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At least 343 records · Page 19Linked to original sources
PubMed1980

Analysis of the different types of leukocyte membrane complement receptors and their interaction with the complement system.

The specificity, distribution, and structure of 8 different types of leukocytes membrane complement (C) receptors (CR1, CR2, CR3, and receptors for C1q, beta 1H, C3e, C3a, and C5a) are discussed. Recent data are reviewed on the synthesis of C components by macrophages and B lymphocytes, and how these components may function in the activation of these two cell type by the C system. Commonly used C receptor assay procedures are evaluated in terms of both specificity and sensitivity. Specific assay procedures are recommended for measuring CR1 (C4b-C3b receptor), CR2 (C3d receptor), CR3 (C3bi receptor), and the beta 1H receptor. Assays include both rosette and fluorescence procedures for detection of C receptors on either mouse or human leukocytes. Primary systems have been selected for optimal sensitivity and specificity, and where possible, acceptable alternative systems that are less sensitive or specific are suggested for laboratories lacking facilities for C purification.

Animals↗
PubMed1984

Purification and characterization of human, guinea pig and mouse C1q by fast protein liquid chromatography (FPLC).

A simple and rapid procedure for the purification of C1q from human, guinea pig and mouse serum is described. This procedure allows the purification of C1q within one and a half days using euglobulin precipitation, chromatography on Superose 6B, followed by chromatography on Mono S by Fast Protein Liquid Chromatography (FPLC). The highly purified, hemolytically active C1q is free of any immunoglobulins. Since the purification of C1q of three different species was performed by the same purification procedure, a comparison of the subunit compositions was made under reducing and non-reducing conditions on SDS-PAGE. The yield was found to be more than 50%.

Animals↗
PubMed1986

New methods for the preparation of the cellular intermediate EAC14.

The cellular intermediate EAC14 was successfully prepared by incubating optimally sensitized sheep erythrocytes (EA) with normal serum diluted in complement diluent buffer containing suitable amounts of inhibitors of EAC142 formation from EAC14 and C2, namely polymyxin E and polymyxin B. EAC14 cells could also be prepared by incubating EA with normal serum diluted in Mg2+ free complement diluent buffer in the presence or absence of sucrose. The cellular intermediates prepared by these methods exhibited characteristic EAC142 formation and decay profiles and were found to be suitable for C2 estimations.

Animals↗
PubMed1987

A substrate amplification system for enzyme-linked immunoassays. Demonstration of its general applicability to ELISA systems for detecting antibodies and immune complexes.

Self recently described a substrate system for alkaline phosphatase (AP)-dependent ELISAs which markedly increased sensitivity, compared to using p-nitrophenyl phosphate. This increase is achieved by having AP, the primary enzyme, produce an activator for a secondary enzyme-substrate system, within which marked amplification occurs. We adapted this technique to study antibodies to casein, bovine serum albumin, ovalbumin, and cardiolipin in the sera of patients with systemic lupus erythematosus (SLE) and normal individuals. The new substrate system yielded titres 30-50-fold higher than those with p-nitrophenyl phosphate (Sigma 104, p-NPP). In addition, when used in a solid-phase C1q binding assay we were able to use a 1 : 100,000 dilution of AP-conjugated anti-human IgG with the amplified substrate, compared to the 1 : 1000 dilution needed with p-NPP. This system is extremely valuable because of its flexibility. It can either be very sparing of limited samples, or if the added sensitivity is not needed, 100-fold less AP conjugate may be used. Thus rare or expensive conjugates can be significantly conserved.

Animals↗
PubMed1987

Detection of C1q-bearing immune complexes by a monoclonal anti-C1q ELISA system.

A monoclonal antibody directed against the collagenous portion of human C1q was used to detect C1q-bearing immune complexes in patients with rheumatic disorders. Sera of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE), osteoarthritis, as well as normal human sera (NHS) used as controls were tested in an ELISA system. C1q-bearing immune complexes were bound to a solid-phase monoclonal anti-C1q antibody, and detected with F(ab')2 antibodies to human IgG. Heat-aggregated human IgG was adjusted to the same concentration as the WHO standard for immune complexes and used for the standard curve in NHS. The mean value in NHS was 19.5 micrograms/ml equivalents of aggregated IgG. Using 2 SD over the mean as the upper limit for normal values, samples greater than 43 micrograms/ml were considered positive. Patients with osteoarthritis were negative; high levels of C1q-bearing immune complexes were detected in patients with rheumatoid arthritis (up to 800 micrograms/ml equivalents of aggregated IgG). With our assay C1q-bearing immune complexes were detected with high frequency (81%) in the sera of patients with rheumatoid arthritis, while a C1q solid-phase binding assay (C1q SPBA) revealed positive results only in 67% of rheumatoid arthritis sera. Compared to NHS, CH50 titers and C1q values of sera from patients with rheumatoid arthritis were frequently high. In contrast, the sera of SLE patients with low CH50 titers and low C1q levels had IgG immune complexes which could be detected only in the C1q-SPBA. C1q-bearing immune complexes were not detectable in the sera of patients with SLE. Since C1q triggers activation of the classical C pathway, this assay with monoclonal anti-C1q antibody appears to be useful for detecting immune complexes in rheumatoid arthritis patients with normal or elevated CH50 and C1q values, especially in the early stage of the disease.

