Quality control of radioimmunoassay for TSH, LH, FSH, C-peptide, prolactin based on NIH-computer program

As there has been no data available on the variation of RIA through a year, we assessed within kit and component of between assay variation of TSH, LH, FSH, C-peptide and PRL kit through a year (more than 12 sequential assays). The average of within kit variation was 6.9% (ranged from 4.7 to 9.8%) and that of component of between assay variation was 11.2% (ranged from 8.9 to 14.5%). These values were almost similar to those obtained for the within kit variation and component of between assay variation employing single lot of kit. These results warrant that quality of kits produced by manufacturers was homogeneous throughout the observation period.     

Rapid enzyme-linked immunoassay for detection of Salmonella in food and feed products: performance testing program

The BIOLINE Salmonella ELISA Test for Salmonella spp., which is a rapid, easy, and convenient assay was evaluated for use in detecting Salmonella in foods and feeds. Each food matrix or feed was artificially contaminated with low levels of Salmonella. Twenty different matrixes were studied and 20 different Salmonella strains from a broad variety of serogroups (B, C, D, E, F, G, H, I, M, O, P, and U) were used. The EUSA Test kit detected levels as low as 1 cfu/25 g sample with at least 4 of the 20 matrixes tested. The test kit is applicable to all sample types tested. The BIOLINE Salmonella ELISA Test kit has been granted AOAC-RI performance tested status.     

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.     

Gluten determination by gliadin enzyme-linked immunosorbent assay kit: interlaboratory study

An interlaboratory study with 10 participants was performed to obtain validation and performance data for an enzyme-linked immunosorbent assay (ELISA) kit developed for quantitative gluten determination in foods. The ELISA kit used for this study is based on 2 monoclonal and 1 polyclonal antibody developed by Immunotech, a Beckman Coulter Co. This kit did not show any false positive results or cross-reactivity with oat, rice, maize, and buckwheat. The gliadin standard from the Working Group on Prolamin Analysis and Toxicity was included in the kit as reference material for calibration. All participants obtained a gliadin ELISA kit with Standard Operational Procedure and a form for recording test results. The study included 13 samples labeled as "gluten-free" and 2 samples spiked by wheat flour. Seven samples had gliadin content below the limit of quantitation (LOQ) of the method, and 1 sample exceeded the highest calibration level. Gliadin content in the range from 10 to 157 mg/kg (1st day) and from 11 to 183 mg/kg (2nd day) was found in 7 samples (including 2 spiked samples). Results of these samples were used for further statistical analysis and evaluation. The Cochran, Dixon, and Mandel statistical tests were applied for detection of outliers. The LOQ of the kit was estimated.     

Human proinsulin-C-peptide radioimmunoassay method; stability of the assay kit

We have been studies time sequent stability each on standard human-C-peptide, human-C-peptide antiserum, 125I-tyrosyl human-C-peptide and the assay kit (all reagent) which is necessary in human proinsulin-C-peptide radioimmunoassay(RIA). Also we measured using this assay system, human proinsulin C-peptide in blood after oral administration of glucose to normal subject. Standard human-C-peptide and human-C-peptide antiserum were very stable on storage at 4 degrees C, 125I-tyrosyl human-C-peptide was unstable as compared with the former two, The stability of the assay kit was influenced by the stability of 125I-tyrosyl human-C-peptide, and was stable at 4 degrees C for ten weeks after preparation. Three lots of the assay kit prepared at different period showed almost same stability. We think this assay system using the assay kit is satisfactory in respect of stability. The measured values of human proinsulin-C-peptide in blood, using this assay system, showed insulin secretory reaction after oral administration of glucose.     

