Establishment of time-resolved fluoroimmunoassay of IgG4 based on magnetic microspheres.

BACKGROUND: The abnormal increase in serum IgG4 level is an important clinical symptom of IgG4-related disease (IgG4-RD), and the detection of serum IgG4 level is a powerful tool for the diagnosis of IgG4-RD. This study was conducted to establish a simple and rapid immunoassay for the determination of human serum IgG4 levels. METHODS: Based on the competition method, a novel immunoassay was established for the determination of human serum IgG4 using a combination of time-resolved fluoroimmunoassay (TRFIA) and magnetic microspheres. IgG4 was coupled with magnetic microspheres and competed with IgG4 in the samples to bind the Eu3+ -labeled anti-IgG4 antibody. The immunocomplex was separated and washed in a magnetic field, and the fluorescence counts were measured according to the number of dissociated europium ions. RESULTS: The analytical sensitivity of IgG4-TRFIA based on magnetic microspheres was 0.006 g/L, and the detection range was 0.006-20 g/L under optimal conditions. The precision, recovery, and specificity of this immunoassay were demonstrated to be acceptable. The clinical application of IgG4-TRFIA based on magnetic microspheres was evaluated and compared with that of immunonephelometry. The results showed that the two detection methods had a good correlation, with a correlation coefficient of .9871. CONCLUSION: IgG4-TRFIA based on magnetic microspheres has the advantages of high sensitivity, wide detection range, and short analysis time and has the potential to become a useful tool for the diagnosis of IgG4-RD.

Evaluation of an Electrochemiluminescent SARS-CoV-2 Antibody Assay.

BACKGROUND: Little is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on Cobas e801/e602 immunoassay analyzers. METHODS: We assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 715 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples [syphilis, rheumatoid factor (RF), antinuclear antibody (ANA), double-stranded DNA (ds-DNA), influenza, dengue, hepatitis B (HBV), hepatitis C (HCV)] and the anti-SARS-CoV-2 kinetics. RESULTS: The assay cut-off index (COI) was linear up to 90.8. The interassay precision was 2.9% for a negative control (COI = 0.1) and 5.1% for a positive control (COI = 3.0). Assay time is 18 min and results are available 1 min later; throughput for 300 samples was 76 min. Only 1 case positive for HBsAg tested falsely positive; specificity was 99.9%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 patients in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to "seroconversion") on average is 3 days (range 1-6 days) and 4 more days (range 1-7) for the anti-SARS-CoV-2 to plateau. CONCLUSION: The Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and seroconversion appears quite early.

Establishment of time-resolved fluoroimmunoassays for detection of growth hormone and insulin-like growth factor I in rainbow trout plasma.

The GH/IGF-I axis influences many aspects of salmonid life history and is involved in a variety of physiological processes that are related to somatic growth (e.g., reproduction, smoltification, and the response to fasting and stress). As such, fisheries studies utilize GH/IGF-I axis components as indicators of growth and metabolic status. This study established time-resolved fluoroimmunoassays (TR-FIAs) for rainbow trout plasma GH and IGF-I using commercially available reagents. For the GH TR-FIA, the ED80 and ED20 were 0.6 and 28.1 ng/mL, the minimum detection limit was 0.2 ng/mL, and the intra- and inter-assay coefficients of variation (%CV) were 4.1% and 13.4%, respectively. Ethanol remaining from acid-ethanol cryoprecipitation (AEC) of plasma samples to remove IGF binding proteins reduced binding and increased variability in the IGF-I TR-FIA. Drying down and reconstituting extracted samples restored binding and reduced variability. The extraction efficiency of IGF-I standards through AEC, drying down, and reconstitution did not vary over the working range of the assay. For the IGF-I TR-FIA, the ED80 and ED20 were 0.2 and 6.5 ng/mL, the minimum detection limit was 0.03 ng/mL, and the intra- and inter-assay %CV were 3.0% and 6.5%, respectively. Biological validation was provided by GH injection and fasting studies in rainbow trout. Intraperitoneal injection with bovine GH increased plasma IGF-I levels. Four weeks of fasting decreased body weight, increased plasma GH levels, and decreased plasma IGF-I levels. The GH and IGF-I TR-FIAs established herein provide a cost-comparable, non-radioisotopic method for quantifying salmonid plasma GH and IGF-I using commercially available reagents.

Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.

Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.

Hydrogel based protein biochip for parallel detection of biomarkers for diagnosis of a Systemic Inflammatory Response Syndrome (SIRS) in human serum.

