Pathological Human Sera Research Articles

Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest.

A Normal Organ Antigen and Its Corresponding Antibodies in Pathological Human Sera

Previous studies on the appearance in pathological human sera of antibodies to an antigen of normal mammalian organs were continued. In gel precipitation reactions, antibodies combining with saline extracts of mammalian organs were found in 20 of 63 cancer sera and in 4 of 15 syphilis sera, but only in 3 of 56 other pathological sera. Furthermore, an identical antigen was demonstrated in 5 of 58 pathological sera. The antigen under study did not belong to any group of known heterophile antigens and it was devoid of organ and tissue specificity.

1,3-Dichloro-5,5-dimethylhydantoin as Triggering Reagent for Chemiluminescent Biochip Based on the Luminol-H2O2 Reaction

A free chlorine donor, 1,3-dichloro-5,5-dimethylhydantoin (DDH), was used for the first time as triggering reagent for the chemiluminescent detection of enzymatically produced hydrogen peroxide in the presence of luminol. A biochip has been developed based on glucose oxidase (GOx) activity as model enzyme. Photopolymer-based sensing layer entrapping luminol immobilized on anion exchanger beads (DEAE-Sepharose) and GOx immobilized on chelating beads (IDA-Sepharose) were spotted as an array of 800 µm spots at the surface of a PVC substrate. Each spot could be incubated separately in a 5 µL solution of DDH and glucose or H2O2, and the different light intensities measured simultaneously on the entire array with a CCD camera. The CL reaction conditions were optimized in terms of DDH concentration: 200 µM and pH: 8.5. Using the selected conditions and with a 3 min measurement duration, hydrogen peroxide could be detected in the range 5 µM–1 mM with a limit of detection of 5 µM. Applied to the glucose determination, the biochip exhibited good performances with a limit of detection of 7.5 µM (Signal/Noise ratio of 3) and a detection ranging over three decades at least. Used for the measurement of glucose concentration in normal and pathological human serum, no interferences problem were encountered for serum dilution of 1:100. In those conditions, the measured concentrations compared well with the expected reference values.

Semiautomated microtiter plate assay for monitoring peptidylprolyl cis/trans isomerase activity in normal and pathological human sera

An UV/VIS spectrophotometric assay technique was developed that was able to routinely monitor peptidylprolyl cis/trans isomerase (PPIase) activity of biological fluids in 96-well microtiter plates. The assay, based on monitoring the cis-to-trans isomerization of succinyl-Phe-cisPro-Phe-4-nitroanilide as substrate in a chymotrypsin-coupled reaction, yields a throughput of 96 samples per 30 min. The assay's capacity was exemplified by dealing with the PPIase activity in several normal and pathological human sera. Reference values of 151 healthy subjects (83 females, 69 males, 17 to 60 years old) were found to possess significant sex-specific differences. PPIase activity factor K of the sera was significantly greater in males (5th, 50th, 95th percentiles: 17, 36, 55 K) than females (14, 30, 48 K). PPIase activities of sera from healthy donors (n = 151) were significantly higher (Mann-Whitney rank-sum test P < 0.0001) than those of patients (n = 47). PPIase activity in serum samples stored at 4 degrees C was stable for at least 20 h.

Kinetic behaviour of the malonaldehyde-thiobarbituric acid reaction. kinetic-fluorimetric determination of malonaldehyde in human serum

For the first time a fluorimetric-kinetic method for the determination of malonaldehyde (MLD) is proposed. The fluorescence emission of the thiobarbituric acid (TBA)-MLD reaction product has been monitored at 553 nm (excitation wavelength 515 nm), and the instrumental and experimental variables have been studied. A selective and fast kinetic-fluorimetric determination of malonaldehyde is proposed. The sensitivity of the method is very high, the detection limit is 0.30 ng ml −1, the application range is between 1.1 and 50 ng ml −1, and the determination time per sample is 500 s. The kinetic-fluorimetric method has been applied in the determination of two types of pathological human serum: rheumatic and hyperlipemic serum. In both cases, perchloric acid is used to precipitate the proteins.

Separation of serum bilirubin species by micellar electrokinetic chromatography with direct sample injection

Four major bilirubin species in serum were separated by micellar electrokinetic chromatography with 25 m M sodium dodecyl sulfate (SDS) and 20 m M sodium tetraborate—boric acid buffer at pH 8.5. Due to the solubilization of the serum proteins by the SDS micelles, serum samples were injected directly into a 50 cm × 75 μm I.D. fused-silica capillary and complete separation of the four bilirubin species was accomplished within ca. 10 min without extensive sample pretreatment. Detection was performed by absorbance at 450 nm and average limit of detection was in the 6.0 μ M concentration range. The usefulness of this method was demonstrated for the separation and detection of a number of bilirubin species present in pathological human serum samples.

