Development and Evaluation of Europium-Based Quantitative Lateral Flow Immunoassay for the Chronic Kidney Disease Marker Cystatin-C.

This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.

Magnetic lateral flow immunoassay test strip development - Considerations for proof of concept evaluation.

Lateral flow immunoassays (LFIA) have grown to become the predominant test device format for the diagnostics and point-of-care industries. The demand for robust and reproducible LFIAs has been facilitated through scale-up production methods using specialized and automated instruments. However, the feasibility of a LFIA device can still be evaluated in a small-scale laboratory setting through controlled manual preparation methods. The advent of super-paramagnetic (SPMP) labels for use in lateral flow has heralded the possibility of highly sensitive and stable LFIAs. The methods used for the preparation of a magnetic LFIA prototype device using a reserved suite of laboratory equipment are described.

PicoMolar level detection of protein biomarkers based on electronic sizing of bead aggregates: theoretical and experimental considerations.

We demonstrate a novel method for electronically detecting and quantifying protein biomarkers using microfluidic impedance cytometry. Our biosensor, which consists of gold electrodes micro-fabricated in a microchannel, detects the differences between bead aggregates of varying sizes in a micro-pore sandwiched between two micro channels. We perform a sandwich immunoassay, where the complementary antibody pairs are immobilized on two different bead types, and the presence of antigen results in bead aggregation, the amount of which depends on antigen quantity. When single beads or bead aggregates pass through the impedance sensor, differences in impedance change are detected. In this manuscript, we perform a comprehensive theoretical study on the limits imposed on sensitivity of this technique due to electronic noise and also mass transfer and reaction limits. We also experimentally characterize the performance of this technique by validating the technique on an IgG detection assay. A detection limit at the picoMolar level is demonstrated, thus comparable in sensitivity to a sandwich ELISA.

[Investigation of 70-kDa heat shock protein in the serum and urine of patients with chronic glomerulonephritis].

AIM: To determine the levels of 70-kDa heat shock protein (HSP70) in urine and anti-HPS70 antibodies (Abs) in serum and to assess their clinical and prognostic value in patients with different forms of chronic glomerulonephritis (CGN). SUBJECTS AND METHODS: Seventy-nine patients with CGN, including 15 with inactive nephritis (Group 1), 35 with active CGN and preserved renal function (Group 2), 14 with the highest CGN activity and transient creatininemia (Group 3), and 15 with persistent proteinuria and chronic renal failure (Group 4) were examined. ELISA was used to estimate urinary HSP70 levels and serum anti-HSP70 Abs in the examined groups. RESULTS: The patients with active CGN were found to have higher excretions of urinary HSP70 and serum anti-HSP70 Abs. Urinary HSP70 excretion was significantly higher in the patients with transient renal function (Group 3) than in those from the other groups. At the same time, there was a decrease in serum anti-HSP70 Abs, which was a poor factor of persistent nephrotic syndrome despite immunosuppressive therapy (IST). Despite long-term IST, the nephrotic syndrome was persistent in 9 (60%) of the 15 patients with low serum Ab titers. At the same time, 8 (80%) of the 10 patients with higher serum Ab titers responded to IST during 9 months. CONCLUSION: The investigation demonstrates the great value of HSP70 as an index of the severity of lesion and the activation of kidney self-defense mechanisms in patients with CGN. Determination of serum anti-HSP70 Abs may be used to assess the prognosis of CGN.

Generation and characterization of a specific single-chain antibody against DSPP as a prostate cancer biomarker: Involvement of bioinformatics-based design of novel epitopes.

