Human Prealbumin Research Articles

A fluorescence immunochromatographic assay for rapid and sensitive detection of human prealbumin in serum

Schematic of the sandwich FMs-LFIA test strip.

Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications.

BackgroundNanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.MethodsWe constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.ResultsA large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 109 CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL.ConclusionThis proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.

Development of nanobody-based flow injection chemiluminescence immunoassay for sensitive detection of human prealbumin

Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000μgL−1 with a detection limit of 0.01μgL−1. The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings.

Electrochemiluminescence of CdSe quantum dots for immunosensing of human prealbumin

We describe a non-labeled electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) for the detection of human prealbumin (PAB, antigen). The immunosensor was fabricated by layer by layer coupled with nanoparticle-amplification techniques. After two gold nanoparticle layers were self-assembled onto the gold electrode surface through cysteamine, anti-PAB (antibody) were conjugated with –COOH groups of both the CdSe QDs and cysteine, which were linked to the gold nanoparticle-modified electrode. The principle of ECL detection was that the immunocomplex inhibited the ECL reaction between CdSe QDs and K 2S 2O 8, which resulted in the decrease of ECL intensity. On the one hand, the immunocomplex increased the steric hindrance. On the other hand, the immunocomplex maybe inhibit the transfer of K 2S 2O 8 to the surface of the CdSe QD-electrode. The PAB concentration was determined in the range of 5.0 × 10 −10 to 1.0 × 10 −6 g mL −1, and the detection limit was 1.0 × 10 −11 g mL −1. The developed CdSe QD-based ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which could be applied in more bioanalytical systems.

Protein standardization I: Protein purification procedure for the purification of human prealbumin, orosomucoid and transferrin as primary protein preparations.

A procedure for the purification of human prealbumin, orosomucoid and transferrin as primary protein preparations has been developed. The procedure describes in detail the chemicals, the fractionation equipment and the purification of the three proteins from the same starting material (a 90-donor plasma pool). The fractionation steps involve methods like salting out, various anion and cation exchange chromatographies, preparative electrophoresis, and finally, size chromatography. Only mild and highly reproducible fractionation methods are used in order to obtain high recoveries. All of these provide the best guarantee that no serious subfractionation has taken place. At the same time, high recoveries are a necessity for the pure protein to be representative of the same protein in vivo. The following recoveries were obtained: prealbumin 55%, orosomucoid 70% and transferrin 85%. The pure proteins are produced as liquid calibrators (primary protein preparations) dissolved in one electrolyte (0.1 mol/l KCl) and stored in sealed glass ampoules at -80 degrees C. These three pure proteins were used as primary reference preparations in the certification of the international reference preparation CRM 470.

A Proton NMR and Nuclear Overhauser Effect (NOE) Study of Human Plasma Prealbumin, Including the Development and Application to Spectral Assignment of a Combined Ring Current Shift and NOE Prediction Program

A Proton NMR and Nuclear Overhauser Effect (NOE) Study of Human Plasma Prealbumin, Including the Development and Application to Spectral Assignment of a Combined Ring Current Shift and NOE Prediction Program

Assignment of the gene for human tear prealbumin (LCN1), a member of the lipocalin superfamily, to chromosome 8q24.

Tear prealbumin, a major protein of the human tear fluid, is also present in several other human body secretions. Recently it was shown to be a member of the lipocalin superfamily, a class of proteins that function as carriers for small hydrophobic molecules. Using a genomic tear prealbumin probe, we have mapped the gene (LCN1) to human chromosome 8q24 by fluorescent in situ hybridization.

Determination of Serum Prealbumin (Transthyretin) by Competitive Enzyme Immunoassay

A competitive enzyme immunoassay (cEIA) to determine the level of serum prealbumin is described. Antiserum to human serum prealbumin conjugated with peroxidase is incubated with solid-phase–bound human serum protein in the presence of sample or standard. The results obtained by the cEIA method (Y) correlate well with those obtained by the radial immunodiffusion method (X); Y = 1.03X + 5.92, r=0.93 for N=40. The coefficent of variations (CVs) of within-run assays (N=20) from three controls (99, 198, and 394 mg/L) are 7.2%, 5.2%, and 3.5%, respectively, and the CVs of between-run assays from the same three controls for 10 days are 8.3%, 5.7%, and 4.3%, respectively. Interference studies by the multiple regression model indicate that 0.2 g/dL (2 g/L) of added hemoglobin, 12 mg/dL (208 μ mol/L) of added bilirubin, and 545 mg/dL (6.2 mmol/L) of added triglycerides do not interfere significantly ( P <.01) with the assay. The assay is rapid (1 hour) and requires only commercially available reagents. Reference ranges of serum prealbumin for infants (aged 0 to 1 year), toddlers (aged 1 to 3 years), and preschool children (aged 3 to 6 years) are 47 to 187 mg/L, 95 to 307 mg/L, and 64 to 280 mg/L, respectively. There is not a significant difference in the serum prealbumin levels between the sexes of the subjects in the age groups studied. However, the reference range for infants is significantly lower ( P <.05) than those for the other two groups.

