Alternative pathway-mediated rebinding of immune complexes to human red blood cells.

Antigen-antibody complexes (Ag-Ab) prepared from 125I-bovine serum albumin (BSA) and guinea-pig anti-BSA were (i) incubated at 37 degrees for 4 min with undiluted normal human serum and autologous red blood cells (RBC) together, or (ii) incubated first at 37 degrees for 30 min with serum diluted optimally for binding and then with RBC. Reactions were stopped by dilution and cooling, and RBC bearing antigen-antibody-complement complexes (Ag-Ab-C) were washed and resuspended in either untreated normal human serum (undiluted or diluted), serum treated with zymosan (SZYM), ethylenediamine tetracetic acid (SEDTA) or ethyleneglycol tetracetic acid-Mg++ (SEGTA), serum heated at 56 degrees for 30 or 120 min (S delta 30 or S delta 120), or buffer alone. The mixtures were placed at 37 degrees and the percentage of Ag-Ab-C dissociated from RBC after progressively increasing times determined. (i) Ag:Ab:C bound to RBC with undiluted serum dissociated more rapidly following resuspension in SZYM, SEDTA, or S delta 30 than following resuspension in untreated serum. Rate of dissociation in SEGTA paralleled that in untreated serum. (ii) Ag-Ab-C bound to RBC with diluted serum dissociated following resuspension in SZYM, SEDTA, or S delta 30, but rebound and dissociated a second time following resuspension in untreated serum or SEGTA. Initial dissociation occurred in less than 1 min in undiluted serum, took place at 0 degrees as well as 37 degrees, and was diminished but detectable in greater than 8- and greater than 64-fold-diluted serum, respectively. Rebinding required 37 degrees, factors B and D and C3, and was maximal at 4-8 min. Subsequent dissociation had similar complement requirements to initial dissociation, but occurred only at 37 degrees and was 90% complete at 15 min. No dissociation of Ag-Ab-C bound to RBC in (i) or in (ii) occurred following resuspension in S delta 120 or in buffer. These findings suggest that after initial binding, release of experimental immune complexes from RBC in whole serum involves concurrent dissociation and alternative pathway-dependent rebinding.     

Differences in interaction between immune complexes and complement receptors in serum and cerebrospinal fluid

Antigen—antibody complexes (Ag—Ab), prepared from ¹²⁵I-radiolabelled bovine serum albumin (BSA) and guinea-pig antibody, were (1) pre-incubated at 37°C for 30 min with serum or cerebrospinal fluid (CSF) in different proportions and then reacted with cells, (2) incubated at 37°C directly with cells suspended in serum or CSF for different time periods, or (3) bound to cells (following incubation with serum in optimal proportions) and the cell-bound immune complexes (IC) incubated with serum or CSF at 37°C for different time periods. When Ag—Ab were pre-incubated with serum or CSF and reacted with unfractionated blood cells or mononuclear cells, binding decreased as serum to Ag—Ab proportion was increased above 1:16, but increased as CSF to Ag—Ab proportion was increased. When serum diluted 200-fold (to approximate the protein concentration of CSF) was used in place of undiluted serum, serum-mediated binding paralleled CSF-mediated binding. Inactivation of serum, CSF, and 1:200 serum in different ways and substitution of human red blood cells (RBC) (known to possess C3b receptors) or sheep RBC (known not to possess C3b receptors) demonstrated that binding was to C3b receptors. Addition of CSF to serum did not alter serum-mediated binding. When Ag—Ab were incubated directly with unfractionated blood cells suspended in serum or CSF, binding increased rapidly in serum, reaching a maximum within 2—4 min, and IC then rapidly dissociated, whereas binding increased gradually in CSF and IC remained associated with cells. When serum diluted approximately 100-fold was used in place of undiluted serum, kinetics of serum-mediated interaction approached that of CSF-mediated interaction. When IC were bound to Raji cells or human RBC and the cell-bound IC incubated in serum or CSF, > 85% of IC dissociated in serum after 30 min, but no dissociation occurred in CSF. Dilution of serum > 1:16 and > 1:64 abolished dissociation from the two cell types, respectively. These results indicate that CSF mediates binding of IC to complement receptors on cells but lacks the activities of serum which convert IC into a non-binding state.     

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.     

Immunochemical and electron microscopic analysis of the high-molecular structures in the tick-borne encephalitis virus

High-molecular structures of tick-borne encephalitis virus were sedimented by centrifugation from virus-containing culture fluid and separated by sucrose concentration gradient centrifugation into 3 main fractions: rapidly sedimenting virions, the fraction sedimenting at a moderate rate, represented by "stellate" structures, and the fraction found on the top of the gradient and represented by structures similar in size and shape to virions, designated slowly sedimenting virions. The latter are first described in the paper. In immunodiffusion, the rapidly sedimenting virions form 1 of the 2 precipitation bands closer to the antigen-containing well, and in immunoelectrophoresis give 2 precipitation bands, anodic and cathodic, which fuse, indicating immunological identify of the 2 subpopulations of rapidly sedimenting virions. The slowly sedimenting virions in immunodiffusion form one of the 2 precipitation bands closer to the antigen well, and in immunoelectrophoresis form the cathodic precipitation band. The material sedimenting in the gradient at a moderate rate gives in immunodiffusion one precipitation band, the 3rd from the antigen well, and in immunoelectrophoresis forms a separate anodic precipitation band. This high-molecular non-virion antigen is found in small amounts also in fractions containing the rapidly sedimenting and slowly sedimenting virions. It is at least partially antigenically identical to previously studied low molecular non-virion ("soluble") antigen of tick-borne encephalitis virus.     

