C1q、C1r和C1s的快速分离

建立了一种同时快速分离纯化C1q及酶原形式C1r和C1s的方法。用IgG Sepharose 4B亲和层析将C1q与C1r2 s2 分离 ,接着用DEAE Sephacel离子交换层析将C1r和C1s分离 ,用C1qMcAb Sepharose 4B亲和层析将C1q进一步纯化。在分离Clr和C1s的整个过程中加入蛋白水解抑制剂PMSF和NPGB ,并控制温度在 4℃、pH 6 1、无二价阳离子的条件下 ,得到高度纯化的C1q、C1r和C1s。     

Isolation and purification of human C1q from plasma

C1q was purified to homogeneity from human plasma by a 3-step purification procedure. Plasma was euglobulin precipitated, and the redissolved precipitate chromatographed on a rabbit IgG-Sepharose column. The 1 M NaCL buffer eluate was passed directly through a rabbit anti-human IgG-Sepharose affinity column. C1q freed of IgG was present in the flow through. The rationale for this scheme to remove IgG free and that bound to C1q is discussed. Overall recovery of C1q was about 40% with IgG less than 4 μg/mg C1q. In SDS-polyacrylamide electrophoresis with non-reducing conditions bands at 52,000 and 42,000 daltons were demonstrated while with reducing conditions bands at 26,000, 24,000 and 20,000 daltons were found as reported by others. C1q was found to be stable at 4°C for 1 year in a 1 M NaCl, 0.4 M Tris, 10% sucrose, 0.005 M EDTA, 0.02% NaN₃, pH 8.6 buffer.     

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.     

A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose

C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10-12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40-45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.     

A specific one step method for the isolation of pancreatic elastase; its use to characterize aortic elastase

Elastase was purified from an acetone-ether powder of porcine pancreas by a one step affinity chromatography procedure on IgG-Sepharose 4B. The IgG was derived from a rabbit immunized with porcine pancreatic elastase and was itself isolated by affinity chromatography on elastase immobilized on the same matrix. The column was calibrated with a known elastase preparation under standardized conditions. Direct isolation of elastase from pancreatic extracts yielded approximately 90 mg from two pancreas. The enzymatic activity and electrophoretic migration in SDS-polyacrylamide gel of the purified elastase were equal to those of the purest commercially available enzymes. Application of this method to aorta of young pigs suggests the presence of active elastase in the aorta.     

Human C1q: rapid isolation and quantitative determination by immunodiffusion

A simple and rapid 2-step procedure for isolating C1q from human plasma at high yields (about 50%) is described. The purification involves diaminopropane precipitation followed by chromatography on IgG-Sepharose. The final product (obtained at a concentration of about 1.5 mg/ml) was electrophoretically and immunochemically pure and stable at -70 degrees C for long periods. The Mancini technique for the quantitative determination of C1q was reinvestigated and the use of gels containing high salt concentrations (1.0 M NaCl) was found to be absolutely necessary. A value of 0.076 mg/ml C1q in pooled human plasma was obtained.     

The fifth component of complement (C5): purification without activation

In the course of our studies on the structural change of C5 by acidification (U. Rother et al., 1978), we found that the C5 preparations purified according to published methods contained more or less activated C56. When added to sensitive target cells (guinea pig or chicken erythrocytes), C5 mediated lysis by C7-C9 without the addition of C6 or any activation procedure. Generation of C56 was probably due to drastic changes in the physicochemical environment during purification. Such changes like high or low pH or high ionic strength were shown to cause activation. A method for purification of C5 is described in which polyethyleneglycol (PEG) or (NH4)2SO4 precipitation, as well as low or high pH, was avoided. As a last step, traces of C6 were removed by affinity chromatography. The resulting preparation was free of C56. Activation by acidification was not possible without the addition of C6. The total recovery of C5 was 12% with almost no loss of specific activity.     

A rapid and efficient method for the purification of the complement subcomponents C1r and C1s in zymogen form using fast protein chromatography

The purification of the subcomponents C1r and C1s of the first component of complement involves multiple steps and is time-consuming. This accounts for the frequently observed partial activation of the subcomponents. In this report we propose a simplified procedure of purification using a batch method and fast protein chromatography avoiding a shift of pH. The method provides C1r and C1s in a yield of 35 and 60% respectively. In addition, this study provides a simple and sensitive test to assess functional purity of C1r and C1s with respect to the other C1 subcomponents.     

Purification of soluble immune complexes from serum using polymethylmetacrylate beads coated with conglutinin or C1q. Application to the analysis of the components of in vitro formed immune complexes and of immune complexes occurring in vivo during leishmaniasis.