The Systemic Inflammatory Response Syndrome (SIRS), a sepsis related inflammatory state, is a self-defense mechanism against specific and nonspecific stimuli. The six most extensively studied inflammatory biomarkers for the clinical diagnosis of SIRS are interleukin 4 (hIL-4), interleukin 6 (hIL-6), interleukin 10 (hIL-10), tumor necrosis factor alpha (hTNF-α), interferon gamma (hIFN-γ) and procalcitonin (hPCT). These biomarkers are naturally present (but usually only at low concentration) in SIRS infected patients [1, 2] and thus the development of a highly sensitive detection method is of major clinical interest. However, the existing analytical techniques are lacking in required analytical sensitivity and parallel determination of these biomarkers. We developed a fast, easy and cost-efficient protein microarray biochip where the capture molecules are attached on hydrogel spots, enabling SIRS diagnosis by parallel detection of these six clinically relevant biomarkers with a sample volume of 25 µl. With our hydrogel based protein microarray biochip we achieved a limit of detection for hIL-4 of 75.2 pg/ml, for hIL-6 of 45.1 pg/ml, for hIL-10 of 71.5 pg/ml, for hTNF-α of 56.7 pg/ml, for IFN-γ of 46.4 pg/ml and for hPCT of 1.1 ng/ml in spiked human serum demonstrating sufficient sensitivity for clinical usage. Additionally, we demonstrated successful detection of two relevant SIRS biomarkers in clinical patient samples with a turnaround time of the complete analysis from sample-to-answer in less than 200 minutes.

Surface plasmon resonance based indirect immunoassay for detection of 17ß-estradiol.

A surface plasmon resonance (SPR) based immunosensor is presented for highly sensitive and selective detection of 17ß-estradiol by the indirect competitive inhibition immuno assay, employing anti-17 ß-estradiol antibody as high molecular weight (HMW) interactant. Immobilization of estradiol-BSA conjugate onto the nano thin gold surface was accomplished by covalent amide linkage through self assembled monolayer. The proposed biosensor is simple to fabricate, reproducible and exhibit excellent sensitivity for estrogen (detection limit,1 pg mL-1) without any significant interference from structurally similar steroidal hormone, progesterone and non-steroidal compound bisphenol-A. The proposed surface displayed a high level of stability during repeated regeneration and immunoreaction cycles suitable for biosensor development.

Cell Culture Analysis of the Phagocytosis of Photoreceptor Outer Segments by Primary Mouse RPE Cells.

The phagocytosis of photoreceptor outer segments (POSs) by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Defects in this process can be caused by mutations in the photoreceptor cells, the RPE cells, or both cell types. This function can be experimentally investigated by performing an in vitro phagocytosis assay, in which cultured RPE cells are challenged with isolated POSs, and subsequently tested for their ability to degrade the POSs. A significant advantage of this approach is that mutant phenotypes can be attributed either to the photoreceptor or the RPE cells, by experimenting with different permutations of mutant and control photoreceptor and RPE cells. In this chapter, we detail the method for a double-immunofluorescence assay for analysis of the binding, ingestion, and subsequent degradation of isolated mouse POSs by cultured mouse primary RPE cells.

Fiber-based fluorescent microsphere immunoassay (FMIA) as a novel multiplex serodiagnostic tool for simultaneous detection and differentiation of all clinically relevant fowl adenovirus (FAdV) serotypes.

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test.

Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as micro plate-, array-, multiplexing- or point of care-format.

Although Enzyme Linked Immuno Sorbent Assay (ELISA) technology is approaching it's 45th year of existence since first described in 1971, it is still the main diagnostic tool in clinical research and routine diagnostics. However, despite its broad usage it suffers from some drawbacks, limiting its use especially in more advanced assay formats like multiplexing platforms, point of care devices or protein arrays. Those limitations result from the need for an enzyme label, a soluble enzyme substrate, washing steps (multiplexing, point care, arrays) and in some cases also insufficient sensitivity, because the majority of circulating proteins and thus potential biomarkers may be found in lower sub-picomolar concentrations. We hereby present a new assay platform based on metal enhanced fluorescence (MEF), that remedies these problems since it eliminates the need for washing steps, for using enzyme labels and allows detection of analytes down to sub-picomolar concentrations. In addition this technology is fully compatible to standard fluorescence reader equipment as it is found in many laboratories nowadays. Since our present work is focused on single biomarker evaluation, we chose a 96 well plate format for convenience, but any other formate like antibody arrays, strip-like point of care devices etc. is feasible too.

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin-producing Escherichia coli strains by a time-resolved fluorescence immunoassay.

