Monoclonal antibodies to complement components without the need of their prior purification. II. Antibodies to mouse C3 and C4

Rat monoclonal antibodies (MAbs) to mouse complement components C3 and C4 were produced by immunizing rats with cell-bound C3 and C4. This principle involves: a) using mouse thymocytes coated with syngeneic rat antibody isotypes that show high affinity to C1q, b) the intercalation of C1q from serum and c) the subsequent activation of the classical complement pathway leading to deposition of cell-bound complement components. Screening for anti-complement antibodies was performed on antibody coating microtiter plates with mouse serum as source of complement. The reactivity of the MAbs was determined by variations of the ELISA screening system using EDTA-serum to inhibit complement activation by C1 dissociation, serum rendered deficient of functionally active C3 by treatment with cobra venom factor (CVF) or serum of genetically C5-deficient mice. The specificity of the MAbs was confirmed by affinity chromatography followed by SDS-PAGE and immunoblotting. We were able to establish a panel of anti-C3 and anti-C4 MAbs of various isotypes.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.     

Monoclonal antibodies to human interferon-gamma. I. Antibodies to a synthetic carboxyl-terminal peptide

Two types of hybridomas secreting monoclonal antibodies (MAB) against human interferon-gamma (HuIFN-gamma) were obtained by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with a conjugate of a synthetic carboxyl-terminal peptide (residues 131-146) of HuIFN-gamma and bovine thyroglobulin. One of the antibodies bound to recombinant HuIFN-gamma produced in E. coli as well as to natural HuIFN-gamma, while the others bound only to recombinant HuIFN-gamma. These 2 types of MAB did not neutralize the anti-viral activity of HuIFN-gamma. They were useful for effectively purifying recombinant HuIFN-gamma and quantitatively determining it by an enzyme-linked immunosorbent assay.     

The mouse C1q A-chain sequence alters beta-amyloid-induced complement activation

In transgenic models of Alzheimer's disease (AD) neuronal loss has not been widely observed. The loss of neurons in AD may be due to chronic activation of complement (C') by beta-amyloid (A beta). A beta has been shown to activate C' by binding to a site on the C1q A-chain. The mouse A-chain sequence differs significantly from human, and a peptide based on the mouse A-chain sequence was ineffective at blocking activation of C' by A beta in contrast to the inhibition seen with the human peptide. Comparison of mouse and human serum showed that human C' was activated more effectively by A beta than was mouse C'. Therefore, additional genetic manipulations may be necessary to replicate in the murine model the inflammation and neurodegeneration that occur in AD.     

The use of [125I]C1q subcomponent for the measurement of complement binding antibodies on cell surfaces.

An assay using 125I-labelled human C1q has been developed for the measurement of complement fixing antibodies bound to cell monolayers or cell suspensions. The method has been adapted for use either during or after sensitisation of the cells with antiserum, is simple to perform and does not require require prelabelling of the target cells.     

C1q rs292001 polymorphism and C1q antibodies in juvenile lupus and their relation to lupus nephritis

C1q deficiency is related strongly to systemic lupus erythematosus (SLE), but very few and inconsistent studies explored the single nucleotide polymorphisms of the C1q gene in relation to juvenile SLE (jSLE) and lupus nephritis (LN). The objective of this study was to analyse whether C1q rs 292001 polymorphism is associated with SLE and disease phenotype, especially nephritis, and to investigate the relation between this polymorphism and clinical data, treatment outcome, serum level of C1q protein and antibodies. Typing of C1q rs292001 polymorphism using restriction fragment length polymorphism and measuring serum levels of C1q protein and antibodies by enzyme-linked immunosorbent assay (ELISA) were performed for 130 children with SLE and 208 healthy controls. The A allele of C1q rs292001 was associated with jSLE and LN (P = 0·005 and 0·013, respectively) and the AA genotype was associated with jSLE (P = 0·036). Low serum levels of C1q protein were found in jSLE and LN (P < 0·001 and 0·009, respectively), and these levels were increased after treatment in patients with LN (P = 0·009) and active renal disease (P = 0·027). Higher titres of C1q antibodies were found in patients with LN (P = 0·015) and correlated negatively with C1q protein level (P < 0·001) and patient age (P = 0·04). The A allele and AA genotype of C1q rs292001 can be considered a susceptibility risk factor and the GG genotype could be considered protective for jSLE and LN in the studied cohort of Egyptian children. Decreased serum levels of C1q protein and increased titres of C1q antibodies may be involved in the pathogenesis of jSLE, especially LN.     