Detection of hazelnuts and almonds using commercial ELISA test kits

Three commercial sandwich enzyme-linked immunosorbent assay (ELISA) test kits for the detection of hazelnuts and almonds were evaluated. Limits of detection and dynamic ranges were determined for hazelnuts and almonds spiked into cooked oatmeal, dipping chocolate, and muffins (baked). The limit of detection values varied from 1 to 38 μg/g, depending on the food matrix and ELISA test kit. Percent recoveries based on the standards supplied with the test kits varied from 10% to 170%. It is impossible to ascertain whether the percent recoveries reflect the performance of the ELISAs or differences between the protein content of the nuts used to spike the samples and the test kit standards. Unfortunately, reference materials do not exist that can be used to compare the results from different test kits and standardize the test kit standards. Also, insufficient knowledge regarding the epitope specificity of the antibodies used in the ELISAs further hinders interpretation of the results generated by the different test kits.     

Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to a porcine thermal-stable muscle protein was developed for detection of pork in cooked meat products. The assay specifically detects porcine skeletal muscle, but not cardiac muscle, smooth muscle, blood, and nonmuscle organs. No cross-reactivity was observed with common food proteins. Validity of the assay was evaluated with laboratory formulated and commercial meat samples. The detection limit was determined as 0.5% (w/w) pork in heterologous meat mixtures. Overall, intra- and inter-assay coefficients of variation were 5.8 and 7.9%, respectively. The accuracy in analyzing market samples was 100% as verified by product labeling and confirmed by a commercial polycolonal antibody test kit.     

Determination of rendering plant sterilization conditions using a commercially available ELISA test kit developed for detection of cooked beef.

A commercially available enzyme-linked immunosorbent assay (ELISA) test kit developed for detection of cooked beef in meat samples was used to determine appropriate heat treatment of rendered materials. An improved extraction procedure increased the absolute difference in R-values between 2 rendered materials treated under different conditions (average temperature 129 and 134 degrees C, respectively). To evaluate the influence of the main sterilization parameters on ELISA results, a factorial design approach was used. The parameters investigated were temperature, time, particle size, and meat composition. Lean meat samples containing beef and pork were sterilized under strictly controlled conditions in a laboratory autoclave. The experiments demonstrated that the R-values obtained with the ELISA test kit for beef are strongly influenced by temperature and time, whereas particle size has a minor influence. The proportion of bovine material did not have any impact on R-values. Autoclave-processed lean meat samples were analyzed by using an ELISA test kit for pork, which was validated in a collaborative trial. The ELISA test kit for pork proved to be more sensitive for the investigated parameters, thus verifying and extending previous investigations.     

Evaluation of a commercial immunoassay for the detection of chlorfenapyr in agricultural samples by comparison with gas chromatography and mass spectrometric detection

A commercially available enzyme-linked immunosorbent assay (ELISA) kit with a high affinity monoclonal antibody was applied to residual analysis of insecticide chlorfenapyr in agricultural samples, and drawn a parallel between the ELISA and gas chromatography (GC) with mass spectrometry (MS). For standards prepared in water containing 5% (v/v) methanol, the sensitivity (I50 value), the dynamic range, and the limit of detection of the ELISA kit were 2.3, 1 - 10, and 0.1 ng/g, respectively. The used monoclonal antibody in the ELISA kit had a high selectivity. The ELISA kit was applied to the determination of chlorfenapyr in two kinds of fruits (apple and peach). The examination of the influence of these matrices on the reliability of the assay performance indicated that the ELISA could determine it in these samples near the regulation values in Japan simply by diluting the methanolic extract or by concentrating it, without any clean-up procedures. Recovery and precision of the proposed ELISA method were assessed by fortifying fruit samples with chlorfenapyr ranging from 0.05 to 1.5 microg/g. Mean recoveries were 94.2 and 90.3% for apple and peach, and coefficients of variation were below 16% in most cases. The results obtained from the proposed ELISA method correlate well the reference GC/MS method for both fruit samples (r > 0.98). These considerations make the ELISA kit very useful analytical tool for monitoring and regulatory programs, without the need of complex and expensive instrumentation.     

