sepharose and Hemolysis

Reptiles are declining worldwide yet our understanding of their immune function lags far behind other taxa. The innate immune system is the primary mode of defense in reptiles, and the serum complement cascade is its major component. We assessed serum complement activity of plasma in two closely related aquatic turtle species, the common snapping turtle (CST; Chelydra serpentina) and alligator snapping turtle (AST; Macrochelys temminckii). We used a sheep red blood cell (SRBC) hemolysis assay to assess serum complement activity. Although the antibacterial activities of the plasma of these turtle species are similar, the hemolytic activity was much stronger in CST than AST. Treatment with inhibitors of the serum complement cascade indicated differences in the mechanisms of complement activation between the turtle species. We subjected plasma from both turtle species to mannan affinity chromatography and analyzed the eluate with SDS-PAGE, which revealed that plasma from the CSTs contained only small amounts of one C-type lectin protein while the AST plasma contained high concentrations of two C-type lectins (31.0 and 35.9 kDa). Edman degradation analyses confirmed that the two AST proteins contained identical N-terminal sequences. Thus, the CST appears to rely more heavily on the alternative mechanism of serum complement activation, while the AST appears to rely more on the lectin-mediated pathway, which is a pattern recognition response to prokaryotes not activated by the SRBCs. These results are unique in that the use of serum complement pathways are generally assumed to be conserved within clades.

Topics: Animals; Complement Activation; Erythrocytes; Hemolysis; Ions; Kinetics; Mannans; Metals; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Turtles

Stings from the hydrozoan species in the genus

Topics: Acetic Acid; Animals; Bites and Stings; Cnidarian Venoms; Erythrocytes; Ethanol; First Aid; Hemolysis; Humans; Hydrozoa; Sepharose; Sodium Bicarbonate; Solutions; Treatment Outcome; Urine

Two novel magnetic agarose bead based assays have been developed to measure complement component C5 interaction with C3b and the Factor I Modules (FIMs) of C7. One innovation was to couple C3b onto the magnetic agarose bead using the alternative pathway C3 convertase, which resulted in a linkage of the ligand by a covalent ester bond. A second innovation was to employ nickel ion charged N,N,N'-tris(carboxymethyl)ethylene-diamine-magnetic agarose to capture recombinantly prepared C7 FIMs that were expressed with an oligo-histidine linker followed by an acidic domain that provided a spacer enabling the C7 modules exposure to C5. Detection was brought about by peroxidase coupled to C5. Both assays exhibited adequate statistics suitable for screening. As examples of the utility of these new methods, we chose to examine influence of natural products on C5 interaction. Fucoidan and β-glucans were observed to inhibit C3b-C5 interaction, and dextran sulfate was similarly active; however, rosmarinic acid had no measurable effect. In contrast only β-glucans from two species of macrofungi were able to interfere with interaction of C5 with the FIMs of C7.

Topics: beta-Glucans; Complement Activation; Complement C3-C5 Convertases; Complement C3b; Complement C5; Complement C7; Complement Hemolytic Activity Assay; Complement Inactivating Agents; Dextran Sulfate; Hemolysis; Humans; Immunologic Techniques; Iron; Magnetics; Polysaccharides; Protein Binding; Sepharose

The influence of sulfated polysaccharides (λ-, κ-, and κ/β-carrageenan and porphyran) - on platelet activation was studied. Carrageenans were much weaker inhibitors of a coagulation process than heparin, while porphyran had not that effect. Results of the aPTT and PT assays suppose that carrageenans affected mostly intrinsic pathway of coagulation, while their effect on the extrinsic pathway is extremely low (λ and κ/β) or absent (κ, LMW derivative of κ-carrageenan). λ-Carrageenan was the most potent anticoagulant agent in TT, aPTT, PT, and anti-factor Xa activity. This sample was also the strongest inhibitor of collagen-induced platelet aggregation in PRP. Generally, the correlation of anticoagulant and antithrombotic action in PRP is preserved for carrageenans but not for heparin. Carrageenans and porphyran affected platelet adhesion to collagen by influencing glycoprotein VI. Low molecular weight κ-carrageenan had a similar effect on platelet adhesion mediated with both major collagen receptors: integrin α2 β1 and glycoprotein VI as native polysaccharide had. Carrageenans resulted in activation of platelets under platelet adhesion mediated by integrin αIIb β3 with less degree than heparin. The least sulfated κ/β-carrageenan that possessed an inhibiting effect on thrombin- and collagen-induced aggregation of washed platelets and on the PT test but it had no significant effect on TT was the weakest promoter of integrin αIIb β3 mediated platelet activation. In summary, our study showed that the polysaccharide action was complex, since it depended on its molecular mass, sulfation degree, and monosaccharide contents (3,6-anhydrogalactose).

