An enzyme-linked immunosorbent assay for detection of mitragynine, other closely related Kratom alkaloids and metabolites was developed using polyclonal antibodies. Mitragynine was conjugated to a carrier protein, cationized-bovine serum albumin using Mannich reaction. The synthesized antigen was injected into rabbits to elicit specific polyclonal antibodies against mitragynine. An enzyme conjugate was synthesized for evaluating its performance with the antibodies produced. The assay had an IC50 of 7.3 ng/mL with a limit of detection of 15 ng/mL for mitragynine. Antibody produced have high affinity for mitragynine (100%), other closely related Kratom alkaloids such as paynantheine (54%), speciociliatine (63%), 7α-hydroxy-7H-mitragynine (83%) and cross-reacted with metabolites 9-O-demethyl mitragynine (79%), 16-carboxy mitragynine (103%), 9-O-demethyl mitragynine sulfate (263%), 9-O-demethyl mitragynine glucuronide (60%), 16-carboxy mitragynine glucuronide (60%), 9-O-demethyl-16-carboxy mitragynine sulfate (270%) and 17-O-demethyl-16,17-dihydro mitragynine glucuronide (34%). It showed cross-reactivity less than 0.01% to reserpine, codeine, morphine, caffeine, methadone, amphetamine, and cocaine. Ten-fold dilution urine was used in the assay to reduce the matrix effects. The recovery ranged from 83% to 112% with variation coefficients in intraday and interday less than 8% and 6%, respectively. The ELISA turned out to be a convenient tool to diagnose mitragynine, other closely related Kratom alkaloids and metabolites in human urine samples.
Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality.
Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.
A simple, non-isotopic in-house enzyme-linked immunoabsorbant assay (ELISA) for human growth hormone (GH) was developed. The assay involved using in-house polyclonal anti-GH adsorbed onto 96-well microtitre plates, commercially prepared mouse monoclonal anti-GH, and goat anti-mouse IgG horseradish peroxidase detection system. Results of recovery and parallelism studies ranged from 95%-106% and 98%-101% respectively, of the expected values. The detection limit of the assay was 0.008 mIU/well or the equivalent to 0.4 mIU/L of undiluted serum. Intra- and interassay coefficients of variations were 4.8%-7.9% and 6.5%-8.7% respectively. Serum GH levels measured in this assay correlated well with those measured in established in-house radioimmunoassays (r = 0.985, p < 0.001) and immunoradiometric assay from NETRIA (r = 0.984, p < 0.001).
A supersensitive ELISA was developed for measurement of thyroid-stimulating hormone (TSH) concentrations in serum using in-house rabbit polyclonal antisera and a commercial monoclonal antibody. The assay was optimised and validated by recovery, linearity and cross-reactivity experiments and further compared to other available assays and EQAS samples. Good precision was obtained with a working assay range of 0.2 to 100 mIU/L with < 10% coefficient of variation (CV) for both intra and interassay. The assay is highly sensitive and specific with a minimum detectable limit of 0.07 mIU/L and negligible cross-reactivities against LH, FSH, HCG and other pituitary peptides. Good correlations were obtained when compared to Abbott hTSH EIA (r = 0.993; p < 0.001; n = 85) and NETRIA IRMA (r + 0.995; p < 0.001; n = 76). The normal reference range established was 0.4 to 4.0 mIU/L (n = 76). TSH levels in serum of thyrotoxic patients (n = 83) were significantly lower (0.07 to 0.20 mIU/L, p < 0.0001) and completely distinct from normal values thereby obviating the requirement of a TRH-stimulation test. Stability studies showed that coated wells can be stored at 4 degrees C for at least 2 months. This highly sensitive in-house hTSH ELISA which is cheap, stable and readily available is useful for diagnosis and management of patients with various thyroid disorders.