A procedure for the isolation of immune complexes from human sera has been developed. Two steps are involved: (1) lipid-free serum is precipitated by polyethylene glycol; (2) the solubilized precipitate is absorbed on a column of polymethylmethacrylate beads coated with conglutinin (K) or C1q; the column is washed, the complexes are then eluted, using 0.02 M EDTA (for K column) or 0.5 M NaCl (for C1q column). This procedure permitted the purification and the characterization of soluble 125I-BSA-anti-BSA, 125 I-tetanus toxoid-anti-tetanus toxoid, and 125-I-HBsAg-anti-HBsAg complexes made in vitro in the presence of fresh human serum. The isolated complexes were shown to contain antigen, antibody, C1q, C1r, C1s and C3. When normal human serum was submitted to such a procedure, no detectable amount of protein was present in the final eluted fraction. Immune complexes formed in vivo were also purified by conglutinin column from the serum of a patient with disseminated leishmaniasis. The isolated material was found to contain IgM, IgG, C1q, C1r, C1s, C3c and C3d. The purified complexes dissociated at acid pH were found to contain anti-IgG and anti-leishmania antibodies.     

The critical concentration of C1-esterase inhibitor (C1-INH) in human serum preventing auto-activation of the first component of complement (C1)

C1-esterase inhibitor (C1-INH) was depleted from normal human serum (NHS) at 4 degrees C by affinity chromatography with a monoclonal anti-C1-INH antibody (mAb 13 E1) coupled to CNBr-activated Sepharose 4B. The C1-INH-depleted serum (C1-INH-depl-HS) had normal levels of C1, C4, and CH 50 and C1-INH concentration was less than 10% of normal (15 microg/ml in C1-INH-depl-HS compared to 230 microg/ml in NHS). C1-auto-activation in C1-INH-depl-HS was followed by measuring C4-consumption in a haemolytic assay and by detection of activated C1s in a C1s-ELISA. After a lag phase of 10-20 min, C1-auto-activation in C1-INH depl-HS occurred and reached its maximum after 40 min at 37 degrees C. In contrast, neutralization of C1-INH activity in NHS by addition of monoclonal antibodies directed against its C1s-binding site, resulted in an immediate, spontaneous C1-activation without a lag-phase and reached its maximum already after 20 min (mAb 140) and 25 min (mAb 88G2). Addition of highly purified C1-INH or NHS as source of C1-INH to C1-INH-depleted serum to a final concentration of 55 microg/ml (22% of normal C1-INH concentration in HS) was sufficient to control spontaneous C1-activation.     

Isolation and purification of HSV-1 specific transfer factor produced by HSV-1 immunized goat leukocyte dialysate.

A herpes simplex virus type 1 (HSV-1)-specific transfer factor (TF), was separated and purified from the leukocyte dialysate of goats immunized with HSV-1 using affinity chromatography on antigen-sorbent and reversed phase high performance liquid chromatography (RP-HPLC). The antigen-specific activities of the starting dialysate and the isolated TF component (s) were examined by 51Cr-labelled leukocyte adherence inhibition (51Cr LAI) assay. The analytical hydrophobic interaction HPLC (HI-HPLC) and isoelectric focusing (IEF) techniques were employed to evaluate the purity and the isoelectric point (PI) of isolated TF component(s). The experiments provided a two-step procedure for purifying the TF material from the starting dialysate. It seems that the purified active TF component (PTFC) was specific for HSV-1. The specific PTFC activity was increased 10,000-fold as compared with the activity of the dialysate. The active moiety appeared as a single band in the IEF gel as demonstrated by silver staining; it was hydrophilic and its PI was pH 4.48.     

A secreted receptor related to M1 protein of Streptococcus pyogenes binds to fibrinogen, IgG, and albumin

An extracellular protein (eMP) of Streptococcus pyogenes M type 1 was isolated by affinity chromatography on albumin- and IgG-Sepharose. The protein was found to bind to the human plasma proteins, fibrinogen, IgG, and albumin. Analysis of eMP by Western blotting demonstrated a major band with a molecular weight of 49 kD which was responsible for binding of the three plasma proteins. The purified protein was found to bind selectively to human and primate polyclonal IgG, human and mouse albumin, as well as human fibrinogen which has been the only fibrinogen tested. Serological investigations revealed a close relation of eMP to streptococcal M1 protein. It showed a reaction of identity with cell-extracted M1 protein in immunodiffusion. Moreover, the 49 kD peptide responsible for binding, was recognized with an antiserum directed against the 20 amino acids comprising synthetic peptide (42VAL-61GLU) of the N-terminal part of the M1 protein sequence. The affinity of M protein to plasma proteins other than fibrinogen opens new approaches to its purification by affinity chromatography.     