Topics: Animals; Blood Coagulation; Blood Platelets; Carrageenan; Cattle; Collagen; Erythrocytes; Factor Xa; Fibrinogen; Hemagglutination Tests; Hemolysis; Humans; Partial Thromboplastin Time; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Polysaccharides; Prothrombin Time; Rhodophyta; Sepharose; Thrombin Time

Biodegradable laminated polymer composites of agarose (A), gelatin (G) and hydroxyapatite (HAp) having 3D network of interconnected pores (1-500 μm) were fabricated without using cross linking agents. The incorporation of HAp to A, G and AG had considerable influence on the swelling behaviour, drug release and haemolytic activity. A-HAp scaffolds demonstrated interconnected porosity with extended drug release. G-HAp scaffolds possessed enhanced mechanical property. AG-HAp scaffolds exhibited extended drug delivery, haemocompatibility and efficacy against Gram positive bacteria compared with G-HAp. Hence, AG-HAp composites could be used us a scaffold for tissue engineering and drug delivery system. This method provides non toxic, versatile and cost effective biodegradable scaffolds which could be used for various biomedical applications.

Topics: Amoxicillin; Biocompatible Materials; Cross-Linking Reagents; Drug Delivery Systems; Durapatite; Elastic Modulus; Freeze Drying; Gelatin; Hemolysis; Microbial Sensitivity Tests; Porosity; Sepharose; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus; Tensile Strength; Tissue Engineering; Tissue Scaffolds; X-Ray Diffraction

Peptide based gene carriers are among the most promising non-viral vectors for gene delivery to eukaryotic cells. We have engineered a new fusion peptide using recombinant technology with the purpose of overcoming the cell barriers to gene delivery. A His- tagged multi-domain peptide was expressed in Escherichia coli BL21 (DE3) pLysS and purified using Ni-NTA resin. The fusion peptide is composed of two repeats of truncated histone H1 peptide to condense pDNA, a fusogenic peptide to disrupt endosome membranes and a nuclear localization signal to enhance translocation of pDNA towards nucleus. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with average sizes of 200 nm. The fusogenic peptide in the vector structure also showed membrane disruptive effect in the endosomal pH. Overall, the transfection efficiency of the vector demonstrated that it holds great promise as a nontoxic and effective non-viral gene carrier.

Topics: Animals; Chloroquine; CHO Cells; Cloning, Molecular; Cricetinae; Cricetulus; Deoxyribonucleases; DNA; Drug Delivery Systems; Drug Design; Electrochemistry; Escherichia coli; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; Hemolysis; Light; Particle Size; Plasmids; Recombinant Proteins; Scattering, Radiation; Sepharose; Transfection

Complement plays an important role in the immune attack against invading microorganisms. However, blood-contacting medical device biomaterials lacking specific complement inhibitors, with free hydroxyl and/or amino groups, or with absorbed antibodies, may inappropriately activate complement. Inappropriate activation by either the antibody-mediated classical or the antibody-independent alternative pathway may have well-known acute or poorly understood chronic effects on the host or device. This article describes methods for screening biomaterials for functional whole complement activation using normal human serum, or specifically for alternative pathway activation using C4-deficient guinea pig serum, or for classical pathway activation using both sera in combination. Detailed protocols are available as standard methodologies from the American Society for Testing and Materials (ASTM F1984, ASTM F2065, and ASTM F2567). Results obtained with these functional tests are confirmed by detection of classical and alternative pathway-specific markers C4d and Bb, respectively. These methods demonstrate dose and time-course activation of complement to beaded agarose, cellulose acetate, and purified alginate as examples of biomaterials. Significant difference in a functional endpoint denoting specific complement pathways is an appropriate screening method for complement activation by medical device biomaterials which might result in adverse events when the device is implanted or contacts human blood.