Enzyme Linked Immunosorbent Assay (ELISA) is a standard assay that has been used widely to validate the presence of analyte in the solution. With the advancement of ELISA, different strategies have shown and became a suitable immunoassay for a wide range of analytes. Herein, we attempted to provide additional evidence with ELISA, to show its suitability for multi-analyte detection. To demonstrate, three clinically relevant targets have been chosen, which include 16kDa protein from Mycobacterium tuberculosis, human blood clotting Factor IXa and a tumour marker Squamous Cell Carcinoma antigen. Indeed, we adapted the routine steps from the conventional ELISA to validate the occurrence of analytes both in homogeneous and heterogeneous solutions. With the homogeneous and heterogeneous solutions, we could attain the sensitivity of 2, 8 and 1nM for the targets 16kDa protein, FIXa and SSC antigen, respectively. Further, the specific multi-analyte validations were evidenced with the similar sensitivities in the presence of human serum. ELISA assay in this study has proven its applicability for the genuine multiple target validation in the heterogeneous solution, can be followed for other target validations.
Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%.
A comparative seroprevalence study on bovine trypanosomiasis and anaplasmosis was conducted. Sera of adult cattle and buffaloes of different breeds from farms from five different states in Malaysia were collected and tested for the presence of Trypanosoma evansi antibodies by CATT and Anaplasma marginale antibodies by c-ELISA. Of the 116 samples, 14.7% tested positive for bovine trypanosomiasis and 77.6% for bovine anaplasmosis.
Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r(2) > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 mug/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.
INTRODUCTION: The aim of this report is to establish an accurate diagnosis of acute dengue virus infection early, in order to provide timely information for the management of patients and early public health control of dengue outbreak.
METHODS: 224 serum samples from patients with a clinical diagnosis of acute dengue infection, which were subsequently confirmed by laboratory tests, were used to evaluate the performance of a commercially-available dengue NS1 antigen-capture ELISA kit.
RESULTS: The dengue NS1 antigen-capture ELISA gave an overall sensitivity rate of 93.3 percent (209/224). The sensitivity rate was significantly higher in acute primary dengue (97.4 percent) than in acute secondary dengue (68.8 percent). In comparison, the virus isolation gave an overall positive isolation rate of 64.7 percent, with a positive rate of 70.8 percent and 28.1 percent, for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 63.4 percent, with a positive rate of 62.5 percent and 68.8 percent, for acute primary dengue and acute secondary dengue, respectively. Of the 224 acute serum samples from patients with laboratory-confirmed acute dengue infection, dengue IgM was detected in 88 specimens, comprising 68 acute primary dengue specimens and 20 acute secondary dengue specimens. NS1 antigen-capture ELISA kit gave an overall sensitivity rate of 88.6 percent in the presence of anti-dengue IgM and 96.3 percent in the absence of anti-dengue IgM.
CONCLUSION: Of the 224 acute serum samples, the sample ages of 166 acute serum samples are known. The positive detection rate of dengue NS1 antigen-capture ELISA, on the whole, was higher than the other three established diagnostic test methods for laboratory diagnosis of acute dengue infection.
Tissue Factor (TF) is a low molecular weight transmembrane glycoprotein that initiates the clotting protease cascade. It is considered to be the principal regulator of the extrinsic coagulation pathway, hemostasis and thrombosis, as well as inflammation and cellular immune response. An in-house two-step direct sandwich ELISA (enzyme-linked immunosorbent assay) for immunological quantification of plasma TF was successfully developed. The assay employed a monoclonal antibody against human TF (1:400 dilution; 1250 ng/ml) and peroxidase-conjugated anti-TF IgG (1:1000 dilution; 2000 ng/ml) as capture and detecting antibodies respectively, whilst tetramethylbenzidine/H2O2 were utilized as substrates. Titration curves of recombinant TF were linear within 10 to 4000 pg/ml, with a detection limit of 36.31 pg/ml. It demonstrated low intra- (2.50 - 9.23 CV%) and inter-assays (5.65 - 13.57 CV%) variability, as well as satisfactory analytical recovery (91.55 - 103.95%) and good parallelism. The assay developed was intended to be applied for measurement of plasma TF levels in patients with thrombotic disorders.
Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.