Isolation of specific single chain antibodies (scFvs) against key epitopes of cancer markers are applied for cancer immunotherapy and diagnosis. In this study following the prediction of the 3D structure of the DSP part of Dentin sialophosphoprotein (DSPP), the epitope was chosen using in silico programs. Panning process was applied to isolate specific human scFv against the epitope. PCR and DNA fingerprinting differentiated the specific clones, which were evaluated by phage ELISA. Following DNA sequencing, the 3D structure of isolated scFv was modeled and Docked on DSP. Results demonstrated the selection of a specific anti-DSPP scFv with 40% frequency, which reacted significantly with the predicted epitope and PCa patients' urines in ELISA tests (P-value < 0.05). The VH and VL of the isolated scFv were from VH1 and VL3 gene families with several amino acid changes in CDRs and FRs domains. The scFv tightly bound to the DSP epitope with the lowest energy level by hydrogen bonds, cation-pi, hydrophobic and ionic interactions demonstrating the specificity of Ag-Ab interactions. The anti-DSPP scFv selected in this study with significant specificity to DSPP antigen offers a promising new agent for both PCa early detection and treatment of cancers with DSPP expression.

Urinary Metabolomics for Noninvasive Detection of Antibody-Mediated Rejection in Children After Kidney Transplantation.

BACKGROUND: Biomarkers are needed that identify patients with antibody-mediated rejection (AMR). The goal of this study was to evaluate the utility of urinary metabolomics for early noninvasive detection of AMR in pediatric kidney transplant recipients. METHODS: Urine samples (n = 396) from a prospective, observational cohort of 59 renal transplant patients with surveillance or indication biopsies were assayed for 133 unique metabolites by quantitative mass spectrometry. Samples were classified according to Banff criteria for AMR and partial least squares discriminant analysis was used to identify associated changes in metabolite patterns by creating a composite index based on all 133 metabolites. RESULTS: Urine samples of patients with (n = 40) and without AMR (n = 278) were analyzed and a classifier for AMR was identified (area under receiver operating characteristic curve = 0.84; 95% confidence interval, 0.77-0.91; P = 0.006). Application of the classifier to "indeterminate" samples (samples that partially fulfilled Banff criteria for AMR; n = 65) yielded an AMR score of 0.19 ± 0.15, intermediate between scores for AMR and No AMR (0.28 ± 0.14 and 0.10 ± 0.13 respectively, P ≤ 0.001). The AMR score was associated with the presence of donor-specific antibodies, biopsy indication, Banff ct, t, ah and cg scores, and retained accuracy when applied to subclinical cases (creatinine, <25% increase from baseline) or had minimal or no transplant glomerulopathy (Banff cg0-1). Exploratory classifiers that segregated samples based on concurrent T cell-mediated rejection (TCMR) identified overlapping metabolite signatures between AMR and TCMR, suggesting similar pathophysiology of tissue injury. CONCLUSIONS: These preliminary findings identify a urine metabolic classifier for AMR. Independent validation is needed to verify its utility for accurate, noninvasive AMR detection.

New haptens and antibodies for ractopamine.

In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 ß-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples.

Urine-based diagnostic technologies.

The worldwide dissemination of infectious agents has created a demand for simple diagnostic tests. Urine-based testing makes use of non-invasive collection of specimens, and there is no need for expensive facilities and equipment, or for highly trained personnel. As urine antibodies retain activity under normal conditions of transport and storage, such tests appear to have widespread application. Urine-based antibody tests have also indicated a compartmentalized antibody response to HIV-1 infection. Urine studies suggest that antibodies to the products of endogenous viral genes may be involved in the pathogenesis of chronic diseases of suspected viral etiology.

Laboratory techniques for detection of urinary tract infection and assessment of value.

Manual methods for diagnosing urinary tract infection have long been under review, modification, and evaluation; thus, methods of collection and interpretation have been found to require more scrutiny. Various screening procedures include chemical, microscopic, and cultural methods, the latter two being highly reliable. In addition, examples of infections due to anaerobic bacteria and Mycoplasma have been documented, with the accompanying need to consider their role in particular situations. There has also been a need for localizing the infection, which has been accomplished with some useful methods. From the literature it is apparent that the tests all have a portion of patients' results that do not fit the true picture. These must be considered carefully in light of other information.

Usefulness of recombinant human thyrotropin in the radiometabolic treatment of selected patients with thyroid cancer.