Topics: Alginates; Animals; Biocompatible Materials; Cellulose; Complement Activation; Complement C4b; Complement Factor B; Complement Pathway, Alternative; Glucuronic Acid; Guinea Pigs; Hemolysis; Hexuronic Acids; Humans; Materials Testing; Peptide Fragments; Rabbits; Sepharose; Serum; Time Factors; Zymosan

Due to the increasing numbers of reported clinical cases of complement deficiency in medical centers, clinicians are now more aware of the role of the complement system in the protection against infections caused by microorganisms. Therefore, clinical laboratories are now prepared to perform a number of diagnostic tests of the complement system other than the standard 50% hemolytic component assay. Deficiencies of alternative complement pathway proteins are related to severe and recurrent infections; and the application of easy, reliable, and low-cost methods for their detection and distinction are always welcome, notably in developing countries. When activation of the alternative complement pathway is evaluated in hemolytic agarose plates, some but not all human sera cross-react to form a late linear lysis. Since the formation of this linear lysis is dependent on C3 and factor B, it is possible to use late linear lysis to routinely screen for the presence of deficiencies of alternative human complement pathway proteins such as factor B. Furthermore, since linear lysis is observed between normal human serum and primary C3-deficient serum but not between normal human serum and secondary C3-deficient serum caused by the lack of factor H or factor I, this assay may also be used to discriminate between primary and secondary C3 deficiencies.

Topics: Animals; Complement C3; Complement Hemolytic Activity Assay; Guinea Pigs; Hemolysis; Humans; Immunocompromised Host; Immunodiffusion; Sepharose

We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B-depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti-C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.

Topics: Absorption; Animals; Complement C1 Inactivator Proteins; Complement C3 Convertase, Alternative Pathway; Complement C3b; Complement Factor B; Complement Factor D; Complement Inactivator Proteins; Complement Pathway, Alternative; Egtazic Acid; Elapid Venoms; Erythrocytes; Glycoproteins; Hemoglobinuria, Paroxysmal; Hemolysis; Humans; Microspheres; Peptide Fragments; Rabbits; Sepharose

Radioimmunotherapy using radiolabeled antitumor antibodies (RAA) is limited by the toxicity of unbound antibodies in the circulation. Removal of excessive antibodies by affinity-adsorption could therefore allow the administration of increased dosages of RAA while decreasing their adverse effects. Recently, avidin-agarose (AA) minicolumns were used in animal experiments for the removal of biotinylated antibodies from whole blood exploiting the high affinity binding of biotin to avidin (pK 1015 M-1). This study was performed to evaluate the ex vivo biocompatibility of AA minicolumns with human blood. Ten ml AA minicolumns were perfused online ex vivo in the single pass mode with fresh blood from 8 healthy donors at a flow rate of 6.25 ml/min. The anticoagulation consisted of 0.5 IU heparin plus 0.0-2.1 mg citrate per ml of blood. In Part 1 of the study (40 min perfusion, n = 4), the optimal anticoagulation was found to be 0.5 IU heparin plus about 1 mg citrate per ml of blood. In Part 2 of the study, four 80 min test-runs were performed. No signs of hemolysis were found, and the thrombogenicity of the AA gel was negligible. Cell counts and column inlet pressures remained constant; toward the end of the 80 min test-runs, some activation of blood cells (elastase, beta-thromboglobulin), the complement system (C3a, C5a) and the plasmatic coagulation (thrombin-antithrombin complex) was detectable. A moderate initial bradykinin release rapidly subsided to very low levels. In summary, AA minicolumns showed good biocompatibility upon contact with human whole blood and merit further investigation in a closed-loop system for a potential application of direct tumor antibody removal by hemoperfusion.

Topics: Adsorption; Antibodies; Antibodies, Neoplasm; Anticoagulants; Antithrombin III; Avidin; beta-Thromboglobulin; Biocompatible Materials; Biotin; Blood Cell Count; Bradykinin; Chromatography, Affinity; Citrates; Complement C3a; Complement C5a; Hemolysis; Hemoperfusion; Heparin; Humans; Ligands; Pancreatic Elastase; Peptide Hydrolases; Pressure; Radioimmunotherapy; Sepharose; Thrombosis

There are several methods for the detection of haemolytic activity in campylobacters. However, we found the haemolytic effect of campylobacters on conventional blood agar plates to be variable, inconsistent and difficult to interpret. Blood agarose plates showed campylobacter haemolytic activity more clearly. The incubation conditions (temperature and gaseous) appear to be important for the expression of this activity. Ninety four percent of the Campylobacter isolates examined were found to be haemolytic by the microplate assay with minimal haemolytic units that ranged from 1 to 64. Haemolytic activity was detected only from live bacterial cultures and not from any of the 50 bacterial culture supernates, which suggests that campylobacters may possess a cell-associated haemolysin. The identification of such haemolytic activity in a large number of campylobacters (94%) suggests its potential role as a virulence factor in campylobacter gastroenteritis.