Detection of microalbuminuria is important in the management of diabetic patients since it is predictive of development of proteinuria and nephropathy. Two sensitive and specific in-house ELISAs for microalbuminuria were established and validated. One of the ELISAs was based on antigen coating while the other employed antibody coating. Recovery and linearity experiments gave acceptable results of 100 +/- 10%, while precision results were <10% for intra-assay and <12% for inter-assay coefficients of variation (CVs). The standard curve ranged from 10-625 ug/l, equivalent to 0.2-12.5 mg/l for urine samples diluted 1:20 fold. When the antibody coated ELISA was compared to antigen coated ELISA, a correlation of r=0.996 was obtained. When compared to commercial kits, the in-house ELISAs gave good correlations of r=0.961 versus the Boehringer Mannheim Micral Test strips and r=0.940 versus Ames Microalb Turbidimetry. The normal microalbumin reference ranges determined for 12h, first morning and random urine samples were 0.7-5.3 mg, 0.1-10.2 mg/l and 0.8-26.1 mg/l respectively. The normal albumin excretion rate (AER) was 1.0-7.3 ug/min while untimed urine samples gave results of 0.1-0.9 and 0.2-1.6 mg/mmol after dividing by creatinine concentrations. The ELISAs were used to detect microalbuminuria in 338 random urine samples from diabetic patients. A high percentage 47.9% was found to be positive for microalbuminuria and 18.0% had macroalbuminuria >25 mg/mmol. Thus screening for microalbuminuria together with creatinine measurements using random urine samples can be used for management of diabetic patients.
The concentration of plasma sialic acid was estimated using the modified chemical method and the more sensitive enzymatic method in 20 subjects with impaired glucose tolerance and 20 control subjects. The mean sialic acid concentration values of the control subjects and subjects with impaired glucose tolerance using the enzymatic method were 1.747 +/- 0.047 and 2.583 +/- 0.070 mmole/l and 1.753 +/- 0.067 and 2.591 +/- 1.02 mmole/l for the chemical method. The intra-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 1.963% and 1.583%, respectively, for the enzymatic assay and 2.728% and 2.431%, respectively, for the chemical assay. The inter-assay coefficient of variation for the control subjects and for the subjects with impaired glucose tolerance were 2.686% and 2.723% for the enzymatic assay, and 3.819% and 3.95% for the chemical assay. Since the values do not differ significantly, the chemical assay is a cost effective method that can be used in large epidemiological studies.
A polymerase chain reaction assay based on the enzyme-linked immunosorbent assay (PCR-ELISA) has been developed to detect Brugia malayi infection in an area of low endemicity in Malaysia. Blood samples from 239 subjects were tested: 192 amicrofilaraemic individuals, 14 microfilaraemic persons and 3 chronic elephantiasis cases from endemic areas and 30 city-dwellers (non-endemic controls). PCR products were examined by ELISA and Southern hybridization. In the PCR-ELISA, digoxigenin-labelled PCR products were hybridized to a biotin-labelled probe. This was followed by incubation in streptavidin-coated microtitre wells and detection using anti-digoxigenin-peroxidase and ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)]. All microfilaraemic samples were positive by PCR-ELISA and Southern hybridization and all samples from non-endemic subjects and chronic elephantiasis patients were negative. The PCR-ELISA detected 12 times as many B. malayi infections as did thick blood film examination. Nineteen of the 194 samples from the endemic area gave positive results by both PCR-ELISA and Southern hybridization, and an additional 5 samples were positive by PCR-ELISA only. The PCR-ELISA was specific and sensitive, detected more infections, and was more reproducible than Southern hybridization.
Rapid diagnosis of dengue infection is essential to patient management and disease control. The development of a rapid (5 min) immunochromatographic test and a 2 h commercial capture enzyme linked immunosorbent assay (ELISA) for anti-dengue IgM and IgG antibodies may lead to more rapid and accurate testing in peripheral health settings and diagnostic laboratories.
A specific monoclonal antibody (AW-3C2) as revealed by ELISA was produced against the adult worm antigens of Parastrongylus cantonensis and used in a sandwich ELISA for the detection of circulating antigens in the sera of parastrongyliasis patients and those with other parasitic diseases. A total of 60 sera was used in this study. Of these, 10 each were from patients with parastrongyliasis, cysticercosis, filariasis, gnathostomiasis, malaria and toxocariasis. The control group consisted of 53 serum samples from normal healthy Thais and Malaysians. The mean +/- optical density (OD) values for the normal Thai and Malaysian groups were 0.126 +/- 0.028 and 0.124 +/- 0.029, respectively. The mean OD values of the parastrongyliasis patient group differed significantly from that of the normal groups as well as those of other parasitic infections. Using a cut-off point of OD +/- 3SD of the control groups as indicating a positive reading, the specificity of the assay with this monoclonal antibody was 100% while the sensitivity was 50%.
The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.