Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PIPET”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed1983

Early screening for hybrid antibodies.

A new system for the screening of hybrid cultures in high number for possible production/secretion of antibodies (immunoglobulin) is presented. In the hybridoma technique for the production of monoclonal antibodies, myeloma cells are fused with B-lymphoblasts. This fusion is induced and often polyethylene glycol is used for this. Soon after fusion, a multitude of small colonies appears. Because of the biochemical characteristics of the myeloma cells, a selection, which only leaves fused cells, is possible. Among these colonies of fused cells, some will be antibody producing and secreting, and some of these will have the correct specificity. This specificity is towards the antigen originally used for immunization of the animal (often mouse). Because of the high number of fused cells, it is important to eliminate competition among different clones, wherefore seeding is done in microtest plates. Even so, more fused cells can end up in the same well, and competition can occur. Therefore, it is important to identify the clones secreting antibody of correct specificity as soon as possible. In the systems employed until now, medium had to be removed from the culture microtest plates. This induces a change in composition with replenishment, and so is not desired too often. Furthermore, this pipetting of small amounts of medium is laborious having 5, 10, or more plates. Therefore, a new system has been developed, which does not remove any medium, that has EIA sensitivity, is fast, and with very few necessary pipettings. The system uses a transferred solid phase, and is called NUNC-TSP-Screening System.

Animals↗
PubMed1984

An evaluation of the Technicon RA-1000 random-access analyzer.

We evaluated the Technicon RA-1000 analyzer, with emphasis on its potential for user-defined method development. Optical linearity and sample pipetting linearity were good. The reagent pipetting system delivered slightly less than the nominal amount, owing to the volumetric effect of added mixing bubbles. Carryover of aqueous solutions was negligible. The instrument had good adaptability for user-defined methods and performed well in method comparisons. The observed dynamic range for enzymes (0-3000 U/L) was excellent. The worklisting software performed its intended functions well, but has limitations. We believe that the RA-1000 represents a significant contribution to the practice of clinical chemistry. A sophisticated benchtop machine, it includes several innovations, along with a few problems that are peculiar to its technology.

Autoanalysis↗
PubMed1995

[Adhesion and penetration of human lymphocytes through allogeneic endothelial cells within the scope of organ rejection].

INTRODUCTION: Cellular rejection mechanisms are characterized by the infiltration of sensitized lymphocytes through the donor endothelium into the transplanted organ. The regulation of cellular adhesion molecules by soluble mediators (cytokines) is thought to play a dominant role in this process. In the present study the kinetics of lymphocyte infiltration were examined using a new established in vitro model and compared to the kinetics of adhesion molecule expression. MATERIALS AND METHODS: Peripheral blood lymphocytes (PBL) of healthy volunteers were pipetted to allogeneic endothelial cell (EC) monolayers and binding rates evaluated after different incubation times. Adherent PBL could be distinguished from penetrated PBL by means of a combined phase contrast- / reflection interference contrast - microscope. RESULTS: 30-35% of all pipetted PBL adhered to unstimulated EC maximally. Out of these cells < 10% penetrated through the endothelium ( = maximum penetration). The cytokines alpha-, beta-, gamma-interferon (IFN) or IL-1 did not enhance maximum adhesion, but accelerated this process. However, a 2 hrs prestimulus of EC by these cytokines was necessary to induce acceleration. Maximum penetration was enhanced by alpha-, beta-, gamma-IFN, but not by IL-1, irrespective whether PBL were added together with cytokines to unstimulated EC or to already prestimulated EC. Immunocytochemical and fluorometric analyses of adhesion molecule expression revealed a cytokine induced upregulation or de novo expression of the adhesion antigens ICAM-1, ELAM-1 and VCAM-1 on EC membranes. Interestingly, PBL adhered to EC before upregulation or de novo expression of adhesion molecules was detected. DISCUSSION: The results showed that PBL adhesion and penetration processes were regulated differentially by cytokines. The early phase of PBL attachment to EC seemed not to be influenced by adhesion antigens but by an activation of the lymphocyte cytoskeleton.

Cell Adhesion↗
PubMed1993

[Migratory ability of cells derived from periodontal tissues and their attachment to root surfaces].

