Homogeneous electrochemical detection of hippuric acid in urine based on the osmium-antigen conjugate.

A homogeneous electrochemical immunoassay is based on the interaction of osmium-antigen conjugate with its antibody. The novelty presented herein is the direct conjugation of the osmium complex to a small antigen and the application of the quantitative analysis of the antigen and its antibody as the electrical signal for homogeneous immunoassay. The small antigen chosen is hippuric acid (HA), a major urinary metabolite in toluene-exposed humans. As a redox mediator, [Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine-HA)Cl](+/2+) (Os-HA antigen) has been synthesized and characterized on screen-printed carbon electrodes. The synthesized Os-HA antigen shows reversible redox peaks at E(½)=0.056 V versus Ag/AgCl. The homogeneous competitive immunoassay relies on the interaction between Os-HA antigen conjugate and free antigen to its antibody, which can generate electrical signals linearly proportional to the free antigen monitored by cyclic voltammetry and differential pulse voltammetry in the range of 10 µg mL(-1) to 5.12 mg mL(-1). The cutoff concentration of HA in urine samples is 2.0 mg mL(-1), so the method can be used to develop a HA immunosensor. Moreover, the proposed homogeneous electrochemical immunoassay method can be applied to detect low concentrations of small antigens found in the healthcare area.

Urinary excretion of foreign antigens and RNA following primary and secondary injections of antigens.

Two soluble antigens, BSA and KLH labeled with sulfanilate-(35)S, when injected intravenously into normal animals, were excreted in the urine to over 70% in 24 hr. Over the next 6 days, 25% more was excreted after which time only a trace could be detected. Much of the antigen remaining from the primary injection appeared in the urine following a secondary injection of the unlabeled protein carrier at 7 days after primary injection. The antigen material found in the urine was quite heterogeneous with respect to physical properties and much of it was associated with RNA material as shown by chromatographic analyses. The main difference between the labeled material released following the primary and secondary injection was the higher degree of association of antigen material with nucleotide material after secondary injection as compared with primary injection. Further study is needed to distinguish qualitative from quantitative changes of the components, antigen and nucleic acid, and also the nature of their association. Possible similarities were found for the RNA-antigen material released from tissue after secondary injection of unlabeled antigen, and the material that was isolated previously from liver.

Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers.

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.

Accuracy of Two Point-of-Care Tests for Rapid Diagnosis of Bovine Tuberculosis at Animal Level using Non-Invasive Specimens.

Bovine tuberculosis (BTB) testing in cattle requires a significant investment of time, equipment, and labor. Novel, rapid, cheaper and accurate methods are needed. The Alere Determine TB lipoarabinomannan antigen (LAM-test) is a World Health Organization-endorsed point-of-care urine test designed to detect active TB disease in humans. The Lionex Animal TB Rapid Test (Lionex-test) is a novel animal specific TB diagnostic blood test. An animal level analysis was performed using urine (n = 141) and milk (n = 63) samples from depopulated BTB-suspected cattle to test the accuracy of the LAM-test when compared to results of positive TB detection by any routine BTB tests (BOVIGAM, necropsy, histology, culture, PCR) that are regularly performed by the United States Department of Agriculture (USDA). The agreement between the urine LAM-test and USDA standard tests were poor at varying testing time points. The same milk samples did not elicit statistically significant agreement with the Lionex-test, although positive trends were present. Hence, we cannot recommend the LAM-test as a valid BTB diagnostic test in cattle using either urine or milk. The Lionex-test's production of positive trends using milk samples suggests larger sample sizes may validate the Lionex-test in accurately diagnosing BTB in cattle using milk samples, potentially providing a quick and reliable field test for BTB.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND: Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS: We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS: In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION: In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Resin-based micropipette tip for immunochromatographic assays in urine samples.

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 microl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 microl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity.

Human glomerular basement membrane. Antigenic determinants in human urine.

Antisera against particulate human glomerular basement membrane prepared from cadaver kidneys were raised in rabbits. It was shown that both normal individuals and patients with glomerular and tubular diseases excrete in their urine several antigens reactive with these antibodies. One antigen crossreacted immunologically with an antigen from human glomerular basement membrane while several others did not. One of the urinary antigens and the antigen crossreacting with the basement membrane were separated from the others by ion exchange chromatography and gel filtration, respectively. The pattern of anttigen excretion differed depending on the underlying renal disease but the multitude of different antigens detected complicates the interpretation of the patterns of excretion in different diseases.