Treatment of persistent/recurrent differentiated thyroid cancer is based on surgery, when feasible, and malignant tissue ablation by 131I administration. This procedure requires levothyroxine withdrawal to obtain high levels of endogenous thyrotropin (TSH) to stimulate radioactive iodine uptake by the malignant tissue. Levothyroxine withdrawal may cause severe adverse effects and complications in patients with concomitant illness or advanced metastatic disease. The recent availability of recombinant human thyrotropin (rhTSH) allows diagnostic whole-body scan (WBS) and thyroglobulin testing without levothyroxine withdrawal. We describe six patients with metastatic differentiated thyroid cancer (DTC) and concomitant illness in whom the use of rhTSH was effective in preventing the complications that patients had previously experienced during hypothyroidism consequent to levothyroxine withdrawal. Our results indicate that rhTSH can be particularly advantageous to avoid signs and symptoms of hypothyroidism and complications because of associated diseases in view of 131I treatment of DTC metastases in selected cases in which levothyroxine withdrawal may be dangerous. Its efficacy to treat advanced metastatic disease should be further investigated.

Immunoaffinity solid-phase extraction for pharmaceutical and biomedical trace-analysis-coupling with HPLC and CE-perspectives.

Immunoaffinity solid-phase extraction (SPE) technique is based upon a molecular recognition mechanism. The high affinity and the high selectivity of the antigen-antibody interactions allow the specific extraction and the concentration of the analytes of interest in one step. In pharmaceutical and biological fields, where most often matrices are complex and analytes at trace-levels, this approach constitutes a unique tool for fast and solvent-free sample preparation. This review presents a general description of this extraction technique and gives numerous examples of its applications in pharmaceutical and biomedical fields. It emphasizes the on-line coupling with chromatographic and electrophoretic separation techniques and introduces new developments. The future directions, especially with regards to the current development of analytical microsystems, are discussed.

Antibody-mediated interference of a homogeneous immunoassay.

We hypothesized that an antibody-mediated interference could arise in a homogeneous immunoassay used to determine the presence of cocaine metabolites in urine. Urine specimens containing benzoylecgonine (BE) at concentrations near the National Institute on Drug Abuse (NIDA) threshold were assayed in replicate determinations by EMIT. Excess reagent protein (containing antibody specific for cocaine metabolites) was added to specimens to test for an antibody-mediated interference. Replicates of the BE-fortified specimens tested by EMIT that did not contain excess reagent antibody were all positive by the assay, while those that contained the excess reagent antibody were all negative. Because it may be difficult to detect excess interfering antibody by using some traditional tests for urine adulteration, we present these findings to illustrate a potential problem for some homogeneous immunoassays in forensic urine drug testing programs.

Oxaprozin cross-reactivity in three commercial immunoassays for benzodiazepines in urine.

Immunoassay methods are commonly used to screen for drugs of abuse and some prescription drug classes as part of drug-testing programs in clinical and forensic toxicology. Oxaprozin (Daypro) is a new nonsteroidal anti-inflammatory drug that is widely prescribed in North America and has been reported to cross-react for benzodiazepines in several different immunoassay methods. The first objective of this study was to characterize the immunoreactivity of oxaprozin standards over a wide concentration range when analyzed by the EMIT dau, Abbott FPIA, and BMC CEDIA urine benzodiazepine assays. The second objective was to measure the immunoreactivity of urine specimens obtained from 12 subjects after receiving a single oral dose (1200 mg) of oxaprozin. Urine oxaprozin standards were prepared in drug-free urine at seven concentrations ranging from 500 to 100,000 ng/mL. The standards gave presumptive positive benzodiazepine results between 5000 and 10,000 ng/mL (EMIT dau) and approximately 10,000 ng/mL (FPIA, CEDIA). With a 200-ng/mL cutoff for benzodiazepines in these assays, all 36 urine specimens collected from the 12 subjects gave positive results by EMIT and CEDIA, and 35 of 36 urine specimens were positive by FPIA. It was concluded that presumptive positive benzodiazepine results by these immunoassays may be due to the presence of oxaprozin or oxaprozin metabolites. It is recommended that all positive immunoassay screening tests for benzodiazepines be confirmed by another technique based upon a different principle of analysis.