Fluorescent derivative of cysteine-10 reveals thyroxine-dependent conformational modifications in human serum prealbumin

Fluorescence studies on the N-(iodoacetyl)- N′-(5-sulfo-1-naphtyl)ethylenediaminelabeled cysteine-10 residue of human prealbumin were carried out to detect conformational changes induced by the binding of the ligand thyroxine to the two structurally identical binding sites. A red shift of the spectrum was observed and the total change was confined to the first ligand. This was interpreted as resulting from a conformational change which increases the exposure of the fluorescent probe moiety. Thyroxine also alters the effect of the collisional quencher, acrylamide, confirming the greater exposure of the probe. This modification in structure is associated with changes in relaxation time which indicate that when thyroxine is bound there is an increase in the rotational freedom of the segment or domain of prealbumin which contains the fluorescent probe.

Effects of Human Thyroxine-Binding Globulin and Prealbumin on the Reverse Flow of Thyroid Hormones from Extravascular Space into the Bloodstream in Rabbits

A plasma fraction rich in thyroid hormone-binding globulin (hTBG, human thyropexin) was injected iv into rabbits in order to see whether thyroid hormone concentrations in plasma would increase by return of T3 and T4 from the extravascular space. For this purpose, both [125I]T3 and [131I]T4 were simultaneously injected. After 1 h, or after 16 h in another series of experiments, 50 mg hTBG were injected iv. Thereafter, the mean radioactivity of both [125I]T3 and [131I]T4 in the plasma rose, and reached its peak 20-30 min after hTBG injection; [125I]T3 and [131I]T4 returned to the preinjection value slowly, after more than 3 h. When hTBG was injected 15-16 h after the radioactive hormones, the mean radioactivity of [125I]T3 reached its peak about 1 h after hTBG injection and returned to the base value after approximately 5.5 h, [131I]T4 reached its peak about 1 h after hTBG injection and returned to the base value within 12 h. After injection of hTBG, total T4 and T3 concentrations in plasma increased about 3- to 5-fold over the base values. At the same time, the percentage of both, free T4 and free T3 dropped instantly whereas absolute free T4 and free T3 values remained almost constant. After injection of 500 mg transthyretin (hTBPA), a similar flux of [125I]T3 and [131I]T4 was observed, whereas 500 mg human serum albumin were ineffective. These marked effects of injected hTBG and hTBPA on the serum levels of [125I]T3, [131I]T4, and total T3 indicate that reentry of T3 and T4 into the intravascular compartment is an important component of thyroid hormone distribution and transport. As can be anticipated from the animal experiments, the efficiency of plasmapheresis or hemofiltration methods may be improved by previous application of large doses of hTBPA or hTBG in cases of thyrotoxicosis.

A Proton Nuclear Magnetic Resonance and Nuclear Overhauser Effect (NOE) Study of Human Plasma Prealbumin, Including the Development and Application to Spectral Assignment of a Combined Ring Current Shift and NOE Prediction Program

Human prealbumin, a homotetrameric protein with an Mr of approximately 55,000 has been characterised by proton magnetic resonance spectroscopy. The pH dependences of the chemical shifts of the 4 histidine residues/subunit are unremarkable; variable temperature studies show that the protein tertiary and quaternary structure is stable to at least 80 degrees C. Assignment of a number of residues was aided by the development of a computer program, PARSNIP (Programme to Analyse Ring current Shifts and nuclear Overhauser effects (NOEs) In Proteins), which employs high resolution x-ray crystal structure atomic co-ordinates (Blake, C.F., and Oatley, S.J. (1977) Nature 268, 115-120, in the case of prealbumin) and several different predictive formalisms to calculate structure-dependent ring current-induced shifts of aromatic and methyl group protons. The program combines these with distance measurements to give plots of expected NOE difference profiles resulting from irradiation at a discrete frequency. The routine should prove applicable to many proteins whose structures have been determined but which are too large for the application of sequential residue assignment techniques. Three well resolved high field shifted methyl groups resonating at 0.18, 0.28, and 0.34 ppm are assigned to Ala-120, Leu-111, and Val-122, respectively. NOEs between these signals and the aromatics, interaromatic effects, and effects involving some of the considerable number of downfield shifted alpha-protons, have been used to identify substituents on several other residues.