Topics: Animals; Campylobacter; Cattle; Culture Media; Hemolysis; Hot Temperature; Humans; Polymyxin B; Rabbits; Sepharose; Sheep; Virulence

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system in Escherichia coli was used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP-Sepharose. The oligomer was folded by one-step dilution in 20 mM ethanolamine/1 mM EDTA supplemented with 1 mM GSH/0.5 mM GSSG. The folded material was processed to a concentrated (> 20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1-3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 x 110 A. SCR(1-3) has an unusual CD spectrum exhibiting a broad maximum at 220-230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.

Topics: Amino Acid Sequence; Animals; Circular Dichroism; Consensus Sequence; Escherichia coli; Gene Expression; Hemolysis; Humans; In Vitro Techniques; Inclusion Bodies; Molecular Sequence Data; Peptide Fragments; Protein Folding; Receptors, Complement 3b; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Sepharose; Sheep; Ultracentrifugation

Human serum was found to contain natural antibodies to the egg-white glycoprotein avidin. Of 270 samples tested, all contained antibodies to different extents, mainly of the IgG and IgM classes. Anti-avidin antibodies could be isolated by affinity chromatography.

Topics: Avidin; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Hemolysis; Humans; Immunity, Innate; Immunoglobulin G; Immunoglobulin M; Sepharose

A gel consisting of agarose and oxyhemoglobin (OxyHb) was developed so that, when placed in the subarachnoid space, OxyHb would be slowly released, simulating lysis of erythocytes after subarachnoid hemorrhage. The system was used to investigate the importance of reactions mediated by free radicals in the genesis of OxyHb-induced vasospasm in monkeys. Seventeen monkeys were randomly assigned to have subarachnoid placement, on Day 0, of one of the following: 1) agarose gel alone (n = 2); 2) agarose plus OxyHb (n = 3); 3) agarose plus OxyHb plus intrathecal administration of superoxide dismutase and catalase (n = 6); and 4) agarose plus OxyHb plus intrathecal administration of placebo (n = 6). Vasospasm was assessed by comparison of angiograms performed on Day 0 and 7 days after subarachnoid placement of compounds, and by electron microscopy. OxyHb alone caused significant reduction in the diameter of the middle cerebral artery (40 +/- 8%, P less than 0.005, paired t test), which was associated with ultrastructural damage to smooth muscle. Treatment with superoxide dismutase plus catalase or with placebo attenuated vasospasm of the middle cerebral artery, although significant narrowing persisted in both groups (27 +/- 12% and 26 +/- 13%, respectively, P less than 0.05, paired t test). Analysis of variance showed no difference in the degree of vasospasm between groups exposed to subarachnoid placement of OxyHb. Cerebrospinal fluid aspirated from the cisterna magna on Day 7 contained elevated activity of superoxide dismutase in animals that received treatment. Malondialdehyde was undetectable in cerebrospinal fluid after subarachnoid placement of agarose alone, although it was present in similar amounts in all groups that received subarachnoid placement of OxyHb. Since intrathecal superoxide dismutase and catalase failed to protect against OxyHb-induced vasospasm, mechanisms mediated by free radicals may not be important in its genesis. As only one combination of doses of superoxide dismutase and catalase was administered, however, it may be that other dosage schedules might be efficacious.

Topics: Animals; Antioxidants; Catalase; Cerebral Angiography; Delayed-Action Preparations; Disease Models, Animal; Free Radical Scavengers; Free Radicals; Gels; Hemolysis; Injections, Spinal; Ischemic Attack, Transient; Macaca fascicularis; Male; Malondialdehyde; Oxyhemoglobins; Random Allocation; Sepharose; Subarachnoid Hemorrhage; Subarachnoid Space; Superoxide Dismutase

A method is presented for screening immobilized dyes applicable to the purification of enzymes from haemolysate (haemolysate can be considered as a nearly pure solution of haemoglobin containing only marginal amounts of enzymes). Haemolysate is loaded on immobilized dye mini-columns until haemoglobin and the studied enzymes are found in the column eluate at the same concentrations as those present in the haemolysate. Such a frontal mode of screening allows those dyes to be selected which, displaying a higher affinity for the enzyme of interest than for haemoglobin, can be used to displace the unwanted protein (haemoglobin) from the column by the enzyme of interest (present at a much lower concentration).