The migratory ability of cells from the periodontal ligament (PDL), alveolar bone and gingiva of monkeys was compared in vitro using a three-dimensional collagen gel model. In addition, the cell attachment to cementum and dentin was examined ultrastructurally. Cultured cells were obtained from monkey periodontal tissues using an explant method. Type I collagen solution was pipetted into the wells of 24 multi-well plates and polymerized. And the cell invasion assay was performed by seeding test cells onto the gel, and subsequent culture for 1, 3 and 7 days. The specimens were fixed and embedded in epoxy resin. Semi-thin sections of 1 micron thickness were obtained and subjected to visual assay under a light microscope. Parameter was two fold; numbers of cells migrating into the gel and the maximum distance from the gel surface to the leading front of migrated cells. Secondly, two kinds of root surface conditioning were performed on extracted monkey teeth, in which included scaling only surface cementum and root planing. Each root fragment was placed into the wells and collagen solution was subsequently pipetted into the well. And the collagen solution was polymerized to prepare the 100 microns-wide gel layers on the root fragments. Test cells were seeded onto the gel and cultured for 2 and 3 weeks. The interaction between the cells and the root surfaces was examined under a transmission electron microscope. The migratory ability of PDL cells was less than that of cells from bone and gingiva. Although the migrated bone and gingival cells produced fibrillar materials on the cementum, the PDL cells could not establish an ideal attachment to either cementum or dentin.

Animals↗
PubMedDate not supplied

Comparison of automated and traditional minimum inhibitory concentration procedures for microbiological cosmetic preservatives.

Minimum inhibitory concentration (MIC) is used to test resistance of microorganisms against antibiotics and to test cosmetic preservatives. This research expanded traditional MIC with automation and application of colorimetric endpoint MIC. All experiments included common cosmetic preservatives and microorganisms used in testing preservative efficacy. An autodilutor using three 96-well microtiter plates processed 6 preservatives against 1 microorganism in 15 min. The unique tip design made it possible to accurately deliver viscous test materials that cannot be dispensed accurately with vacuum or fluid-filled systems. Tetrazolium violet, a redox indicator, provided a visual color change from clear to purple at the MIC. Optimum concentration of tetrazolium violet was 0.01% with addition of 0.2% glucose to Mueller-Hinton broth for both gram-positive and gram-negative bacteria. The colorimetric endpoint was evident after 24 h from previously cryogenically stored organisms that were thawed before use and after 4 h for 18-24 h broth cultures subcultured from agar plates. The autodilutor accurately pipetted viscous cosmetic products such as hand lotion and shampoo, which cannot be pipetted with a traditional micropipetter.

Azoles↗
PubMedDate not supplied

Influence of centrifugation regimes on motility, yield, and cell associations of mouse spermatozoa.

Mouse sperm are exceptionally sensitive to mechanical forces associated with pipetting and mixing. This characteristic raised the question of the sensitivity of mouse sperm to centrifugation, a step necessary in the removal of cryoprotectants and a common component in the general manipulation of sperm suspensions for experimental purpose. Epididymal spermatozoa from ICR mice were isolated and manipulated to minimize pipetting and mixing damage. The centrifugal accelerations studied were 200, 400, 600, and 800 x g (measured with a stroboscope) for 5, 10, or 15 minutes of centrifugation time. The number of cells and the number of motile cells were counted. The percent motility and longevity, total yield, and motile yield were calculated. Centrifugation at 200 and 400 x g for short times (5 minutes) caused only a small loss in either immediate or 2.5-hour motility, but centrifugation at 600 and 800 x g for 15 minutes produced up to a fivefold loss. Low speed/short time centrifugation pelleted only about half of the cells; the others were lost when the supernatant was removed. The maximum number of motile sperm (motile yield) was obtained at intermediate centrifugal forces (approximately 400 x g for 10-12 minutes), and it is the total number of motile sperm (and not the percent motility) that is important in the use of cryopreserved sperm to regenerate cryopreserved mutant lines. Relative centrifugal force and centrifugation time exhibit reciprocity (e.g., 200 x g for 10 minutes produces similar results to 400 x g for 5 minutes). The spermatozoa must be centrifuged under carefully defined conditions to minimize the damage and to maximize the recovery of viable cells.

Animals↗
PubMed1999

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.

CD4 Lymphocyte Count↗
PubMed2000

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

Antigens, CD34↗
PubMed2001

High lane density slab-gel electrophoresis using micromachined instrumentation.

In this paper, micromachined pipette arrays (MPAs) and microcombs were studied as a means of enabling high lane density gel electrophoresis. The MPA provide a miniaturized format to interface sub-microliter volumes of samples between macroscale sample preparation formats and microscale biochemical analysis systems. The microcombs provide a means of creating sample loading wells in the gel material on the same center-to-center spacing as the MPAs. Together, the two micromachined instruments provide an alternative to current combs and pipetting technologies used for creating sample loading wells and sample delivery in gel electrophoresis systems. Using three designs for the microcomb-MPA pair, center-to-center spacings of 1.0 mm, 500 microm, and 250 microm are studied. The results demonstrate an approximate 10-fold increase in lane density and a 10-fold reduction in sample size from 5 microL to 500 pL. As a result, the number of theoretical plates has increased 2.5-fold, while system resolution has increased 1.5-fold over the conventional agarose gel systems. An examination of changes in resolution across the width of individual separation lanes in both systems revealed dependence in the case of the conventional gels and no dependence for the gels loaded with the micromachined instrumentation.