Purification of Clostridium difficile toxin A by affinity chromatography on immobilized thyroglobulin.

An efficient, single-step method for isolating highly purified toxin A from Clostridium difficile culture filtrates is described. The purification procedure was based on the affinity binding and release of toxin A to bovine thyroglobulin conjugated to agarose beads. The toxin strongly bound at 4 degrees C to the carbohydrate binding determinant Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence which occurs on bovine thyroglobulin. Toxin bound to thyroglobulin at 4 degrees C, allowing its separation from the culture filtrate and contaminating proteins during the purification scheme. The toxin was eluted by increasing the temperature to 37 degrees C. The toxin-binding capacity was related to the amount of thyroglobulin immobilized on the gel: an affinity column containing 15 mg of bovine thyroglobulin per ml of gel bound 0.53 mg of toxin A per ml of gel. The percent recovery of purified toxin ranged from 56 to 80% and was inversely related to the amount of thyroglobulin coupled to the gel. The affinity-purified toxin was homogeneous as judged by crossed immunoelectrophoresis and gradient polyacrylamide gel electrophoresis and was immunologically identical to toxin A purified by conventional methods as determined by immunodiffusion analysis. The biochemical, hemagglutinating, and toxic properties of the toxin were preserved after affinity chromatography and were comparable with those of toxin A purified by conventional methods.     

Measurement of the association constants of the complexes formed between intact C1q or pepsin-treated C1q stalks and the unactivated or activated C1r2C1s2 tetramers

The association constants between C1q and C1r2C1s2 and between C1q and C1r2C1s2 were measured in solution using a new technique which employs sucrose gradient ultracentrifugation to estimate thermodynamic association constants. In this technique, zones of dilute, radioiodine-labeled C1q were sedimented through uniform concentrations of either C1r2C1s2 or C1r2C1s2. The zones remained intact, indicating that the dynamic equilibrium was rapid compared with the time of centrifugation. The observed increases in the sedimentation coefficients of the C1q zones were assumed to be directly proportional to the fraction of C1q bound in the dynamic equilibrium. Binding curves were constructed by performing the measurements at many C1r2C1s2 and C1r2C1s2 concentrations. The association constants were estimated from the midpoints of the binding curves and found to be 6.7 X 10(7)M-1 for C1r2C1s2 binding to 125I-C1q. After activation of the C1r2C1s2 the association constant decreased 10-fold to 7.1 X 10(6)M-1. These association constants refer to solvent conditions of pH 7.35, 1 mM Tris, 5 mM Ca2+ and 150 mM NaC1, pH 7.35. Similar measurements were performed with the collagenous peptic fragment of C1q and both 125I-C1r2C1s2 and 125I-C1r2C1s2. The association constants were independent of the state of activation and both found to be about 2 X 10(7) M-1, suggesting that most if not all of the interactions between C1q and C1r2C1s2 were confined to the collagenous portion of C1q.     

Isolation and properties of a novel IgG-binding protein from streptococci of serological group U

A nonimmune binding of immunoglobulin (Ig) G has been detected in streptococci of group U. The group U Fc-binding site differed from the five previously known types of staphylococcal and streptococcal Fc-binding sites by its strong affinity for murine IgG, with dissociation constants in nanomolar range for rat and mouse IgG, as well as for mouse IgG subclasses 1, 2a, 2b and 3. It also differed from other binding sites by the high sensitivity towards trypsin. The Fc-binding protein could be solubilized from the streptococci of group U with papain and purified by gel filtration on sephacryl S-200 and by subsequent affinity chromatography on human IgG-Sepharose. The purified binding protein was homogenenous on SDS-polyacrylamide gel electrophoresis and had a molecular weight of approximately 58,000 daltons. It retained its binding activities for murine IgG subclasses as revealed by western blotting. Coupled to CNBr-activated sepharose, the purified Fc-binding protein could be effectively used for the isolation of murine IgG subclasses by affinity chromatography.     

A simple two-step procedure for the preparation of the first component of human complement (C1) in its native form

Native human C1 was purified from fresh human serum by affinity chromatography on protein A-bound Sepharose in the presence of 4-nitrophenyl-4-guanidinobenzoate hydrochloride (NPGB) taking advantage of the successive binding of IgG to protein A followed by C1 binding to IgG. After elution the C1 preparation contained IgG as a major contaminant as shown by SDS-PAGE. C1 was further purified by gel filtration. The yield of C1 was 12% and less than 4% of this C1 was activated during purification as assessed by a C4 consumption assay.     