Diagnostic aspects of alkaline phosphatase: separation of isoenzymes in normal and pathological human serum by high-performance liquid chromatography

Diagnostic aspects of alkaline phosphatase: separation of isoenzymes in normal and pathological human serum by high-performance liquid chromatography

Distinctive expression of neoantigenic C3(D) epitopes on bound C3 following activation and binding to different target surfaces in normal and pathological human sera

Binding of C3 to sheep erythrocytes in a serum-free milieu (EAC14 oxy2, EAC142) has previously been shown to mimic the antigenic change that occurs upon denaturation of C3 in sodium dodecyl sulphate (SDS), whereby neoantigenic C3(D) epitopes are exposed. The present paper deals with C3 bound to various target surfaces which are known to modulate the functional properties of C3 in different ways. Bound C3 fragments on serum-treated human aggregated gammaglobulin, zymosan, rabbit and sheep erythrocytes, and on circulating immune complexes isolated from sera of patients with rheumatoid arthritis and systemic lupus erythematosus, were shown to be mainly in the iC3b form. By RIAs, employing polyclonal antibodies, the range of C3(D) antigenic epitopes of 125I-labelled SDS denatured C3 expressed by the particle-bound iC3b was monitored. The physiologically bound iC3b on all tested particles expressed wide ranges of C3(D) epitopes and each type of particle-bound C3 exposed its individual range. By competition ELISA specific C3(D)α epitopes were monitored, employing monoclonal antibodies. A distinct difference in the expression of these epitopes was observed in iC3b bound to various test particles in the presence of normal serum and in iC3b present on circulating immune complexes from pathological sera. Considering that the neoantigenic C3(D) epitopes have been shown to be associated with different functions of C3, the distinctive antigenic expression of each type of serum-treated particle might reflect different functional forms of the protein.

Unusual Precipitins in Pathological Human Sera

In conducting double diffusion gel precipitation tests, unexpected, strong precipitation reactions were noticed between a serum originating from a renal graft recipient and many pathological human sera. The highest frequency of positive reactions were produced by sera of patients with infectious mononucleosis (83%), chronic Epstein-Barr virus infections (72%), rheumatoid arthritis (57%), lepromatous leprosy (57%), and AIDS (44%). The precipitin in these pathological sera was identified as an antibody of IgM variety and the precipitinogen in the transplantation serum was shown to be a thermostable component with beta-globulin mobility. No explanation of the nature of reactions observed can be given at present.

A Nonimmunoglobulin Precipitin to Tissue Extracts in Pathological Human Sera

An alpha-globulin component was noted in pathological human sera, which produced gel precipitation reactions with extracts of human and animal liver. The highest incidence of the precipitin was found in malaria (95%), renal graft rejection (81%), and rheumatoid arthritis (57%). The precipitinogen was thermostable and ethanol soluble; of two precipitation lines formed by this component, one merged into identity reaction with a line produced by commercial lecithin of bovine origin. The possible diagnostic application of the reactions noted was considered.

Enzyme immunoassay for IgG rheumatoid factor combining with homologous IgG.

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')2 of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%-85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.

A specific enzyme-linked immunosorbent assay (ELISA) procedure for detection of heterophile Hanganutziu and Deicher (HD) antibodies

A specific, relatively sensitive, quantitative and standardized enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of heterophile Hanganutziu and Deicher (HD) antibodies which are occasionally elevated in pathologic human sera. The HD antigen-active molecule used for the assay was a ganglioside ( N-glycolylneuraminyllactosylceramide, abbreviated as NeuGc-LacCer) previously purified from horse erythrocyte membranes. The test used antigen-coated plastic microtiter plates and anti-human immunoglobulin G (IgG, Fab fragment) conjugated with alkaline phosphatase. Fifty-four normal human sera gave ELISA values ranging from −2 to 2%. Random sera from hospitalized patients were first screened by the horse erythrocyte hemagglutination (HA) test, whereby 5.7% (76 cases) gave abnormal HA titers of 128–4096 compared to titers in normal sera equal to or less than 64. Ninety-seven % of the patients' sera gave abnormal ELISA values (3–200%). They were classified into 3 groups: cancer (42 cases), infection (10 cases), and others (24 cases). The potential value of this ELISA method is discussed.

