Isolation of human complement subcomponents C1r and C1s in their unactivated, proenzyme forms

We have modified a standard isolation procedure for C1r and C1s, which employs IgG-Sepharose affinity chromatography followed by DEAE chromatography. As usual, all steps were performed at low temperature and two proteolytic inhibitors, PMSF and NPGB, were added during affinity chromatography on IgG-Sepharose. The novel condition was to keep the pH at pH 6.1 during the entire procedure, where activation was markedly depressed. In addition, purification was improved by washing the IgG-Sepharose column with a buffer free of added divalent cations immediately prior to elution of the C1r and C1s with EDTA. The final yields of highly purified C1r and C1s were about 20%; little or no activated material was detected in these highly purified fractions.     

Biosynthesis of the subcomponents C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures

Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 X 10(3) effective C1q molecules/cell/24 h and 34 X 10(3) effective C1r/C1s molecules/cell/24 h. The synthesis of C1q and C1r/C1s could be reversibly inhibited by cycloheximide (50 micrograms/ml). Furthermore, to demonstrate de novo synthesis of the C1q subcomponent, endogeneous labeling with 3H-proline (or 14C-proline) was performed. The immunoprecipitated C1q from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig C1q from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize C1 subcomponents, whereby the synthesis of C1q and C1r/C1s occurs independently.     

A rapid FPLC method for purification of the third component of human and guinea pig complement

A method is described for the purification of human and guinea pig C3 from small amounts of serum. This procedure requires only two steps--polyethylene glycol (PEG) precipitation and fast protein liquid chromatography (FPLC) Mono Q HR 10/10 ion exchange chromatography. The protocol takes less than two hours to complete and yields 4-6 mg of purified C3. Similar results, in terms of antigenic and functional recovery, were obtained for both human and guinea pig components. About 67% of C3 antigen was recovered from eluted fractions with fully preserved specific activity. Isolated C3 was over 95% pure as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography; this level of purity was confirmed by the absence of any observable contamination as assessed by immunoelectrophoresis using high titer anti-whole human serum. This method allows rapid and reproducible purification of fully active human or guinea pig C3 on a daily basis.     

A rapid and efficient method for purification of rat B cells for complement-dependent cytotoxicity testing

The development of an effective technique for the enrichment of rat B cells is described. This protocol is an adaptation of the ‘panning’ technique employing anti-Ig-coated plastic dishes. Rat B cells are bound directly to anti-rat Ig-coated wells in Terasaki test plates where they are typed by complement-dependent cytotoxicity. This technique enriches class II (Ia-like) antigen-positive B cells, facilitating detection with antisera specific for rat class II alloantigens. The utility of this technique in the cell surface phenotyping of minor lymphocyte subpopulations is discussed.     

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。     

Dynamic equilibria between subcomponents of C1, the first component of human complement.

C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53-66 (1983)]. We have used the fluorescence anisotropy (A) of fluorescein-labeled C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta S = -26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.     

A Neoepitope-Based Enzyme Immunoassay for Quantification of C1-Inhibitor in Complex with C1r and C1s

Monoclonal antibodies (MoAb) recognizing neoepitopes exposed on activation products of complement proteins but hidden in the native components have been used for quantification of activated complement. A previously produced and characterized mouse MoAb, recognizing a neoepitope on the human plasma protein C1-inhibitor complexed with its substrates, was used to design an enzyme immunoassay for detection of C1-inhibitor complexed with C1r and C1s. These complexes are indicators of early classical complement pathway activation. The standard was serum activated with heat aggregated IgG defined to contain 1000 arbitrary units (AU)/ml. The lower detection limit was ≈0.05 AU/ml corresponding to 0.005% of fully activated serum. The reliability of the assay, including day-to-day variation, was tested. Intra-assay variation coefficients were 12% for low plasma control and 13% for high plasma control ( n  = 12 for both). Inter-assay variation coefficients were 12% for low control ( n  = 6), 19% for high control ( n  = 6) and 15% for the normal plasma control ( n  = 9). A 2.5–97.5 percentile reference range (normal blood donors) was 16–33 AU/ml. Two patients with systemic lupus erythematosus had considerably elevated plasma levels of the activation product (56 and 62 AU/ml), and six patients with hereditary angioedema had normal plasma levels despite considerably reduced C1-inhibitor concentration. We conclude that the present method is sensitive and reliable for detection of early classical pathway activation and superior to previously published methods by utilizing neoepitope specificity and non-radiolabelled reagents.     

Technique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.     

A rapid procedure for the purification of IgA1 and IgA2 subclasses from normal human serum using protein G and jackfruit lectin (jacalin) affinity chromatography

Immunoglobulin A (IgA) from pooled normal human sera was purified using antibody and protein G affinity chromatography and gel filtration high-pressure liquid chromatography (HPLC). This high purity product was separated into IgA1 and IgA2 subclasses utilizing the agarose-bound lectin 'jacalin'. Evaluation of product homogeneity by immunological testing confirmed greater than 95% purity. The total IgA1 and IgA2 recovered from sera was approximately 26% of the initial antibody present.     

A rapid method for the purification of cytoplasmic paramyxoviral nucleocapsids

A rapid method for the purification of cytoplasmic paramyxoviral nucleocapsids using a tabletop ultracentrifuge is described. The method yields materials equivalent in purity to materials purified by previously published procedures but requires only a fraction of the time (65 min vs 7-41.5 h). When used in conjunction with modern hypersensitive analytical techniques, the procedure provides equivalent or greater amounts of nucleocapsid for subsequent analysis or study.     

A rapid and efficient method for generating anti-variable region monoclonal antibodies using type-1 interferons as immune modulators

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.     

A rapid method for purification of herpesvirus DNA.

A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during isopycnic centrifugation, so that phenol/chloroform extractions can be omitted. The method was used for the purification of DNA from several members of the Alfaherpesvirinae subfamily.     

A fast and efficient method for sequential cone-beam tomography.

Sequential cone-beam tomography is a method that uses data of two or more parallel circular trajectories of a cone-beam scanner to reconstruct the object function. We propose a condition for the data acquisition that ensures that all object points between two successive circles are irradiated over an angular span of the x-ray source position of exactly 360 degrees in total as seen along the rotation axis. A fast and efficient approximative reconstruction method for the proposed acquisition is presented which uses data from exactly 360 degrees for every object point. It is based on the Tent-FDK method which was recently developed for single circular cone-beam CT. The measurement geometry does not provide sufficient data for exact reconstruction but it is shown that the proposed reconstruction method provides satisfying image quality for small cone angles.     

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.     

A rapid method for purification of human granulocytes using percoll. A comparison with dextran sedimentation

A simple one-step method for purification of human granulocytes, based on the use of a discontinuous gradient of Percoll, is described. The cell yield is 55% and the purity of the granulocyte fraction is 97%. The cells' ability to exclude trypan blue and phagocytic function are not altered during the purification process. We conclude that centrifugation in Percoll is superior to conventional dextran sedimentation; it is also less time-consuming.     

A rapid and highly efficient method for PCR-based site-directed mutagenesis using only one new primer

We present a rapid, cheap and highly efficient method for site-directed mutagenesis using the polymerase chain reaction (PCR). This method is applicable to every DNA fragment which has to be cloned into the multiple cloning site of any vector, or vector pair, in two different orientations. It requires only two primers, one new and specific mutagenic primer and one of the usual sequencing primers. In the first PCR, a mutagenic DNA fragment is synthesized which is amplified exponentially in the second PCR. In contrast, wild-type sequences are only linearly amplified resulting in an efficiency of mutagenesis of nearly 100%.     

A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.