Insulin-like insulinase-resistant material, distinguishable from normal insulin, in juvenile diabetes.

This study compares some properties of the immunoreactive insulin-like material extracted from the urine of children with overt diabetes with that from normal children. Insulin-like species were fractionated by gel filtration and by isoelectric focusing and were tested for sensitivity to an insulin-specific degradative enzyme. Insulin concentration was measured by radioimmunoassay. The major insulin-like component from the urine of ten normal children and fifteen untreated juvenile diabetics and from the urine of four and the serum of one latent diabetics behaved (on gel filtration) as normal insulin, was sensitive to insulinase, and (in all cases studied) had an identical isoelectric point (resolution 0.1 pH units). A proportion of the immunoreactivity extracted from urine (0-4 per cent from normal children, 5-30 per cent from twelve of the thirteen nonobese untreated diabetic children) eluted from the gel filtration column before insulin. This material from diabetic urine was of two size classes, "proinsulin-like" and "mid-insulin," both resistant to degradation by insulinase. Insulinase-resistant immunoreactivity from one patient was analyzed by isoelectric focusing. Urine samples from two obese children with overt diabetes and four children with latent diabetes contained normal proportions (less than 4 per cent) of immunoreactive species larger than insulin. The possible nature and significance of the present insulinase-resistant species are briefly considered.

Immunoreactive somatomedin B in urine.

Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 +/- 4.4 mug between 1000 h and 1400 h compared to a mean plasma concentration at 1200 h of 5.9 +/- 0.9 mug/ml. Free somatomedin B in urine averaged 85.9%, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.

Immunoreactive luteinizing hormone, follicle stimulating hormone and their subunits in human urine following gel filtration.

Urine collected from postmenopausal women, normal men, women and children was prepared by vacuum dialysis and kaolin extraction prior to chromatography on Sephadex G-100. Specific radioimmunoassays for LH, alpha subunit, LHbeta, FSH and FSHbeta were utilized to define the elution patterns of the urinary filtrates, extracts, 2nd IRP-HMG and postmenopausal serum. The following findings were obtained: 1) FSH eluted as a distinct immunoreactive peak whereas LH eluted more diffusely, 2) free alpha subunit was present in all urinary preparations, the 2nd IRP-HMG and postmenopausal serum, 3) free FSHbeta but not LHbeta was detectable in postmenopausal urine, and 4) an LHbeta immunoreactive fragment with an approximate molecular weight of 5000 daltons was present in all urinary preparations and the 2nd IRP-HMG but not in postmenopausal serum.

Immunoassay with stable polystyrene latex particles.

Three variations of a new immunoassay which used physicochemically stable latex particles (LP) are described. New features include (1) separation of free and agglutinated LP by centrifugation after the addition of sucrose, and (2) combined use of two or three types of LP. Assay results for anti-rabbit IgG (anti-RG), human chorionic gonadotrophin (HCG), C3 components and insulin indicated excellence of these methods in sensitivity, reproducibility and preservability of reagents.

Development of monoclonal antibodies to proteins separated by two-dimensional gel electrophoresis.

We have developed techniques for the production of monoclonal antibodies using Coomassie blue-stained protein spots cut from high resolution 2-dimensional polyacrylamide gels. The gel spots were homogenized with Freund's adjuvant and injected sub-cutaneously into a mouse, at several places along the flank. After boosting twice the spleen cells were hybridized by standard methods. Hybrids, clones and ascitic fluids were also screened with antigen prepared from 2-dimensional gel spots. The spots were cut from gels, homogenized in the presence of guanidinium chloride, and extracted by shaking overnight. The acrylamide was removed, the sample dialyzed to remove denaturant and the protein labeled with 125I. An alternative method for the production of screening antigen using column chromatography is described. These techniques allow the production of monoclonal antibodies to specific protein components of complex mixtures, even in the presence of other immunodominant proteins.

Urine as an antigen reservoir for diagnosis of infectious diseases.

