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.

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.

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.

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.

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.

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.

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.

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.

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.

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk.

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Development of a dual-label time-resolved fluoroimmunoassay for the detection of α-fetoprotein and hepatitis B virus surface antigen.

Time-resolved fluorometry of lanthanide chelates is one of the most useful non-isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time-resolved fluoroimmunoassay (TRFIA) to quantify α-fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two-site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co-coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu(3+)- and Sm(3+)-McAbs were added and fluorescence signals of Sm(3+) and Eu(3+) tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP-TRFIA and HBsAg-TRFIA were 1-1000 mIU/L and 0.2-150 µg/L, respectively. Intra- and inter-assay coefficients of variation of AFP-TRFIA were 3.3-4.1% and 5.7-7.2% and for HBsAg-TRFIA were 2.9-3.9% and 4.9-6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu-labeled McAbs were stable for at least one year at -20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use.

Facile synthesis and biosensing application of hybrid zinc nanoparticles.

Hybrid zinc nanoparticles were synthesized by adding thioglycolic acid (TGA) into a ZnO-particle synthesis procedure. Compared to the ZnO particles prepared without TGA, the hybrid nanoparticles are markedly different in their morphology, chemical composition, and growth kinetics. Moreover, they display colloidal stability and appropriate surface properties for bioconjugation. To demonstrate their biosensing application, the hybrid nanoparticles were conjugated and applied as biolabels or signal transducers in a sandwich immunoassay for mouse IgG. The immunoassay fluorescence signal was obtained by releasing zinc ions from these nanoparticle labels and further incubating the released zinc ions with zinc-sensitive fluorescence indicator Fluozin-3. The immunoassay presents a dynamic detection range from 10 pM to 1 nM.

Magnetic fluorescent composite nanoparticles for the fluoroimmunoassays of Newcastle disease virus and avian virus arthritis virus.

A new detection format for multiplexed analysis based on the use of magnetic fluorescent composite nanoparticles was presented in this paper. Two different antigens, Newcastle disease virus (NDV) antigen and Avian virus arthritis virus (AVAV) antigen, were conjugated to two kinds of magnetic fluorescent composite nanoparticles of different luminescent colors, while red-emitting CdTe QDs were attached to the antibody of NDV and AVAV. Both CdTe QDs-labeled antibodies and magnetic fluorescent composite nanoparticles labeled antigens were used to form a typical immunoreaction system for the detection of NDV and AVAV. Also a typical mixed separation format was realized, which showed the outstanding magnetic properties of composite nanoparticles and the broad application in immunoseparation.

A rapid and sensitive fluoroimmunoassay based on quantum dot for the detection of chlorpyrifos residue in drinking water.

A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (cFLISA) method based on quantum dots as the fluorescence label coupled with secondary antibody (Ab(2)) for the detection of chlorpyrifos in drinking water has been developed. The cFLISA method allowed for chlorpyrifos determination in a liner working range of 15.2-205.5 ng mL(-1). The 50 % inhibition value (IC(50)) and the limit of detection (LOD) of the cFLISA were 50.2 ng mL(-1) and 8.4 ng mL(-1), while the IC(50) and the LOD of the conventional enzyme linked immunosorbent assay (ELISA) were 95.3 ng- mL(-1) and 16.2 ng mL(-1), respectively. When the concentrations of chlorpyrifos were 200, 100 and 50 ng mL(-1), the recoveries ranged from 90.8 % to 108.2 % with a coefficient of variation (CV) of 7.5 %-15.2 %. In water sample analysis, the results of cFLISA were similar to those obtained from a cELISA and a high performance liquid chromatography (HPLC) method, while the detection time by cFLISA was reduced 0.5 h compared with ELISA. It showed that cFLISA could be used as a new screening method for the detection of pesticide residue.

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.

Capillary waveguide fluoroimmunosensor with improved repeatability and detection sensitivity.

An optical capillary waveguide fluoroimmunosensor based on glass capillaries internally coated with an ultrathin poly(dimethylsiloxane) (PDMS) film is presented. The evaluation of the capillaries developed was done in comparison with aminosilanized [3-(aminopropyl)triethoxysilane, APTES] glass and poly(methylpentene) (PMP) capillaries by immobilizing rabbit gamma-globulins on the internal capillary wall. Following reaction with (R)-phycoerythrin-labelled antibody, the capillary was scanned with a laser beam and the fluorescence waveguided through the capillary wall was detected by a photomultiplier placed at one of its ends. The capillaries developed provided considerably improved protein coating homogeneity (intracapillary coefficients of variation 2.9-6.6%) and repeatability (intercapillary coefficients of variation 2.1-5.0%) compared with APTES-treated ones (7.9-13.4 and 8.5-15.2%, respectively). With use of these capillaries in a sandwich-type immunosensor for the determination of rabbit gamma-globulins, the assay detection limit was improved eightfold (4.4 ng/mL) compared with that obtained using PMP capillaries (35.3 ng/mL), whereas the assay repeatability was improved threefold (intra-assay coefficients of variation 5.9-13.1%) compared with APTES-treated capillaries (15.6-36%).

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.

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.

Enhanced microfiltration devices configured with hydrodynamic trapping and a rain drop bypass filtering architecture for microbial cells detection.

Ultra-fine (<1 microm) microfilters are required to effectively trap microbial cells. We designed microfilters featuring a rain drop bypass architecture, which significantly reduces the likelihood of clogging at the cost of limited cell loss. The new rain drop bypass architecture configuration has a substantially lower pressure drop and allows a better efficiency in trapping protozoan cells (Cryptosporidium parvum and Giardia lamblia) in comparison to our previous generation of a microfilter device. A modified version displaying sub-micron filter gaps was adapted to trap and detect bacterial cells (Escherichia coli), through a method of cells labeling, which aims to amplify the fluorescence signal emission and therefore the sensitivity of detection.

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.