Development of a screening method for anti-6 beta-hydroxycortisol antibody using an enzyme-linked immunosorbent assay (ELISA) and its applications.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of anti-6 beta-hydroxycortisol (6 beta-OHC) antibody has been developed. After immunization of 6 beta-OHC protein conjugates in New Zealand White rabbits, specific polyclonal antibody to 6 beta-OHC was detected by ELISA in which the wells of microtiter plates were coated with 6 beta-OHC conjugated to protein. The rabbit anti-6 beta-OHC antibody titer was above 1:500,000 dilution. Cross-reactivity with other structurally related steroids such as cortisol hydrocortisone was less than 5%. The sensitivity of the polyclonal antibody was comparable to previous studies reported, and was within the accepted detection limit for 6 beta-OHC in man and in laboratory animals. The assay has a low detection limit of 1 ng/ml, an intraassay variation of 3.1% and an interassay variation of 5.2%. The application of the anti-6 beta-OHC-based-ELISA to detect urinary 6 beta-OHC was tested by measuring the concentration of 6 beta-OHC in man before and after enzyme induction by rifampicin treatment. The mean 24 h urine output of 6 beta-OHC in man subjects was 370 +/- 105 micrograms and 1350 +/- 201 micrograms before and after rifampicin administration, respectively. This polyclonal anti-6 beta-OHC antibody-based ELISA can be modified to detect mouse anti-6 beta-OHC IgG with equally good precision and specificity which should be useful in screening positive clones of a 6 beta-OHC IgG secreting mouse hybridoma currently being developed for detecting enzyme induction of CYP3A4 in man and laboratory animals.

Immunoreactivity between venoms and commercial antiserums in four Chinese snakes and venom identification by species-specific antibody.

We studied the immunoreactivity between venoms and commercial antiserums in four Chinese venomous snakes, Bungarus multicinctus, Naja atra, Deinagkistrodon acutus and Gloydius brevicaudus. Venoms from the four snakes shared common antigenic components, and most venom components expressed antigenicity in the immunological reaction between venoms and antiserums. Antiserums cross-reacted with heterologous venoms. Homologous venom and antiserum expressed the highest reaction activity in all cross-reactions. Species-specific antibodies (SSAbs) were obtained from four antiserums by immunoaffinity chromatography: the whole antiserum against each species was gradually passed through a medium system coated with heterologous venoms, and the cross-reacting components in antiserum were immunoabsorbed by the common antigens in heterologous venoms; the unbound components (i.e., SSAbs) were collected, and passed through Hitrap G protein column and concentrated. The SSAbs were found to have high specificity by western blot and enzyme-linked immunosorbent assay (ELISA). A 6-well ELISA strip coated with SSAbs was used to assign a venom sample and blood and urine samples from the envenomed rats to a given snake species. Our detections could differentiate positive and negative samples, and identify venoms of a snake species in about 35 min. The ELISA strips developed in this study are clinically useful in rapid and reliable identification of venoms from the above four snake species.

Immune response biomarker profiling application on ProtoArray protein microarrays.

The development of autoantibodies is observed in autoimmune disorders and numerous cancers. Consequently, autoantibodies form the basis of potential diagnostic and prognostic assays, as well as approaches for monitoring disease progression and treatment response. The effective use of autoantigen biomarkers for these applications, however, is contingent upon the identification of not one but multiple biomarkers. This is a consequence of the observation that the development of autoantibodies to any given protein is typically seen only in a fraction of patients. We have previously demonstrated the utility of functional protein microarrays containing thousands of different human proteins (ProtoArrays) for discovering novel autoimmune biomarkers in serum and plasma. Here, we describe a protocol for detecting autoantibodies in urine.