Charge-based binding of complement component C1q to the Alzheimer amyloid beta-peptide.

Activation of the classical pathway in Alzheimer''s disease derives from the binding of the first protein, subcomponent C1q, to the amyloid beta-peptide (A beta). Analysis of the binding of C1q to A beta by competitive enzyme-linked immunosorbent assay shows that A beta fragments 1-16 and 1-28 but not 12-28 and 17-42 are capable of inhibiting the A beta/C1q interaction, implicating the A beta 1-11 region as the C1q binding site. Binding is also shown to be inhibited by conditions of high ionic strength, suggesting that charged side chains in the A beta 1-11 region are critical to the A beta/c1q interaction. Ultrastructural evidence of binding is provided by platinum replica electron microscopy. Along with a previous demonstration of the 14-26 region of the C1q A chain as the A beta binding site, these findings suggest that attractions between a negative charge cluster in A beta 1-11 and a positive charge cluster in C1qA14-26 mediate the binding of A beta and C1q.     

Monoclonal Antibodies Specific for Sendai Virus I. Production and Characterization of Monoclonal Antibodies

Twelve monoclonal antibodies specific for Sendai virus were prepared by fusing immune LOU rat spleen cells with Y3 or FO myeloma cells. Six antibodies bound the viral glycoprotein HN, and six the viral protein F. Among the six HN-specific monoclonal antibodies, five reacted with the very same epitope and inhibited viral haemagglutination. Two antibodies against the F protein recognized the same epitope, but all the others reacted with different epitopes. All monoclonals were characterized with regard to specificity, biological function, epitope recognition, isotypes, and pI.     

Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.     

Rapid three-step purification procedure for isolation of the C1q component of human complement and its use in a solid-phase C1q binding assay

A new procedure for the isolation of the C1q subcomponent of complement from human sera has been devised. The 3-step protocol employs DEAE Sephadex A-50, hydroxyapatite and Sephacryl S-200 chromatographies and can be performed within 9 h. It yields immunoglobulin-free homogeneous C1q protein with about 80% recovery. The isolated C1q protein is biologically active and may be used for the detection of circulating immune complexes in sera by the solid-phase C1q binding assay.     

Monoclonal antibodies to subgroup 1 rotavirus.

Subgroup 1-specific monoclones were analyzed and used successfully in an enzyme-linked immunosorbent assay to recognize certain subgroup 1 rotaviruses.     

Monoclonal antibodies to human interferon-gamma. II: Antibodies with neutralizing activity

Two types of hybridomas secreting monoclonal antibodies (MAB) with neutralizing activity against human interferon-r (HuIFN-r) were obtained by somatic cell hybridization between mouse myeloma P3UI cells and spleen cells from BALB/c mice immunized with purified recombinant HuIFN-r and a conjugate of a synthetic amino-terminal peptide (residues 4-21) of HuIFN-r and bovine thyroglobulin. One of these MAB recognized the amino-terminal region (residues 4-21) of HuIFN-r, and the other recognized some region in the internal part (residues 22-130) of the molecule. These results suggest that another region, in addition to the amino-terminal region, contributed to the biological activity of rHuIFN-r. A combination of one of these MAB and a previously described MAB directed to the carboxyl-terminal region (residues 131-146) of HuIFN-r enables a quantitative and sensitive assay method of biologically active rHuIFN-r.     

Effect of Pseudomonas aeruginosa elastase and alkaline protease on serum complement and isolated components C1q and C3.