Soluble or particulate microbial antigens are excreted in the urine in many systemic infectious processes. The ease with which urine antigens can be concentrated has facilitated their detection by immunologic methods. The group and type-specific bacterial polysaccharides are among the best studied examples of urinary excretion of microbial antigens. These polysaccharides are often present in the urine as low molecular weight fragments (70,000 daltons or less) and in some instances may represent degradation products of the native polysaccharides. Urine polysaccharides are sufficiently immunoreactive to be detectable by simple precipitin and agglutination techniques in a large percentage of patients with infections due to certain pyogenic bacteria including Haemophilus influenzae and group B streptococci. Both polysaccharide and protein antigens have been detected in the urine by immunologic methods in numerous other infections including parasitic, viral, and spirochetal diseases. Detection of a thermostable antigen in the urine of patients with Legionnaires' disease by radio- and enzyme-linked immunoassays represents an important recent advance. The exact role of immunologic tests for etiologic diagnosis in infectious diseases is not established, but will undoubtedly be influenced by developments such as monoclonal antibody technology and better availability of standardized immunologic reagents.

Urinary excretion rate of antithrombin III related antigen in cerebral stroke.

The urinary excretion rate of antithrombin III related antigen (AT III RA) was examined in cerebral stroke. The excretion rate of AT III RA in cerebral hemorrhage (CH) was 12.33 +/- 1.61 X 10(-4) ml/min. The patients with CH were further classified into two groups: in group CH-I, whose consciousness state was stupor or further deteriorated including coma on admission, the excretion rate of AT III RA was 18.08 +/- 2.50 X 10(-4) ml/min. In group CH-II, whose consciousness state was clear on admission, the excretion rate of AT III RA was significantly lower than that in CH-I (6.20 +/- 1.56 X 10(-4) ml/min). The excretion rate in cerebral thrombosis (CT) was 1.96 +/- 0.25 X 10(-4) ml/min, which was significantly lower than that in CH. The excretion rate of AT III RA in both CH and CT was significantly higher than that in the healthy control group (0.29 +/- 0.04 X 10(-4) ml/min). Thus, AT III may change dynamically in cerebral stroke.

Tissue factor pathway inhibitor (TFPI) levels in the plasma and urine of children with meningococcal disease.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the TF-dependent coagulation system. In meningococcal disease, up-regulation of tissue factor expression on blood monocytes and possibly on endothelial cells has the potential to trigger the activation of the TF-dependent pathway of coagulation. Intravascular coagulation is considered to be a major pathogenic factor in meningococcal disease. We postulated that imbalance between TF expression and TFPI concentration might lead to uncontrolled coagulation in meningococcal disease. The aim of this study was to assess the levels of total TFPI in the plasma of patients with meningococcal disease and assess whether increased leaking of the TFPI was occurring. TFPI antigen levels and activity were measured in the plasma of 54 patients with meningococcal disease, and 13 healthy control children. TFPI antigen level were also determined in the urines of 14 of the 54 and 9 healthy control children. Plasma TFPI activity was reduced in the meningococcal diseased patients (mean of 0.503 +/- 0.341 U/ml; control, 1.010 +/- 0.199 U/ml: p <0.0001), as was the TFPI antigen levels (mean of 54.85 +/- 35.05 ng/ml; Control, 94.51 +/- 11.44 ng/ml; p <0.0001). In contrast, TFPI antigen levels were increased in the urine of these patients when compared to the levels found in the urine of the healthy control children (mean of 12.96 +/- 5.392 ng/mmol creatinine; Control, 0.239 +/- 0.191 ng/mmol creatinine; p <0.035). A lack of correlation between TFPI-activity and TFPI-antigen plasma levels was observed (r = 0.002, p = 0.85). This data is consistent with the hypothesis that in meningococcal disease there is increased inactivation of plasma TFPI by the up regulation of tissue factor expression but in addition increased clearance of TFPI in urine is occurring.

Factor XII and other hemostatic protein abnormalities in nephrotic syndrome patients.

Factor XII clotting activities and antigen levels were assayed in 14 plasma samples from 10 patients with nephrotic syndrome; the group was heterogeneous clinically and histologically. Factor XII was low at initial sampling in 7 of the 10 patients; in 7 of the 14 samples, factor XII antigen was in excess over clotting activity. Inhibition of factor XII could not be demonstrated; excess plasma antigen and urinary antigen (when present) had normal patterns on crossed-immunoelectrophoresis, indicating no major changes in charge or size. In 3 patients tested more than once, plasma levels of factor XII were increased up to 6fold in steroid-induced remission. Of other hemostatic factors assessed for comparison, factor VIII was elevated in 11 of the 14 samples; eight of these had elevated factor VII levels as well. Eight samples from six patients showed low antithrombin III levels; one of these patients had recurrent thromboses. Antithrombin III levels correlated with the serum albumin concentration. Only two of the eight urines tested had detectable factor XII antigen; a third had factor IX and prothrombin and no factor XII. Plasminogen and antithrombin III were readily demonstrated in all urine samples with higher concentrations in those patients with less selective proteinuria. Urinary and plasma levels were not correlated, suggesting that increased consumption or turnover was not simply related to increased filtration.