Topics: Coloring Agents; Enzymes; Erythrocytes; Glucose-6-Phosphate Isomerase; Hemolysis; Humans; NADP; Phosphogluconate Dehydrogenase; Sepharose

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

Topics: Bacterial Toxins; Chromatography, Affinity; Chromatography, Gel; Complement Fixation Tests; Digitonin; Electrophoresis, Polyacrylamide Gel; Hemolysin Proteins; Hemolysis; Sepharose; Ultracentrifugation

The haemolytic activity of human C3 was destroyed by freezing and thawing or by treatment with methylamine. This partially denatured C3 (C3i) was coupled to activated Thiol-Sepharose via its single SH group. The fixed C3i was capable of reversibly binding C5 and of supporting C5 cleavage in the presence of factors B and D. Soluble C3i lacked these properties. C3i was also fixed to sheep red cells which had been supplied with activated thiol groups by treatment with N-succinimidyl-3-(2-pyridyldithio)-propionate. The E-C3i thus obtained were lysed by factors B, D and late complement components C5-C9. The experiments demonstrate that upon fixation, C3i becomes C3b-like in functional activity directed to C5 utilization.

Topics: Binding Sites; Complement Activation; Complement C3b; Complement C5; Disulfides; Hemolysis; Humans; In Vitro Techniques; Protein Conformation; Protein Denaturation; Sepharose

Whole blood hematocrit was determined by an approach which depends on the diffusion of an inert probe, to which red blood cells are impermeable, from a small agarose gel into a stirred, much larger blood sample. Blood cells influence the diffusion rate of the probe by, on the average, physically blocking a fraction of the gel surface. The blocking effect increases with the hematocrit. Cyanocobalamin (B-12) was found to be a suitable probe because it did not penetrate, bind to, or lyse blood cells and was not bound by plasma solutes. The loss of B-12 from gels in contact with blood was monitored by determination of the absorbance change at 540 nm of gels which had been quickly rinsed. The visible spectrum of B-12 in agarose gels was identical to the spectrum in water. Beer's Law was obeyed in 1-mm thick agarose gels over a concentration range of 0.1-0.8 mM. Based on the results from 48 blood samples covering the hematocrit range 25-69, a least-squares line was generated with a slope, -3.46 X 10(-3) delta A/hematocrit unit, a Y intercept of 0.295, and a correlation coefficient of 0.971. The precision of the technique was +/- 9.7%. The assay was insensitive to mean corpuscular volume and sample volume as long as the latter was 50-fold larger than the gel volume. The diffusion coefficient for B-12 in 1% agarose gels was found to be 1.4 +/- 0.2 X 10(-6) cm2 sec-1.

Topics: Blood Glucose; Diffusion; Gels; Hematocrit; Hemolysis; Humans; Models, Chemical; Polysaccharides; Sepharose; Serum Albumin; Vitamin B 12

Topics: Chromatography, Gel; Chromatography, Ion Exchange; Glycoproteins; Hemagglutination; Hemolysis; Immunodiffusion; Liposomes; Microscopy, Electron; Molecular Weight; Neuraminidase; Parainfluenza Virus 1, Human; Sepharose; Viral Proteins

Two cobra-venom factors, one from Naja n. naja (CVFn), the other from Naja h. haje venom (CVFh), have been purified and compared, functionally and structurally. Both factors interacted with human factors B and DS to form a potent C3 convertase, CVFBb. However, while the convertase formed with CVFn did also efficiently cleave C5, CVFhBb had very little C5-cleaving potency only, in particular when human C5 was used as substrate. Studies with agarose-linked CVF preparations indicated that CVFh has only low binding affinity for C5gp and C5hu whereas CVFn binds to both C5 species with much higher affinity. Since C5-binding (to CVF or to C3b) is a prerequisite for its cleavage by C3/C5 convertases, the difference in binding potency explains the different C5-cleaving activity of the two CVF preparations. When a ligand for C5, surface-fixed C3b, is present, CVFhBb is also capable of cleaving C5. The difference in activities of CVFn and CVFh is reflected in their different potency to interfere with immune haemolysis and in causing indirect lysis by their complexes with activated factor B. By gel chromatography of the CVF preparations in C5-containing medium, a stoichiometric complex CVFn-C5 (1 + 1) could be demonstrated. An analogous complex of C5 was neither found with CVFh, nor with C3hu or soluble C3bhu. Structural differences between CVFn and CVFh were revealed by immunodiffusion analysis and by polyacrylamide-gel electrophoresis in presence of SDS. The data available so far provide, however, no clear information about the structure of the C5 binding site.