Antibodies, Monoclonal↗
PubMed1988

Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification.

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.

Avidin↗
PubMed1988

An ELISA technique for the measurement of C1q in cerebrospinal fluid.

Determination of the C1q content of cerebrospinal fluid (CSF) may be of value in understanding the immunological reactions occurring within the central nervous system (CNS). A double sandwich ELISA method has been developed for the detection of C1q in human serum and CSF. It uses polyclonal antibodies and is sensitive in the nanogram range. The mean concentrations of C1q were determined to be 127 micrograms/ml in serum and 0.4 microgram/ml in CSF. These results suggest that increased levels of C1q in the CSF play a role in some neurodegenerative disorders.

Adolescent↗
PubMed1988

An analysis of the fluid phase C1q binding assay. The effect of endogenous C1q on the precipitation and detection of an immune complex model.

We examined the effect of endogenous C1q on the sensitivity of the fluid-phase C1q binding assay (C1qBA) in detecting an immune complex (IC) model, heat-aggregated IgG (HAIgG), at concentrations of 10-10,000 micrograms/ml sample. Results in normal human serum (NHS) or plasma (NHP) were compared with those in heat-inactivated NHS (NHS/56) in which most endogenous C1q was depleted by heat denaturation. Higher HAIgG concentrations were required in NHP and NHS to produce the same 125I-C1q precipitation seen in NHS/56. This decreased sensitivity varied from 70% at low HAIgG concentrations to 0% at high concentrations, as predicted for a large pool of endogenous C1q, in equilibrium with 125I-C1q, but in excess of that which could bind to all but the highest concentrations of IC model. In serum depleted of functional C1q on an immunoadsorbant of HAIgG, the precipitation of radiolabeled HAIgG under C1qBA conditions was concentration dependent and generated a saturation curve, showing that only a fraction of IC are usually precipitated in this assay. HAIgG precipitation was enhanced 1.4-fold in NHS/56 (8 micrograms C1q/ml) and three-fold in NHS (67 micrograms C1q/ml) suggesting that IC size is increased by endogenous C1q. In dual label experiments using 131I-HAIgG, the precipitation of 125I-C1q in NHS/56 was directly proportional to IC model precipitation, but markedly discordant in NHP, showing the measurement of IC in heat-inactivated sera superior to that in native serum. A comparison of the C1q:HAIgG ratio in PEG precipitates with that in samples, indicated that equilibrium was established between C1q and IC model. Thus the precipitation of 125I-C1q in the C1qBA represents (1) the fraction of total C1q bound to IC, and (2) the fraction of IC precipitated by PEG.

Antigen-Antibody Complex↗
PubMed1988

Determination of circulating immune complexes by chicken anti-human C3 and anti-human C1q microELISA.

The present study reports on an anti-human C1q and an anti-human C3 microELISA for measuring circulating immune complexes (CIC). Affinity-purified chicken anti-human C3 and anti-human C1q were used as capture antibodies and protein A-alkaline phosphatase conjugate for detection. Chicken antibodies do not activate mammalian complement, do not react with rheumatoid factor and are not bound to anti-mammalian IgG antibodies or protein A, which often are used for detection in ELISA. They are therefore suitable as capture antibodies in CIC assays. We have tested Bell's palsy patients and found an increase in both anti-C3- and anti-C1q-containing CIC in acute and convalescent sera compared with the normal.

Animals↗
PubMed1989

Binding of complement components C1q, C3, C4 and C5 to a model immune complex in ELISA.

When normal human serum is added to microELISA plates coated with monomeric or aggregated IgG various complement components become bound and can be detected with specific chicken anti-C1q, anti-C3, anti-C4 and anti-C5 antibodies. Using such assays we found increased C1q- and decreased C3- and C4-binding in sera from patients with SLE. In contrast, sera from patients with rheumatoid arthritis showed decreased C3 binding but normal C1q binding. The decreases in C3 and C4 binding observed in the sera from patients with SLE were larger than the corresponding decreases determined by radial immunodiffusion. Comparing these results with those of the CH50 assay, the correlation coefficient between CH50 and the C3-binding assay was 0.48. There was no correlation between the results of the CH50 and those of the C1q-, C4- or C5-binding assays.

Antigen-Antibody Complex↗
PubMed1992

Mechanisms of oligodendrocyte interaction with normal human serum--defining the role of complement.

The interaction of human serum with oligodendroglia was investigated in vitro using purified cultured neonatal rat oligodendrocytes. Previous evidence for antibody independent classical pathway complement activation was confirmed; the results also showed a deficit in the protection of rat oligodendrocytes from complement attack suggesting a deficiency in the expression of terminal regulatory proteins of the complement cascade. Thus, rat oligodendrocytes are selectively sensitive to normal serum due both to complement activation and impaired protection from terminal complement attack.

Animals↗
PubMed1982

C1q.

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Animals↗