Bromphenol Blue↗
PubMedDate not supplied

Enhanced chemiluminescent immunoassay for aldosterone.

A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100 fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%-7.3% in the concentration range 140-1180 pmol/l. The assay showed a significant correlation (r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit.

Aldosterone↗
PubMed1997

Quantitative determination of oLAb titers in various animal species.

It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously. Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37 degrees C. After washing, a protein A horse-radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1:20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer. Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37 degrees C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2-4 degrees C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti-species IgG conjugates. The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system.

Animals↗
PubMed1983

Origins of the differences in function of rat adrenal zona glomerulosa cells incubated as intact tissue and as collagenase-prepared cell suspensions.

While in vitro incubation of dispersed cell preparations of adrenal cell types has been widely used as an experimental model, few studies have addressed the possibility that the enzymic and mechanical treatments involved may affect tissue functions. Using rat adrenal whole capsule tissue, consisting of glomerulosa cells still attached to the connective tissue capsule together with some fasciculata cells, and dispersed glomerulosa cell preparations formed by a variety of enzymic and incubation treatments, striking differences have been demonstrated between the functions of the various preparations in vitro. Under ACTH stimulation, whole capsules produced (ng per pair +/- s.e.) 405 +/- 35 ng aldosterone, 650 +/- 60 ng 18-hydroxycorticosterone (18-OH-B) and 850 +/- 90 ng corticosterone. In cells dispersed by collagenase incubation followed by repeated pipetting and filtration, aldosterone and 18-OH-B yields under ACTH stimulation fell to values less than 10% of those produced by whole tissue, whereas corticosterone values were unchanged. Omitting the filtration step gave a less well marked decline in aldosterone and 18-OH-B to 50% of intact tissue values. When the tissue was not dispersed after collagenase incubation, aldosterone and 18-OH-B outputs were similar in the two preparations. The decline in aldosterone and 18-OH-B is not attributable to loss in cell-cell contact alone, since short term culture of collagenase dispersed cells on contracting collagen discs did not restore the capacity to produce these steroids, and a decline in their output also occurred in similar culture of intact capsule tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

18-Hydroxycorticosterone↗
PubMed2003

Gliding movement in Peranema trichophorum is powered by flagellar surface motility.

A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.

Animals↗
PubMed1982

Topography between the entorhinal cortex and the dentate septotemporal axis in rats: I. Medial and intermediate entorhinal projecting cells.

Retrograde tracing experiments were performed to clarify the topographic projection from medial (area 28m) and intermediate (area 28i) divisions of the entorhinal cortex to the dentate gyrus. Pipets filled with horseradish peroxidase (HRP) were positioned by electrophysiologic guidance at one of several septotemporal (S-T) levels in the dentate molecular layer of anesthetized rats; the tracer was expelled iontrophoretically to minimize its spread. Retrograde labeling of neurons within areas 28m and 28i was analyzed in relation to cytoarchitectonic as well as spatial features of the region (obtained by histologic reconstruction). Regardless of the S-T level, ejections of HRP which were confined to the dentate gyrus labeled only layer II neurons of each area. Following septal pole ejections, labeled neurons were located in the posterolateral, extreme posterior, and posteromedial parts of both areas 28m and 28i. Mid S-T ejections produced not only a ventral, but also an anteromedial, shift in the location of entorhinal projection cells; no cells were labeled posterolaterally. After temporal dentate ejections labeled neurons occupied the most anteromedial part of these entorhinal areas. For both areas, but especially for area 28i, convergence of entorhinal efferents upon a single S-T level in the dentate gyrus occurred from neurons which lay in a dorsoventral (i.e., frontal), and to a lesser extent a rostrocaudal, plane. The efferent axes of both areas 28m and 28i thus appear to be curved and are therefore best described in three dimensions. The entorhinal axes begin in a posterodorsolateral location, wrap around the posterior cortical convexity, and end in an anteroventromedial position. The results provide a useful map for in situ exploration of entorhinodentate connections in the rat, emphasize the parallel innervation of the dentate gyrus by distinct entorhinal fiber systems, and reflect the importance of the S-T axis as a framework for interpreting hippocampal organization.

Animals↗
PubMed1994

A disposable-chamber temperature-regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital-imaging microscopy.

Utilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the [Ca2+]i transient involved in T-cell activation. We wished to extend these studies to single-cell transients using florescence digital-imaging microscopy (DIM). However, the most currently available temperature-regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual-temperature regulation system that will maintain Nunc, eight-well, coverglass-bottom, disposable chambers, and three disposable addition pipets at 37 degrees C for physiological studies on an inverted digital-imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX-inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow-cytometric measurement indicated. The correlation between flow cytometry and DIM permits the amalgamation of these results in the interpretation of studies on the regulation of T-cell activation.

Calcium↗
PubMed2006

Toward complete laser ablation of melanoma contaminant cells in a co-culture outgrowth model via image cytometry.

BACKGROUND: Contaminant cancer cells in autologous transplant tissue can cause relapse and the rates are unknown. A method capable of removing all contaminant cells with a high probability detected by cytomic analyses would be useful. Neither 100% cell purging nor techniques for measuring the probability of success have been developed. Here, we report a method for removing 100% of the cells under ideal staining conditions and quantify the probability of success. METHODS: Laser ablation was combined with previously reported automated microscopy to purge contaminant cells and evaluate 100% ablation in a co-culture model of prestained mouse melanoma cells mixed with mouse NIH-3T3 cells. Melanoma passage efficiency was measured by: (1) micropipetting single cells into microtiter wells and (2) ablating all but one melanoma cell in co-cultures. RESULTS: (74 +/- 5)% of single melanoma cells pipetted into microtiter plate wells divided at least once. With ablation of all but one contaminant cell in co-cultures, melanoma dominated in (62 +/- 8)% cultures in 21 days. With 100% ablation in six additional experiments, no melanoma outgrowth was observed, giving a >99.1% probability that all contaminant melanoma cells were purged. CONCLUSIONS: We successfully demonstrated a model for complete ablation within a defined probability using automated high-content image cytometry with ideal staining conditions. The results show that the instrumentation is capable of delivering 100% ablation at a defined probability and establishes the basis for further studies with clinical models wherein pretherapeutic cytomic analyses of unique cellular expression and/or morphological characteristics will be key for contaminant cancer cell identification.

Animals↗
PubMed1989

Mites in pulmonary cytology specimens.

Mites were observed in 55 pulmonary specimens examined routinely over a 4.5-yr period. The 55 specimens included 33 spontaneous and 12 aerosol-induced sputum samples, nine bronchial washings, and one fine-needle aspirate. Two specimens contained only fragments of mites; however, in most cases the mites were relatively intact. Many were identified as Tyrophagus sp, but a few were definitely not Tyrophagus sp. Although only a single mite was noted in most instances, eight specimens contained more than one, with the maximum being six. No patient was found to have a mite in more than one specimen. Mite eggs were found in five of the specimens containing mites. One specimen contained adults and a larval form. Mites appeared to be surrounded by acute inflammatory cells in 13 cases. Specimens were processed by the Saccomanno blending technique with smears prepared by pipetting a portion directly onto a slide or by utilizing a cytocentrifuge. No mites were found in nonpulmonary specimens. Both a seasonal variation and an interobserver variability were found. Mites often resemble specimen contaminants, such as food, and may be overlooked on routine screening. Mites are known to represent or produce a respiratory allergen, but the significance of their presence in pulmonary cytology specimens has not been determined.

Adult↗
PubMed1994

Specific, sensitive and accurate quantification of albumin, retinol binding protein and transferrin in human urine and serum by zone immunoelectrophoresis assay (ZIA).

For zone immunoelectrophoresis assay (ZIA) glass tubes, ID 2 mm and 90 mm high, are filled to 2/3 with buffer containing agarose and antibodies against the protein to be quantified, each sample being pipetted on top of separate agarose gel rods. On electrophoresis at 35-150 V for several hours, the sample proteins enter the gel with resultant immunoprecipitates, visualized by staining. The extension of each immunoprecipitation zone from the upper gel surface (measured with a ruler) is directly proportional to the amount of protein in each sample and can easily be quantitated by comparison with a linear calibration curve. ZIA can be used for quantification of several proteins in blood serum and plasma as well as in urine, as is illustrated for albumin, retinol-binding protein (RBP) and transferrin. The recovery of the pure proteins added to urine is often close to 100%. ZIA has many advantages: (i) simple apparatus and procedure (no gel punching nor cooling), (ii) minimal antiserum consumption (1 mL may allow > 1000 assays), (iii) electrophoresis can be performed within a few hours or overnight, (iv) low coefficient of variation (often < 4%), (v) linear calibration curves, (vi) low detection limit (< 20 ng/mL), (vii) wide concentration ranges, (viii) no kits nor unique antisera preparation are required, and (ix) good agreement with the results from other methods.

Albuminuria↗