The present study was undertaken to examine and compare the direct effect of two Pseudomonas enzymes, elastase and alkaline protease, on the serum hemolytic complement as a whole, and on the two recognition molecules of complement, C1q and C3 in particular. The results of our study show that incubation of serum with 0-50 micrograms/ml elastase or protease (60 min, 37 degrees C) resulted in a dose-dependent depletion of hemolytic complement with the protease being 3-4 times more efficient than elastase. Incubation of highly purified C3 (20 hr, 37 degrees C) with protease (2% w/w) resulted in the conversion of the 190-kDa molecule to a 120-kDa fragment. When analyzed by SDS-PAGE under reducing conditions, the 120-kDa piece yielded three distinct bands: an intact 75-kDa beta-chain and two alpha-chain pieces of approximately 41- and 26-kDa. NH2-terminal end sequence analysis localized the 26-kDa fragment within the cysteine-rich 41-kDa, COOH-terminal piece. This in turn suggests that the 70-kDa fragment which is not accounted for on SDS-PAGE is derived from the NH2-terminal end of the alpha-chain molecule which is completely degraded into small fragments. While the degradation pattern obtained with elastase is similar to that of protease, the latter enzyme was found to be more efficient. Exposure of C1q (0-5 hr, 37 degrees C) to protease or elastase on the other hand appears to reveal preferential sensitivity of the 28-kDa A-chain and 24-kDa C-chain, of the C1q molecule, with the protease being more potent than the elastase. Since both C1q and physiologic fragments of C3 (C3b, iC3b, and C3dg) are important opsonins of varying efficiencies, degradation of these molecules by Pseudomonas enzymes may, in part, facilitate the survival and proliferation of the organism in plasma. Furthermore, degradation of the key recognition molecules of complement, C1q and C3, would enhance the virulence of this organism by aborting complement-mediated bacterial killing. In addition the results imply that during Pseudomonas bacteremia, PaAP may be a much more destructive enzyme than PaE with regards to C3 and C1q but combined, the synergistic effect may overwhelm not only the proteins of the complement system, but other proteins of the humoral immune defense system as well.     

Antibodies against C1q in anti-glomerular basement membrane nephritis.

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.     

Binding of soluble immune complexes to Raji lymphocytes. Role of receptors for complement components, C1q and C3-C3b.

We have found that, although the binding of particulate antigen-antibody complement complexes such as EAC to lymphoblastoid Raji cells is mediated largely through receptors for C3b, the binding of complement-containing soluble complexes such as those prepared with aggregated human IgG (AHG) occurs also via receptors for C1q. Evidence supporting this conclusion included: (1) Binding of AHG to Raji cells takes place after incubation in EDTA serum; (2) Binding of AHG does not occur in C1q deficient EDTA serum but does take place after addition of C1q; (3) The extent of binding of AHG in EDTA serum is a function of the amount of C1q present; (4) Raji cells can bind up to 5-4 times 10(5) molecules of 125I C1q per cell which can be blocked by unlabelled C1q; (5) AHG pre-incubated with C can bind to a T-cell line MOLT, which lacks receptors for C3b but possesses receptors for C1q to the same extent as Raji cells; (6) Immunoassays for immune complexes in human sera yield similar results whether Raji cells, MOLT cells or C1q precipitation is used for assay; (7) EAC-Raji cell rosettes can be inhibited with inulin-treated, C1q deficient serum containing C3b or C3d whereas binding of AHG or immune complexes in patient samples to Raji or MOLT cells is not inhibited by this reagent. We conclude that receptors for C1q on certain B and T lymphocytes may play an important role in physiologic functions of lymphocytes depending on binding of soluble immune complexes to their surfaces.     

Monoclonal antibodies to Escherichia coli beta-galactosidase and their use for detection and purification of recombinant expression products.

A panel of seven mouse monoclonal antibodies (BG-01-BG-07) was prepared against beta-galactosidase derived from E. coli. The antibodies are beta-galactosidase specific, show no cross-reactivity with other E. coli proteins and can be used for identification and characterization of beta-galactosidase fusion proteins expressed in lambda expression vectors. One of the antibodies allows a simple, one-step isolation of the fusion proteins directly from the crude bacterial lysates using immunoaffinity chromatography.