Immunohistochemistry of vacuoles occurring in rat hepatocytes after retinol administration.

The vacuoles occurring in rat hepatocytes after intraportal injection of retinol (33 or 67 micrograms) were examined immunohistochemically using respective antibodies against rat albumin, human retinol-binding protein, human ceruloplasmin, human alpha 1-antitrypsin, human transferrin, and human prealbumin as representative plasma proteins. The occurrence of the vacuoles reached a numerical maximum 30 min after injection of 67 micrograms retinol, followed by a temporal decrease. Hepatocytes from control rats, which had been intraportally injected with either blood plasma diluted to 2/3 concentration or with retinol palmitate solvent (castor oil) dissolved in blood plasma, showed immunoreactive fine granules without the occurrence of vacuoles in the cytoplasm. Identical vacuoles in serial sections appeared immunohistochemically either immunoreactive or non-immunoreactive for all the antibodies used, with rare exceptions. The occurrence of several rare exceptions suggested that 2 kinds of vacuoles might be formed in different cytoplasmic compartments. A zonal distribution of vacuoles was apparent in the hepatic laminae (or acini) within the liver lobules. The vacuoles were predominantly distributed in zone 2, and to a lesser extent in zone 3 and zone 1 in that order.

4816388 Human prealbumin and related methods and products: Jean Sipe, Alexander Whitehead, Alan S Cohen, Martha Skinner assigned to Children's Medical Center Corporation

4816388 Human prealbumin and related methods and products: Jean Sipe, Alexander Whitehead, Alan S Cohen, Martha Skinner assigned to Children's Medical Center Corporation

Human chorionic prealbumin: Identification, physicochemical properties and its detection in blood serum in trophoblastic diseases

Human chorionic prealbumin: Identification, physicochemical properties and its detection in blood serum in trophoblastic diseases

Uptake of thyroxine by the perfused rat liver: implications for the free hormone hypothesis.

To investigate the mechanism by which thyroxine (T4) in plasma enters hepatic cells, we measured the rate constants for uptake of free T4 by the perfused rat liver and for dissociation of T4 from its plasma binding proteins. Quantitative autoradiography of liver lobules after perfusion with [125I]T4 indicated an apparent rat constant for removal of free T4 from the sinusoids of at least 1.1 +/- 0.2 s-1. Single-pass extraction of T4 from human serum was 10.6 +/- 1.7% at physiological flow rates (1 ml.min-1.g liver-1). Rate constants for dissociation of T4 from plasma binding proteins at 37 degrees C (determined by rapid filtration) were 0.017 +/- 0.002 s-1 for human thyroid hormone-binding globulin, 0.080 +/- 0.015 s-1 for human thyroid hormone-binding prealbumin, and greater than 0.5 s-1 for human albumin. To investigate the factors that determine the concentration of T4 within hepatic cells, we analyzed the above data together with data reported in the literature on the equilibrium-binding constants and the rate constant for cellular metabolism of T4. Analysis of all of these data using a previously published mathematical model leads to the following conclusions for the physiological state: 1) metabolism, not uptake, is rate limiting to removal of T4 from plasma by the liver; 2) binding equilibrium is present in the intrahepatic plasma; 3) intracellular T4 is in equilibrium with the free T4 pool in plasma (and maintenance of this equilibrium may be an important function of plasma thyroid hormone-binding proteins); and 4) the concentration of T4 within the liver is proportional to the concentration of free T4 in the plasma. Our data do not allow us to determine definitively whether hepatic uptake of T4 occurs only from the free T4 pool in plasma or also from the protein-bound pool by interaction of one or more of the binding proteins with the liver cell. However, mathematical analysis indicates that this distinction is irrelevant to steady-state intracellular hormone concentrations when equilibrium exists between the plasma and cytosolic pools of hormone.

Origin of the extra chromosome in trisomy 18. A study on five patients using a restriction fragment length polymorphism.

The parental origin of an extra chromosome in five patients with trisomy 18 was traced using a restriction fragment length polymorphism (RFLP) of the human prealbumin (PA) gene, localized to 18p11.1-q12.1, as a genetic marker. MspI digests of the genomic DNAs of the five patients, their parents and normal controls were hybridized with the PA-cDNA. Densitometric analysis on the gene dose of the polymorphic fragments of these patients revealed that three had originated from a maternal meiotic error. The other two patients were uninformative for the parental origin of trisomy 18. Our results indicate that nondisjunctional errors leading to trisomy 18 may occur predominantly at the maternal meiosis, consistent with the results of previous studies on the parental origin of trisomies 21 and 13.

Calcitonin-like immunoreactivity of amyloid fibrils in medullary thyroid carcinomas. An immunoelectron microscope study.