Non-Organ Specific Thermostable Tissue Antigens

Thermostable ethanol insoluble antigens (BE antigens) were identified that occur in all human tissues and human dispersed cells, but which are absent from normal human serum. In contradistinction to previously described organ-specific BE antigens, these antigens were referred to as non-organ-specific tissue antigens (NOST). Antisera obtained by immunization of rabbits with BE preparations of human organs had been selected for being devoid of any organ specificity and had been absorbed by BE preparations of pooled human serum. Such antisera could be used as reagents for detection of NOST BE antigens in pathological human sera. Inhibition of enzyme immunoassay proved to be a convenient procedure for these studies. Inhibition of 35% or more was only exceptionally noted in studying sera of normal subjects 20-40 years old, but inhibition exceeding 35% (positive results) was noted in 48% of sera from subjects at the age of 70 years or more. A high incidence of positive results was also encountered in sera of patients with end-stage renal disease (30%), renal graft recipients (18%), and in patients with lymphoma or leukemia (44%), but interestingly enough, no positive tests were noted in patients with lepromatous leprosy.

Antiglycolipid antibodies in normal and pathologic human sera and synovial fluids.

Antiglycolipid antibodies were measured in normal and pathologic sera and synovial fluids by means of a modified microplate method of complement-mediated immune lysis of fluorescent dye-trapped liposomes. All sera of normal subjects had antibodies against globopentaosylceramide (IV3 GalNAcGbOse4Cer), ganglioside GM1, gangliotriaosylceramide, gangliotetraosylceramide, and galactosylneolactotetraosylceramide antigens. Most sera of normal subjects had antibodies against lactotriaosylceramide, N-glycolylneuraminosyl-neolactotetraosylceramide (NeuGcnLcOse4Cer), GM3 ganglioside with N-glycolylneuraminic acid (NeuGcGM3) and GD1a antigens. Differences of titers against IV3GalNAcGbOse4Cer, neolactotetraosylceramide, NeuGcGM3 and NeuGcnLcOse4Cer antigens were observed between sera of normal subjects and pathologic sera from cases of leukemias, lymphomas, several autoimmune diseases and liver diseases.

Quantitative liquid-chromatographic estimation of bilirubin species in pathological serum.

Earlier we described a "high-performance" liquid-chromatographic procedure for separating the four distinct fractions of bilirubin (unconjugated, monoconjugated, diconjugated, and protein-bound) in pathological human serum (J. Chromatogr. 226: 391-402, 1981). We have modified the prechromatography precipitation of the serum globulins required in that method and have measured the bilirubin content of the precipitate spectrophotometrically. On average, the precipitate contained less than 10% of the total bilirubin in the serum samples. Adding the value obtained for the precipitate to that obtained by chromatography for the individual bilirubin fractions gave an estimate of the concentration of the total bilirubin in the sample. For 357 samples from 132 patients, this total value correlated well with that obtained by the Jendrassik-Gróf diazo procedure (slope = 1.00; r = 0.995, linear least-squares fit). The CV for the total and fractional bilirubin measurements was, on average, less than or equal to 5% for pathological sera. Serum sampled at different times from the same patient showed significant changes in the distribution of bilirubin among the four fractions.

Clinically Significant Effect of Protein Concentration on Ion-Selective Electrode Measurements of Ionised Calcium

Others, using an Orion SS-20 ionised calcium analyser, noted that the ionised calcium concentration of a native serum sample was 8% greater than that of its ultrafiltrate. The experiments described here, using a Nova 2 ionised calcium analyser, confirmed a positive protein interference which was greater for human albumin than for IgG. Uncharged dextran showed no positive interference but dextran sulphate, which is highly charged and binds calcium, showed a large effect. Thus the interference is related to macromolecular charge. Dialysis experiments with normal and pathological human serum samples indicated that the ionised calcium of diffusible plasma water was overestimated by an average of 9.6% at an albumin concentration of 40 g/l and by 4.8% at 20 g/l. It is concluded that the measurement of ionised calcium with existing analysers can be clinically misleading in patients with abnormal plasma proteins. Hypocalcaemia is likely to be overdiagnosed and hypercalcaemia underdiagnosed in the presence of hypoalbuminaemia.