Fundamental studies on thyroid stimulating hormone immunoradiometric assay kit (TSH RIABEAD II)

We have reported fundamental studies on the TSH immunoradiometric assay, using TSH RIABEAD II kit (Dainabot). The sensitivity of the assay was 0.03 mu IU/ml and its C.V. was 27.2%. Intra- and inter-assay C.V. were less than 5%. Dilution test and recovery test were good. Serum TSH level was 0.3-4.0 mu IU/ml in normal subjects, less than 0.03 mu IU/ml in untreated Graves' disease and subacute thyroiditis. Therefore, it was found that the clear difference exist in serum TSH levels between normal subjects and patients with untreated Graves' disease. There was a well correlation on the serum TSH levels between this method and TSH radioimmunoassay kit (Amerlex TSH, r = 0.983). Especially, the measurement of serum TSH levels, using immunoradiometric assay kit, was useful for the diagnosis of patients with Graves' disease.     

Biosensor analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.     

Basic evaluation of highly sensitive thyroid stimulating hormone immunoradiometric assay kits

Usefulness of three kinds of TSH kits by immunoradiometric assay (IRMA) was evaluated. They were able to measure low levels (less than 0.1 microIU/ml) in serum thyroid stimulating hormone (TSH) with incubation of short time (4 hours). In particular, RIABEAD II kit had a highly specific affinity for TSH and the normal range (+/- 2 S.D.) using it showed from 0.20 to 3.50 microIU/ml in 150 normal subjects. In patients with hyperthyroidism and in patients with hypothyroidism, the values of TSH were lower and higher than those of normal subjects, respectively. Another kits showed similar results. These results indicate that these TSH-IRMA kits are useful to evaluate serum TSH levels exactly.     

Evaluation of radioimmunoassay of the blood tobramycin level for the purpose of setting the dosage based on the clinical pharmacokinetic theory and a comparison of radioimmunoassay with other assay methods

With the purpose to use for therapeutic drug monitoring (TDM), blood concentrations of tobramycin (TOB) in each patient were measured by radioimmunoassay (RIA). A RIA kit of TOB (Clinical assay-Japan Travenol) was evaluated for precision and recovery, in that partial improvement of the method was made, in order to measure low level of TOB. The RIA was compared with high-performance-liquid-chromatography (HPLC), bioassay (BA) and 2 kinds of enzyme immunoassay (EIA) (EMIT and SLFIA). The RIA of TOB revealed high precision (1.8-2.4% in C.V.) and high reproducibility (5.0-6.9% in C.V.). It was found that this RIA kit can be used for measuring low level of serum TOB concentrations by a modification of the method. The total range of measurable blood level is from 0.1 to 16.0 micrograms/ml. The nearly one to one correspondence was observed between RIA and other 4 methods, when 154 samples obtained from 18 cases were measured. A representative case of TDM for TOB was demonstrated, in which predicted concentrations agreed fairly well with actual measured values at steady state. It was concluded that the RIA kit is useful for clinical application of TDM for the adequate dosage regimen of TOB. Modification of the method for rapid assay of a small number of samples will increase the clinical usefulness.     

Evaluation and comparison of high-sensitivity immunoradiometric assay kits for thyroid stimulating hormone

Fundamental and clinical characteristics of 3 kinds of high-sensitivity immunoradiometric assay (IRMA) kits for thyroid stimulating hormone (TSH). i.e., RIA BEADS II (kit A), TSH kit Daiichi II (kit B) and Ab tube TSH 'Eiken' (kit C) and one conventional radioimmunoassay (RIA) kit, i.e., TSH kit Daiichi (kit D), were studied. In the recovery test and the reproducibility test, there was no significant difference between the 4 kits. The sensitivities of kits A, B and C were much higher than that of kit D, and those IRMA kits were sensitive enough to distinguish hyperthyroidism from normal samples. For low concentrations of TSH (less than 5 microU/ml), the data from kits D, B, C and A tended to show higher values in that order. The correlation between the data measured by kits B and D, and the tendency of kit A toward lower values agreed well with other reports.     