Using 3 polyclonal antisera directed against synthetic human calcitonin, we investigated at the electron microscope level the intra-or-extra-cellular fibrillar/filamentous aggregates found in 4 amyloid-rich medullary thyroid carcinomas (MTC) and in a number of other endocrine polypeptide tumours with or without demonstrable amyloid deposition. The antisera were applied by the immunogold procedure on ultrathin sections of glutaraldehyde-fixed, usually osmium-postfixed, tissues. In MTC cases, a strong labelling was present over two types of aggregates: one composed of rigid, criss-crossing fibrils 7-10 nm in diameter, suggestive of amyloid, and the other consisting of loosely arranged fibrils, 4-7 nm in width, often wavy or poorly defined. In both cases, the labelling was closely associated with that part of the sectioned fibril exposed to the antiserum. Amorphous material was sometimes present adjacent to the later aggregates, but did not bind the calcitonin antibodies. In contrast, no labelling occurred over the amyloid deposits found in two non-calcitonin-producing endocrine tumours of the pancreas, nor over the cytoskeletal filaments stored in various endocrine polypeptide tumours. The specific value of the labelling for calcitonin-like immunoreactivity was assessed by control tests, such as absorption of the antiserum by excess calcitonin and comparative use of normal serum and antisera directed against human IgG and P component. No immunoreactivity of the MTC amyloid fibrils was found using antibodies directed against katacalcin and human prealbumin. We conclude that in tumour tissues conventionally processed for electron microscopy, MTC amyloid fibrils of varying morphology can be selectively and specifically labelled for calcitonin-like immunoreactivity.

Theoretical determination of the CD of proteins containing closely packed antiparallel β‐sheets

AbstractProteins containing two closely packed β‐sheets comprise an important class of biopolymers. Rotational and dipole strengths have been determined by the excition coupling model for interacting pairs of two idealized flat β‐sheets and for the double β‐sheets of seven globular proteins: plastocyanin, human prealbumin, immunoglobulin VREI, concanavalin A, Cu,Zn superoxide dismutase, staphylococcal nuclease, and elastase. The effects of various geometrical factors on the CD spectra were investigated. Results for the idealized sheets indicate that two sheets display through‐space interactions that are large at distances of 5–7 Å and remain significant even at distances typical of the intersheet separations in globular proteins (12–15 Å). The CD spectra are sensitive to the angle (Ω) between the strand directions of the two sheets, with maximum intersheet contributions at Ω = ±45°. Both the intrastrand and interstrand twisting were determined in the seven proteins, and their effects on the calculated CD are discussed. This work represents the first theoretical CD study on the interactions of two regular protein secondary structures, including rotational strength calculations on large sections (up to 135 residues) of globular proteins.

Senile cardiac amyloidosis with myocardial dysfunction. Diagnosis by endomyocardial biopsy and immunohistochemistry.

Senile cardiac amyloid discovered at autopsy is usually regarded as an incidental finding. However, in immunohistochemical studies of autopsy material, three distinct forms of senile cardiovascular amyloid have been characterized, including a systemic form that diffusely infiltrates the cardiac ventricles. The systemic form can be identified immunohistochemically with use of antiserum to human prealbumin. We diagnosed senile systemic amyloidosis causing cardiac dysfunction in five men (57 to 72 years old) by using antiserum to prealbumin in myocardial biopsy tissue. Clinically, the five patients were indistinguishable from patients with nonsecretory immunoglobulin-derived primary amyloidosis with cardiac involvement; only immunohistochemical staining of myocardial tissue distinguished between the two entities. This distinction is important, because the treatment and prognosis of the two disorders are different. We recommend immunohistochemical staining of myocardial tissue for prealbumin in patients with biopsy-proved cardiac amyloid in whom no monoclonal immunoglobulin light chain is detectable in the serum or urine.

Cerebral amyloid angiopathy in the aged.

Cerebral amyloid angiopathy (CAA) was found in 57% of 123 autopsy brains removed from patients aged 59-101 years. The incidence of CAA increased with age. CAA was seen most frequently in the occipital cortex. Immunohistochemically, amyloid of CAA was positive for amyloid P component and negative for human AA protein and human prealbumin. The presence and severity of CAA were significantly correlated with the number of senile plaques and neurofibrillary tangles. The incidence of CAA in 17 patients with dementia of Alzheimer type (DAT) was estimated to be 88% and was significantly higher than that in 26 patients with dementia of non-Alzheimer type. CAA had a pathogenetic relationship with both brain ageing and DAT. Lobar cerebral haemorrhage was found in 3 patients with CAA of marked or moderate degree. Lobar cerebral haemorrhage in the aged and in patients with DAT suggest the presence of CAA.