Topics: Animals; Binding Sites; Chromatography, Gel; Complement Activation; Complement C3; Complement C3b; Complement C5; Complement Factor B; Elapid Venoms; Guinea Pigs; Hemolysis; Humans; Rabbits; Sepharose; Sheep

The first component of complement has been purified by using affinity chromatography on Sepharose-bound IgG. Unlike earlier procedures that yield the activated form of C1, in this method C1 is maintained in the native form by the protease inhibitor p-nitrophenyl, p'-guanidinobenzoate (NPGB). The procedure requires only two steps and yields pure C1 as judged both by SDS-PAGE analysis and by effective molecule calculations. The yields have varied from 30 to 50% in over 50 preparations. The functional properties of the purified native C1 correspond to those of C1 in serum. The dose-response activity profile is nonlinear, but becomes linear when C1 IS ALLOWED TO SELF-ACTIVATE. From SDS-PAGE analysis of the self-activated C1, all the C1r and C1s subcomponents are converted to the activated split products, indicating that all C1 molecules are biologically active. The recovery of C1 activity is dependent on the use of a heterologous source for the IgG on the affinity absorbant. The conditions of binding and elution from the Sepharose-IgG column are critical, indicating that immunoglobulin-bound C1 is rapidly inactivated under physiologic conditions by serum inactivators. The activation of the purified C1 in fluid phase has been explored both in the presence and absence of C1-inhibitor.

Topics: Benzoates; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Complement Activation; Complement C1; Complement C1 Inactivator Proteins; Electrophoresis, Polyacrylamide Gel; Guanidines; Hemolysis; Humans; Immunoglobulin G; Molecular Weight; Sepharose; Sucrose

Topics: Amyloid; Binding Sites; Calcium; Complement C1; Edetic Acid; Hemolysis; Humans; Immunoglobulin G; Macromolecular Substances; Sepharose

A simple method is described for the production of human serum deficient in the complement component C1. This C1-deficient serum can be used for the assay of C1. If the amount of C1 is expressed in terms of its subcomponent C1q, the method can detect C1 when the C1q subcomponent content is only 10 pg.

Topics: Animals; Antibodies; Chemical Precipitation; Complement C1; Complement Pathway, Classical; Hemolysis; Humans; Immunoglobulin G; Immunologic Techniques; Rabbits; Sepharose; Sheep

A simple one-step radial hemolytic assay for properdin has been devised. In this assay, the test material is introduced into a well in an agarose plate containing optimal concentrations of normal human serum immunochemically depleted of properdin (RP), EGTA, magnesium ions and unsensitized guinea pig erythrocytes. Following radial diffusion, the area of the hemolytic zones resulting from the activation of the alternative complement pathway and bystander lysis of guinea pig erythrocytes was directly proportional to the concentration of properdin in the test material. The assay is specific reproducible and sensitive and the correlation with the radioimmunoassay for properdin is very good. The assay has been used to measure properdin activity in animal sera.

Topics: Animals; Complement Factor B; Complement Factor D; Dose-Response Relationship, Immunologic; Erythrocytes; Guinea Pigs; Hemolysis; Hot Temperature; Humans; Magnesium; Polysaccharides; Properdin; Sepharose

Topics: Animals; Chromatography; Erythrocytes; Guinea Pigs; Hemolysis; Hydrocarbons; Mice; Osmotic Fragility; Sepharose; Species Specificity; Structure-Activity Relationship

Topics: Binding Sites; Erythrocytes; Hemolysis; Protein Binding; Sepharose; Staphylococcus aureus; Toxins, Biological

Topics: Acids; beta-Aminoethyl Isothiourea; Complement System Proteins; Erythrocytes; Hemoglobinuria, Paroxysmal; Hemolysis; Humans; Sepharose; Spectrophotometry; Sucrose