A rapid and sensitive two-step time-resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin-producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu3+ ) chelate was then used as a detector, followed by fluorescence measurements using time-resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1-1000 ng/ml). The intra- and inter-assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2-specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2-specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation-based platform and aid in the accurate and prompt diagnosis of STEC infections.

Jack Aviv and brains of children.

Both lead intoxication in early childhood and deficient bilirubin-binding capacity (BBC) of blood in jaundiced neonates indicate risk for brain damage. Zinc protoporphyrin (ZPP) is a biomarker for lead intoxication (PbI) as well as well as for iron deficiency. Under the leadership of Jack Aviv, Aviv Biomedical, Inc. developed robust hematofluorometers for point-of-care assays of ZPP in blood and for the high-affinity BBC of blood. These assays use just drops of whole blood and are simple, fast and inexpensive. ZPP by hematofluorometry has been used world-wide as a primary screen for lead intoxication since 1979. Recent clinical studies enabled by an Aviv Biomedical, Inc. bilirubin hematofluorometer have renewed interest in BBC-based assessment of neurotoxicity for improved management of neonatal jaundice. This article sketches Jack Aviv's contribution to the development and application of hematofluorometry.

Evaluation of Sofia Fluorescent immunoassay analyzer for pneumococcal urinary antigen detection in hospitalized patients with community-acquired pneumonia.

The Sofia Streptococcus pneumoniae FIA® test was prospectively evaluated in non-concentrated urine samples of 106 hospitalized patients with community-acquired pneumonia. The test detected pneumococcal urinary antigen in 24/31 (77.4%) confirmed pneumococcal community-acquired pneumonia episodes. The specificity of the test was 86.7% (92% after urine heating).

Bulk immunoassays for analysis of extracellular vesicles.

There is increasing clinical interest in extracellular vesicles (EV) for diagnostic and treatment purposes. This review provides an overview of bulk immunoassays to analyse EV. Western blot and enzyme-linked immunosorbent assay are still the two predominant bulk immunoassays. Recently, new assays have become available that can detect exposure to EV concentrations that are up to 10,000-fold lower. This is advantageous for applications that detect rare EV. Other important parameters are the detectable concentration range, the required sample volume, whether simultaneous presence of different antigens on a single EV can be detected, size selectivity of each assay and practical considerations. In this review, we will explain the working principles of the traditional and novel assays together with their performance parameters. The most sensitive assays are micro-nuclear magnetic resonance, surface plasmon resonance, and time-resolved fluorescent immunoassay.

Dual FITC lateral flow immunoassay for sensitive detection of Escherichia coli O157:H7 in food samples.

A pattern of signal amplification lateral flow immunoassay (LFIA) for pathogen detection, which used fluorescein isothiocyanate (FITC) labeled antigen and antibody for dual FITC-LFIA was developed. Escherichia coli O157:H7 (E.coli O157:H7) was selected as the model analyte. In the signal amplification LFIA method, FITC was mixed with sample culture medium, with the presence of E.coli O157:H7 in the samples, the bacteria could emit a yellow-green fluorescence after incubation, creating a fluorescent antigen probe. This antigen probe was added to LFIA, which already contained E.coli O157:H7 monoclonal antibodies-FITC (McAb-E.coli O157:H7-FITC) dispersed in the conjugate pad. Another E.coli O157:H7 McAb was the test line, and goat anti-mouse IgG antibody was the control line in nitrocellulose (NC) membrane. The visual limit of detection (LOD) of the strip for qualitative detection was 10(5) CFU/mL while the LOD for semi-quantitative detection could down to 10(4) CFU/mL by using scanning reader. Signal amplification LFIA was perfectly applied to the detection of food samples with E.coli O157:H7. The LOD was substantially improved to 1 CFU/mL of the original bacterial content after pre-incubation of the bread, milk and jelly samples in broth for 10, 8 and 8h respectively. The results of this method was more sensitive by 10-fold than the conventional colloidal gold (CG) based strips and comparable to the traditional ELISA. This simple, low-cost and easy to be popularized method served as a significant step towards the development of monitoring food-borne pathogens in food-safety testing.

Magnetically-refreshable receptor platform structures for reusable nano-biosensor chips.

We developed a magnetically-refreshable receptor platform structure which can be integrated with quite versatile nano-biosensor structures to build reusable nano-biosensor chips. This structure allows one to easily remove used receptor molecules from a biosensor surface and reuse the biosensor for repeated sensing operations. Using this structure, we demonstrated reusable immunofluorescence biosensors. Significantly, since our method allows one to place receptor molecules very close to a nano-biosensor surface, it can be utilized to build reusable carbon nanotube transistor-based biosensors which require receptor molecules within a Debye length from the sensor surface. Furthermore, we also show that a single sensor chip can be utilized to detect two different target molecules simply by replacing receptor molecules using our method. Since this method does not rely on any chemical reaction to refresh sensor chips, it can be utilized for versatile biosensor structures and virtually-general receptor molecular species.

Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips.

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.

Multi-color quantum dot-based fluorescence immunoassay array for simultaneous visual detection of multiple antibiotic residues in milk.

Antibiotic residues, which are among the most common contaminants in animal-based food products such as milk, have become a significant public health concern. Here, we combine a multicolor quantum dot (QD)-based immunofluorescence assay and an array analysis method to achieve simultaneous, sensitive and visual detection of streptomycin (SM), tetracycline (TC), and penicillin G (PC-G) in milk. Antibodies (Abs) for SM, TC and PC-G were conjugated to QDs with different emission wavelengths (QD 520 nm, QD 565 nm and QD 610 nm) to serve as detection probes (QD-Ab). Then a direct competitive fluorescent immunoassay was performed in antigen-coated microtiter plate wells for simultaneous qualitative and quantitative detection of SM, TC, and PC-G residues, based on fluorescence of the QD-Ab probes. The linear ranges for SM, TC and PC-G were 0.01-25 ng/mL, 0.01-25 ng/mL and 0.01-10 ng/mL, respectively, with detection limit of 5 pg/mL for each of them. Based on fluorescence of the QD-Ab probes, residues of the three antibiotics were determined visually and simultaneously. Compared with a commercial enzyme-linked immunosorbent assay kit, our method could achieve simultaneous analysis of multiple target antibiotics in multiple samples in a single run (high-throughput analysis) and improved accuracy and sensitivity for analysis of residues of the three antibiotics in authentic milk samples. This new analytical tool can play an important role in ameliorating the negative impact of the residual antibiotics on human health and the ecosystem.

Portable smartphone quantitation of prostate specific antigen (PSA) in a fluoropolymer microfluidic device.

We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with<7% precision in 13 min using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 min total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.

Fluorescent immunodetection of epigenetic modifications on preimplantation mouse embryos.

A common problem in research laboratories that study the mammalian embryo after nuclear transfer is the limited supply of material. For this reason, new methods are continually developed, and existing methods for cells in culture are adapted to suit this peculiar experimental model. Among them is the fluorescent immunodetection. Fluorescent immuno-detection on fixed embryos is an invaluable technique to detect and locate proteins, especially nuclear ones such as modified histones, in single embryos thanks to its specificity and its sensitivity. Moreover, with specific fixation procedures that preserve the 3D shape of the embryos, immunostaining can now be performed on whole-mount embryos. Target proteins are detected by specific binding of first antibody usually nonfluorescent, and revealed with a second antibody conjugated with a fluorochrome directed specifically against the host animal in which the first antibody was produced. The result can then be observed on a microscope equipped with fluorescent detection. Here, we describe the 3D fluorescent immunodetection of epigenetic modifications in mouse embryos. This procedure can be used on nuclear transferred embryos but also on in vivo-collected, in vitro-developed and in vitro-fertilized ones.

Evaluation of the analytical and clinical performances of time-resolved fluoroimmunoassay for detecting carcinoma antigen 50.

We developed a TR-FIA kit for quantitative detection of CA50. This study aims to evaluate the analytical and clinical performances of this kit. Precision, accuracy, specificity, sensitivity, stability, and endogenous interference of this kit are evaluated. Reference range is established. Coincidence rate and correlation between TR-FIA and RIA are evaluated. ROC is adopted to evaluate the diagnostic performance. This kit shows excellent precision with a coefficients of variation (CVs) ranged from 2.2-9.3%, accuracy (average recovery, 98.5%), sensitivity (minimum detectable concentration is 0.2 U/mL), specificity (all cross-reactivity is less than 0.1% except CA199, which is 0.175%), and storage stability (recoveries, 90.8-100.4%). Bilirubin, hemoglobin, and triglyceride dose not interfere with CA50 detection (recovery, 97.13-109.1%). The range from 0-25 U/mL is chosen as the reference range. There are good correlation (r = 0.804) and coincidence (p = 0.608, kappa = 0.924) between TR-FIA and RIA. Diagnostic performance of this kits, which based on RIA results, is perfect (AUC = 0.996), and the diagnostic accuracy for malignancy diagnosis is in moderate degree (AUC, 0.802-0.861). The TR-FIA (CA50) kit performs well in analytical and clinical performances, and can be employed in the clinical diagnosis of malignancy.