Standardized method comparison for ACS:180 plus and Immulite sensitive PSA (sPSA) measurement methods

The aim of this study was to compare two immunoassay measurement methods, the ACS:180 Plus (Chiron Diagnostics) kit and the Immulite sPSA (DPC) kit. Method comparison analysis was performed according to EP9-A; approved guideline of NCCLS 51. Serum samples having a wide range of total prostate-specific antigen (PSA) concentrations were evaluated in split-sample analysis. F-test, t-test analysis and regression statistics were performed. In Deming regression analysis the coefficients were as follows; the slope=0.967; y-intercept=-0.148, r=0.989. An acceptable bias was seen since the systematical error was calculated to have a value less than the total allowable error calculated from biological variations. Non-parametric evaluation of the area under ROC curves for ACS:180 Plus and Immulite sPSA were 0.997 and 0.987, respectively. Diagnostic accuracy was at the level of p= 0.000 and no statistical difference was found between the two assay methods.     

Immunochemical analytical methods for the determination of peanut proteins in foods

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.     

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic assay for enrofloxacin in biological matrices

Enrofloxacin has been increasingly used in veterinary medicine to treat microbial infections. A simple and reliable analytical method for this drug is required. The current determination by high performance liquid chromatography (HPLC) is sensitive but labor-intensive. This paper reports an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) and the development of a rapid test kit based on immunochromatography. The detection limits using the ELISA were 10 ppb for chicken liver and muscle, and 1 ppb for cattle milk, respectively. The mean recovery values were 77.3-96.0% for chicken liver, 72.4-92.0% for chicken muscle and 84.0-99.0% for cattle milk. The detection limits using the kit were ca. 100 ppb for chicken muscle and ca. 10 ppb for cattle milk, respectively. All ELISA results for assay of chicken liver, chicken muscle and cattle milk were confirmed using HPLC which is used as the routine assay. The HPLC (x) and ELISA (y) results showed close correlation for chicken liver (y = 8.7 + 0.85x, r2 = 0.99, n = 25), chicken muscle (y = -3.9 + 0.94x, r2 = 0.98, n = 25) and cattle milk (y = 18.4 + 0.92x, r2 = 0.99, n = 25).     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods

The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.     

Several concerns about the primer design in the universal molecular beacon real-time PCR assay and its application in HBV DNA detection

A universal hepatitis B virus (HBV) DNA detection kit is appealing for the worldwide diagnosis and monitoring of the treatment of different mutant types of hepatitis B virus. A sensitive and reproducible real-time PCR assay based on the universal molecular beacon (U-MB) technique was developed for the detection of HBV DNA in serum. The U-MB probe used in the assay has no interaction with the HBV DNA sequence. The U-MB technique not only reduced the cost of HBV detection but also had the potential for the development of a universal detection kit for different mutant HBV types and other DNA systems. To demonstrate its clinical utility, 90 serum samples were analyzed using the U-MB real-time PCR method. In the experiments we found that several crucial factors needed to be considered in the primer design, such as the avoidance of formation of severe primer–dimer and primer self-hairpin structure. With the optimized primer sets, satisfactory results were obtained for all the tested samples. We concluded that this assay would be an excellent candidate for a universal HBV DNA detection method. Principle of the U-MB real-time PCR method for HBV DNAdetection     

基于甲基苯丙胺和吗啡的胶体金免疫层析试剂盒检测法的可靠性评价

通过固相萃取-液相色谱-多级质谱(SPE-LC-MS/MS)联用技术和毒品胶体金免疫层析试剂盒检测法对13种中药及调味品样品中甲基苯丙胺及吗啡分别进行定量分析,依据LC-MS/MS检测结果,对毒品胶体金免疫层析试剂盒检测法进行可靠性评价。实验结果表明:型号1试剂盒对甲基苯丙胺和吗啡的特异性均不高,检测准确率分别为57.7%与78.8%;型号2试剂盒对甲基苯丙胺的特异性不足,准确率为73.1%,但对吗啡的检测准确率达到100%。在利用毒品胶体金免疫层析试剂盒进行毒品快速筛查时,应注重排除干扰因素以提高免疫胶体金层析试剂盒的检测准确度。