Localization of human seminal plasma No. 7 antigen (Ferrisplan) in accessory glands of male genital tract and spermatozoa.

The establishment of a hybridoma (1C4) producing sperm immobilizing monoclonal antibody to human seminal plasma No. 7 antigen (HSP No. 7 Ag.) and the isolation of the pure antigen by immunoaffinity chromatography bound monoclonal antibodies have been reported previously. In the present investigation, HSP No. 7 Ag. has been termed 'Ferrisplan' and its distribution in male genital organs, spermatozoa and body fluids has been studied. The amount of Ferrisplan in the body fluids was determined by radioimmunoassay. Large amounts were detected in seminal plasma and milk, trace amounts in saliva, and none in the serum and urine. The concentration of Ferrisplan was highest in the seminal plasma of azoospermic patients and gradually decreased from oligospermia to normospermia. Using an immunofluorescent method with anti-Ferrisplan monoclonal antibody, strong staining was observed on the epithelial layers of human seminal vesicles, no staining on testes and bright staining on the post-nuclear cap and mid-piece segment of spermatozoa. These results indicate that Ferrisplan is excreted mainly from the seminal vesicle and adheres to the post-nuclear cap and mid-piece of the spermatozoa as a sperm-coating antigen.

Quantitative counterimmunoelectrophoresis for urinary fibrinogen-related antigens.

A quantitative procedure for urinary fibrinogen-related antigens, applying counterimmunoelectrophoresis to serial dilutions of concentrated samples against standards of known fibrinogen concentrations, is described. It is fast, convenient, sensitive enough (r = 0.8729, p less than 0.001), and can be easily incorporated into routine laboratory work.

Detection of fibrinogen antigens with two latex techniques applied to urine concentrates.

Fibrinogen antigens were measured either with an agglutination inhibition method (using latex particles coated with fibrinogen; Diagen test) or with a direct agglutination technique (using latex particles coated with a mixture of anti-D and anti-E antibodies; Thrombo-Wellcotest). Both methods were compared with the tanned red cell haemagglutination inhibition immunoassay (TRCHII) during progressive degradation of fibrinogen with plasmin and using purified fibrinogen fragments or urine concentrates from chronic glomerulonephritis or transplanted patients. Due to the different sensitivity of the two latex techniques to fibrinogen and its plasmin derivatives, their combined use may be helpful to distinguish the nature of the fibrinogen-like material excreted in urine.

Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection.

OBJECTIVES: Hyperglycosylated human chorionic gonadotrophin (hCG) is an hCG variant with extra-large O-linked oligosaccharides, produced by phenotypically invasive cytotrophoblast cells in choriocarcinoma and pregnancy. It is the principal form of hCG produced in the first weeks of gestation. We investigated the importance of hyperglycosylated hCG in pregnancy testing and its detection by current hCG tests. DESIGN AND METHODS: We measured the concentration of hyperglycosylated hCG and total hCG in 512 pregnancies throughout gestation. We assessed and compared the abilities of 14 commonly used commercial laboratory hCG tests and 18 home pregnancy tests to detect regular and hyperglycosylated hCG. RESULTS: Hyperglycosylated hCG is the principal source of hCG-related immunoreactivity in early pregnancy. In the week following missing menses, hyperglycosylated hCG measurements may be more sensitive than regular hCG measurements in detecting pregnancy. Of 14 commercial laboratory hCG tests, 3 appropriately detected hyperglycosylated hCG standard. Of 18 different home pregnancy products 11 poorly or very poorly detected this key antigen. CONCLUSIONS: Hyperglycosylated hCG may be the key molecule in the detection of early pregnancy. However, the majority of tests poorly detected or failed to detect this key antigen. New pregnancy tests are needed that either solely detect hyperglycosylated hCG or equally detect regular hCG and hyperglycosylated hCG.