Detection of antibodies to gangliosides in pathologic human sera. Serum-sickness type heterophile antibodies.

The production of heterophile antibodies in sera of patients receiving therapeutic injections of foreign serum was first described by HANGANUTIZIU (1924) and DEICHER (1926). Thereafter, these antibodies were called serum-sickness or H-D antibodies. The antibodies differ in specificity from other kinds of heterophile antibodies, such as FORSSMAN (1911) antibodies and PAUL-BUNNELL (1932) antibodies (P-B antibodies) of infectious mononucleosis. H-D antibodies react with erythrocytes and sera of various animal species (horse, sheep, ox and rabbit) and are absorbed by sediment of guinea-pig kidney homogenate (DAVIDSON and WALKER, 1935). Recently, “H-D antibodies” were detected in sera from almost of all patients who received γ-globulin fraction of goat anti-human thymocyte serum (PIROFSKY, RAMIREZ-MATEOS and AUGUST (1973) and also in sera from some patients, suffering from various diseases, who had never received a therapeutic injection of foreign serum (KASUKAWA et al., 1976). The nature of H-D antigen was unknown, except for some properties: heat-stable, extractable with hot ethanol (SCHIFF, 1937) and precipitable by 75% ethanol solution (KASUKAWA et al., 1976). Our previous studies (HIGASHI et al., 1977) succeeded in the isolation of H-D antigen from bovine and equine erythrocytes and demonstrated that this antigen is a ganglioside with N-glycolylneuraminic acid (GcNeu). The antigen of equine erythrocytes was identified as GcNeu-hematoside (YAMAKAWA and SUZUKI, 1951), and the antigen of bovine erythrocytes was identified as GcNeu-sialosylparagloboside (WIEGANDT and SCHULZE, 1969). The structures of both the gangliosides are shown in Table 1.

Immunoglobulins G, A and M in normal and pathologic human sera determined with the spot immunoprecipitate assay (SIA) in multi-assay plates.

A simple rapid spot immunoprecipitate assay (SIA) is presented. The antigen--antibody reaction takes place in a field of alternating current; small amounts of specific antisera are used, and the influence of diffusion or electromobility of the antigen is avoided. Determinations of IgG, IgA and IgM in sera from healthy individuals correlated highly significantly with radial immunodiffusion (RID) results. The reaction conditions in SIA, particularly the availability of a sufficiency of specific antibodies, may permit more accurate estimates of monoclonal forms of Ig than in RID, where the combination depends on diffusion of one reactant. The precision of SIA quantitation of IgG and IgA was within +/- 10% and +/- 13% for IgM. Working ranges of the standard curves were from about 25--150 ng for G and A and 75--300 ng for IgM; least squares regression coefficients for four sets of standards on three SIA plates were 0.995 for IgG, 0.969 for IgA, and 0.986 for IgM. IgA at 7.5 ng in a 2.5 mg/l dilution was at the lower limit of demonstrability. Photometric registrations and estimates made by visual inspection compared highly significantly (P less than 0.001), differing by +/- 3%.

Immunological assay for a carboxyterminal peptide of the fibrinogen Aα-chain in normal and pathological human sera

A radioimmunoassay has been established to determine the reactive antigen related to human fibrinogen CNBr peptide F-CB3 in human serum. The amount of antigen found in healthy subjects was in the range of 1 to 10 pmoles per milliliter. A 10 to 30 fold increase in antigen level could be demonstrated in a group of patients with various haemorrhagic disorders and in patients having undergone cesarean section. Plasma levels of fibrinogen and of soluble fibrin monomer complexes were also increased in these patients.

Antigen of “serum sickness” type of heterophile antibodies in human sera: Identification as gangliosides with N-glycolylneuraminic acid

Antigen of “serum-sickness” type of heterophile antibodies in pathologic human sera was purified from equine and bovine erythrocyte stroma. The chemical nature of this antigen was glycosphingolipids with N-glycolylneuraminic acid. The antigen of equine erythrocytes was identified as hematoside with N-glycolylneuraminic acid, GlNeu(α, 2–3)Gal(β, 1–4)Glc(β,1-1) ceramide and the antigen of bovine erythrocytes was N-glycolylneuraminyl-paragloboside, GlNeu (α,2–3)Gal(β,1–4)GlcNAc(β,1–3)Gal(β,1–4)Glc(β,1-1) ceramide. The results indicate that “serum-sickness” antibodies react with a common disaccharide moiety of non-reducing end of the both glycosphingolipids.