from 5.93 μg·L−1 to 59.15 μg·L−1 by BA-ELISA. Satisfactory recoveries (89.19–112.33%) and variation coefficient values (5.81–9.43%) were successfully obtained. The consistency between the results of BA-ELISA and gas chromatography–mass spectrometry (GC–MS) was 97.48%, which further confirmed that the proposed BA-ELISA immunoassay is reliable, rapid, sensitive, and accurate for monitoring DEP in wine samples.

An indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay for the determination of dimethyl phthalate (DMP) in milk and milk products

After the “plasticizer event” in Taiwan, phthalic acid esters (PAEs) have been listed in “Inedible materials possibly added into food illegally” and “Commonly abused food additives.” As one of the PAEs family, DMP has long been a problem of great concern due to its potential impacts on human health. In order to detect DMP with high sensitivity and specificity, a sensitive indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established in this study. A high-titer rabbit polyclonal antibody (pAb-DMP) targeting DMP was obtained, and the procedures of BA-ELISA were optimized for the determination of DMP in milk and milk products. Under optimal conditions, good linearity was achieved within a range of 0.024 to 6.027 μg L−1, with low cross-reactivity values for DMP structural analogues (lower than 10%). The median inhibitory concentration (IC50) was 0.356 μg L−1 and the limit of detection (LOD) was 0.0082 μg L−1. Finally, the concentrations of DMP in milk and milk products ranged from 1.03 μg kg−1 to 7.23 μg kg−1 by BA-ELISA. Satisfactory recoveries (90.26–112.38%) and coefficient of variation (CV) values (5.08–8.46%) were obtained. These results were consistent with those using gas chromatography-mass spectrometry (GC-MS), which further confirmed that the proposed BA-ELISA was accurate, specific, reliable and rapid for routine monitoring trace DMP residues in foodstuff, especially milk and milk products.

Biotin-Streptavidin Enzyme-linked Immunosorbent Assay for the Determination of Dibutyl Phthalate in Beverages and Drinking Water Using a Specific Polyclonal Antibody

A specific polyclonal antibody targeting dibutyl phthalate (DBP) was obtained, and a sensitive indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay (BA-ELISA) was developed for the determination of DBP in beverages and drinking water. Under optimal conditions, good linearity was achieved within a range of 0.02 to 8.97 μg · L−1. The limit of detection (IC10) was 5 ng · L−1, and the median inhibitory concentration (IC50) was 0.36 μg · L−1. The BA-ELISA was highly selective, with lower cross-reactivity values with DBP analogues (lower than 4 %). Finally, the concentrations of DBP in beverages and drinking water by this method ranged from 0.45 to 7.06 μg · L−1. Satisfactory recoveries (89.5 to 109.5 %) and variation coefficient values (6.0 to 8.7 %) were obtained. The consistency between the results obtained from BA-ELISA and gas chromatography-mass spectrometry (GC-MS) was 98.8 %, which further confirmed that this proposed BA-ELISA immunoassay was reliable, rapid, sensitive, and accurate for monitoring DBP in the environmental samples.

A sensitive heterogeneous biotin–streptavidin enzyme-linked immunosorbent assay for the determination of di-(2-ethylhexyl)phthalate (DEHP) in beverages using a specific polyclonal antibody

Di-(2-ethylhexyl)phthalate (DEHP) is one of the long-chain or high-molecular-weight compounds of the phthalic acid diester (PAEs) family, which is the most commonly used plasticizer and additive.

Biotin–streptavidin enzyme-linked immunosorbent assay for detecting Tetrabromobisphenol A in electronic waste

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant. A sensitive and selective indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed for detecting TBBPA. The optimal hapten of TBBPA was 2-(2,6-dibromo-4-(2-(3,5-dibromo-4-hydroxyphenly)propan-2-yl)) acetic acid. Several physiochemical factors that influence assay performance, such as optimal coupling concentration of immunogen and antibody, organic solvent, ionic strength, and pH, were studied and optimized. The limit of detection (IC10) was 0.027 ng/mL and the median inhibitory concentration (IC50) was 0.58 ng/mL. The BA-ELISA was highly selective, with low cross-reactivity with TBBPA analogs. Finally, the assay was used to detect TBBPA in electronic waste samples. The results are consistent with those using liquid chromatography, which proves that the proposed immunoassay is accurate and receptive. This BA-ELISA method is suitable for the rapid and sensitive screening of TBBPA in environmental monitoring.

Clinical research on correlation between CT-1 and cardiac function in patients with diabetic cardiopathy

Objective To study the eorrelation between the plasma cardiotrophin-1(CT-1)level and cardiac function of patients with diabetic cardiopathy.Methods Using the Biotin-StreptAvidin-enzyme-linked immunosorbent assay(BSA-ELISA),the level of plasma CT-1 jn 35 normal controls,40 patients with type 2 diabetes and 60 patients with diabetic cardiopathy was measured.The relation to heart function was observed.Results The difference of plasma CT-1 level in the three groups was significant(all P＜0.05).The level of plasma CT-1 in diabetic cardiopathy group was apparently higher than that of type 2 diabetes group and normal controls(all P＜0.01);The level in type 2 diabetes group was significantly higher than that of normal controls(P＜0.01).The level of plasma CT-1 elevated with the worsening of heart failure(NYHA classification).The level of plasma CT-1 was correlated with EF(r=0.669,P＜0.01);LAD(r=0.528,P＜0.01);PVE(r=0.502,P＜0.01);CK-MB(r=0.312,P＜0.01);TG(r=0.187,P＜0.05);DBP(r=0.158,P＜0.05)o Stepwise regression analysis revealed that EF and LAD were the most significant agents affecting the plasma CT-1.Conclusion The plasma CT-1 level could reflect the state of cardiac function of diabetic cardiopathy patient,it could help to diagnose diabetic cardiopathy earlier. Key words: Diabetic; Cardiopathy; Cardiotrophin-1; Cardiac function

Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay

A modified sandwich enzyme-linked immunosorbent assay using biotin-streptavidin system (BS-ELISA) was developed to determine levels of tumor necrosis factor-alpha (TNF-α) in serum samples of children infected with dengue virus ( n=99) and healthy controls ( n=41). The minimum detectable concentration of TNF-α by the BS-ELISA was 3.3 pg/ml. The mean TNF-α level was highest in those patients with dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF) grade III (37.44±42.0 pg/ml). Lower levels were found in DHF grade I (28.44±42.7 pg/ml), DHF grade II (24.21±25.4 pg/ml) and dengue fever (DF) (14.10±24.0 pg/ml). TNF-α in the sera of DF and DHF patients could be detected on days 2–6 after the onset of fever, the high level occuring on day 5. TNF-α was detected in 41.4% (24.01±35.2 pg/ml) of dengue virus infected patients and 7.3% (4.2±15.6 pg/ml) of control subjects. The sera of patients contained significantly higher levels of TNF-α than the sera of controls, P-value<0.001. DHF patients had significantly higher levels of TNF-α than DF patients ( P-value=0.020) but no difference in the TNF-α levels from sera of DHF grades I–III patients was observed ( P-value=0.295). The results indicate that the BS-ELISA is a very sensitive method for determining TNF-α in serum samples of DF and DHF patients. The TNF-α levels might be associated with dengue virus infection and related to disease severity of DHF.

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.

Immunoglobulin M-capture biotin-streptavidin enzyme-linked immunosorbent assay for detection of antibodies to dengue viruses.

A biotin-streptavidin system was adapted to an IgM-capture ELISA for detection of dengue antibodies in human sera. To develop this assay, high titers of antibodies to flavivirus were purified by ion-exchange chromatography (DEAE-cellulose) and labeled with biotin. Heavy chain-specific goat anti-human IgM was first bound to the wells of a polystyrene microtiter plate, followed by binding of IgM in test specimens, and the use of tetravalent dengue antigens (dengue 1-4), biotin-labeled anti-flavivirus IgG, and streptavidin-peroxidase conjugate. The sensitivity and specificity of the IgM-capture biotin-streptavidin ELISA (IgM-BS-ELISA) in acute sera were 83.3% of patients with dengue infection and 95.3% of nondengue-infected cases, respectively. The positive predictive value was 92.4% and the negative predictive value was 89.2%. The efficiency of test was 90.4%. In convalescent sera, the sensitivity and specificity of IgM-BS-ELISA were 100% and 92.6%, respectively. The predictive values of positive and negative results were 90.3% and 100%, respectively. The efficiency of test was 95.6%. The agreement rate of IgM-BS-ELISA and standard hemagglutination inhibition test was good: kappa (kappa) values were 0.79 for acute sera and 0.91 for convalescent sera. The correlation between two methods was quite good, with correlation coefficients (r) of 0.76 for acute sera and 0.85 for convalescent sera (P < 0.001). The results indicate that the IgM-BS-ELISA is highly sensitive, specific, simple to perform, and rapid.

Prevalence of genital herpes simplex infection and abnormal vaginal cytology in late pregnancy in asymptomatic patients

A study was undertaken to determine the prevalence of genital herpes simplex virus (HSV) infection in 809 asymptomatic, late pregnant women attending the antenatal clinic (ANC) at Ramathibodi Hospital. There was only one case where the cervico-vaginal swab specimen was positive for HSV by cultivation and the biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA), for detection of viral antigens. This gave a prevalence rate of 0.12%. The anti-HSV IgM in cord blood of the infant born to this mother was negative. The Papanicoulaou (Pap) smear was performed in 554 cases, including the above patient, but none showed evidence of HSV infection. The high prevalence of lower genital tract infection in these subjects was noted in both Pap smear (31.05%) and wet preparation of the cervico-vaginal discharge (46.72%). Thus, examination of the cervico-vaginal discharge during late pregnancy should be of benefit to the patients, in revealing asymptomatic infections of the lower genital tract.

Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay.

A sensitive enzyme-linked immunosorbent capture assay with biotin and streptavidin (capture B/SA ELISA) was developed to detect herpes simplex virus (HSV) antigen. Rabbit anti-HSV antibody (immunoglobulin G fraction) was coated on flat-bottom, irradiated, 96-well polystyrene microtiter plates and served to capture HSV antigen. Clinical specimens from patients with genital herpes were added. Biotin-linked rabbit anti-HSV immunoglobulin G was used as the second antibody. The antigen-antibody complex was detected with alkaline phosphatase-conjugated streptavidin, which linked to the biotin. With clinical specimens, the test had a sensitivity of 95.6% and a specificity of 91.4% when compared with the tissue culture method. The presence of HSV antigen in specimens devoid of infectivity was confirmed by blocking the reaction with unlabeled rabbit and human antibody to HSV. The level of antigen detected by the capture B/SA ELISA did not necessarily correlate with the infectivity titer of the specimens. HSV antigens could be detected by the capture B/SA ELISA when the virus infectivity was destroyed at 37 degrees C, by UV irradiation, or by Triton X-100 treatment, but not when hypochlorite treatment was used. Greater sensitivity was obtained when HSV-1- and HSV-2-specific antibody reagents were used simultaneously in each test. The capture B/SA ELISA provides a relatively rapid method (4.5 h) which is quite sensitive and specific when compared with other non-tissue culture, direct assay methods.