Direct radioimmunoassay of rat cystatin C: increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. The two-steps purification procedure included a Carboxymethyl-papain affinity chromatography and anion exchange chromatography. The purified protein was identified as rat cystatin C by the following criteria: firstly retained on a Cm-papain affinity column, secondly an apparent molecular weight of 15 kDa and pI of 10.2. Antisera raised in rabbits against our purified rat cystatin C did not cross-react with other urinary proteins such as rat albumin and rat kallikrein, but partially cross-reacted with human cystatin C. A direct radioimmunoassay was developed and it enabled 8.32 fmol/ml of rat cystatin C to be detected. The detection range was between 0.125 and 62.5 ng/ml, with 10% intra-assay variation and 14% inter-assay variation. Physiological rat cystatin C excretion (40 +/- 18 micrograms/24 h) was found by the direct assay. In the chromate-intoxicated rat, urinary excretion increased twenty-fivefold (1017 +/- 391 micrograms/24 h) and returned to normal level one week after intoxication. This RIA will allow the study of rat cystatin C metabolism particularly during renal dysfunction.     

Influence of serum complement and rheumatoid factor on detection of immune complexes by the C1q and monoclonal rheumatoid factor solid-phase assay

Soluble purified monoclonal and polyclonal rheumatoid factor, total serum complement, and soluble C1q all inhibit the detection of model tetanus toxoid/anti-toxoid immune complexes in the solid-phase C1q assay. The binding of these immune complexes to solid-phase monoclonal rheumatoid factor is less inhibited by soluble C1q and by total serum complement, but clearly decreased by soluble monoclonal or polyclonal rheumatoid factor. Serum complement does not reduce the size of these model complexes. We recommend the use of low ionic strength EDTA (10 mM) to partly neutralize the complement-mediated inhibition. This procedure is shown to be superior to currently used higher EDTA concentrations and to the use of IgG-Sepharose.     

Characterization of the activation of the human C1r complement molecule

The proenzyme form of C1r was isolated by sequential chromatography from the euglobulin fraction of human serum on DEAE-Sepharose 6B-CL, CM-Sepharose 6B-CL and Sepharose S-300-CL. This C1r had the tendency to spontaneously activate within 60-90 min of incubation at 37 degrees C in presence of EDTA and more slowly in the presence of Ca2+. The spontaneous activation of C1r was found to be a bimolecular process and could be completely inhibited by DFP in the pH range 6-9 and in the presence of Ca2+ without affecting the hemolytic C1r activity. [14C]DFP bound to trace proteins in the 60-90 kD range, but not to C1r proenzyme. The spontaneous activation of C1r was diminished in the presence of EDTA by DFP, but could not be completely suppressed. EDTA acts by removing Ca2+ from C1r, thereby changing the conformation of the protein and causing an increased digestibility of the C1r H-chain. At temperatures above 0-4 degrees C this influence destroyed the ability of C1r proenzyme and enzyme to form macromolecular C1 and thereby abolished its hemolytic activity. We conclude from these results that the spontaneous C1r activation in the pH range 6-9 and in the presence of Ca2+ is due to contaminant proteases. C1r activated also spontaneously at higher pH values between pH 9 and 13.2, but the spontaneous activation ceased abruptly at pH 13.4. An intramolecular process of activation cannot be excluded at these high pH values. It is, however, not clear, whether this activation is a suitable model for the C1r activation in the C1 molecule, because the hemolytic activity of C1r was substantially diminished under the high pH conditions.     

C1 inhibitor removes the entire C1qr2s2 complex from anti-C1Q monoclonal antibodies with low binding affinities.

Evidence is presented for a new C1 Inhibitor (C1 INH) function. C1 INH was capable of dislodging the entire C1qr2s2 complex from C1-activating substances that bound weakly to the globular heads of C1q. Two different mouse IgG1 monoclonal antibodies with different affinities for C1q globular heads were compared for their complement-activating properties in the presence of normal human serum. As expected the higher affinity monoclonal antibody (Qu) was more effective in binding C1q and causing C1-mediated C4b deposition. Unexpectedly, time responses of C1 (C1q) binding to immobilized 3C7 reached a peak then gradually decreased. However, C1q remained constantly bound to immobilized Qu. These results indicated that after C1 activation in human serum, the entire C1 complex (including C1q) was dislodged from 3C7, but not from immobilized Qu. The addition of purified C1 INH to purified C1, which had bound to immobilized 3C7, resulted in removal of C1 (C1q). Removal of the entire C1qr2s2 did not occur when C1 INH preparations were first neutralized by the addition of purified activated C1s. In summary, it is suggested that C1 INH plays a prominent role in dislodging the entire C1qr2s2 from immunoglobulin preparations which have a low binding affinity for the globular heads of C1q.