Effect of age on physico-chemical properties of the uterine nuclear estrogen receptors of albino rats

Estrogen receptors (ER) were extracted with high ionic strength buffer from the nuclei of uteri of young (21 weeks) and old (89 weeks) rats. Following the analysis of these receptors on a sucrose gradient and a Sephadex column, two peaks representing the two forms of receptors were obtained. The minor peak sedimented at 6.8 S with Stokes radius (RS) = 7.2 nm, molecular weight (Mr) = 204 K and frictional ratio (f/f0) = 1.71. On the other hand, the major peak sedimented at 4.1 S with RS = 3.3 nm, Mr = 57 K and f/f0 = 1.18. These properties of nuclear ER were similar in both ages. Also, the half life of ER complexes from both ages at 25 degrees C and 37 degrees C were 135 min and 30 min, respectively. However, these complexes were retained for longer periods in the nuclei of young than old rats. Furthermore, the dissociation constant of the binding of nuclear receptors to estrogen remained constant, but the number of binding sites decreased from 1.56 in young to 1.05 pmol/mg DNA in the old. In young rats, about 61% of nuclear receptors bound to DNA-cellulose. Out of this 2/5 was eluted with 0.15 M and the remaining 3/5 with 0.5 M KCl. On contrary, only 37% of total receptors bound to DNA-cellulose in the old. Out of this 3/5 was eluted with 0.15 M and the remaining 2/5 with 0.5 M KCl. These data suggested that despite the similarity in different physicochemical properties, the number of estrogen binding sites and the retention time of ER complexes in nuclei and the ability of these complexes to bind to DNA decrease in the uterus of old age.     

Interaction of Complement-Solubilized Immune Complexes with CR1 Receptors on Human Erythrocytes. The Binding Reaction

The binding of complement (C)-solubilized ¹²⁵I bovine serum albumin (BSA) anti-BSA immune complexes (IC) to CR1 receptors on human red blond cells (RBC-CR1) was studied. The binding of IC to CRI was strongly dependent on the molar antigen lo antibody ratio, and IC formed in moderate antigen excess showed no binding. IC solubilized, in 50% human serum in the presence of autologous RBC bound rapidly lo RBC-CRI, with maximal binding within less than 1 min at 37°C. Release of CRI-hound IC under these conditions occurred slowly, requiring more than.30 min. Only binding of 'partially' solubilized, e.g., anti C3c (C4c) and conglutinin-reactive 1C occurred, whereas fully solubilized complexes (IC-C3dg. C4d) showed virtually no binding. Solubilization of IC in the presence of Mg-EGTA or in C2-deficient serum resulted in a markedly delayed binding of IC ti RBC, indicating the importance of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solobilized IC could be absorbed to solid-phase conglutinin or antibody to C3c abd C4c, and tgese kugabds were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect on the binding of presolubilized IC to RBC-CR1, whereas the binding was unaffected by the addition of monosaccharides or by the concentration of Ca²⁻ or Mg²⁻ ions.     

Release of Immune Complexes Bound to CR1 on Erythrocytes; Suramin Inhibits Factor I in the Presence of EDTA

An experimental model was established in order to study the release of immune complexes (IC) bound by complement C3b receptors (CR1) on human erythrocytes (RBC). Soluble tetanus toxoid anti-tetanus toxoid complexes were incubated with RBC in the presence of autologous serum at optimal conditions for binding. The RBC carrying complement-opsonized complexes were incubated with appropriate serum reagents, and it was shown that factor I was required for release of the complexes, which occurred without loss of CR1. Suramin was, irrespective of factor I, found to induce release of CR1-bound IC in the absence of EDTA, whereas factor I-mediated release was inhibited by suramin in the presence of EDTA. EDTA probably interfered through a charge-dependent interaction. These observations are decisive for the interpretation of in vitro experiments involving these reagents. The combination of EDTA and suramin was found inappropriate for use in quantitative determination of in vivo CR1-bound IC.     

Complement fixation by small, DNase-resistant DNA-anti-DNA immune complexes

125I-ds DNA-anti-DNA immune complexes (IC) formed at antibody excess and containing DNA of 300-350 base pairs (bp) fixed complement, incorporated C3b and bound to the C3b receptors (CR1) on human red blood cells (RBC). When the IC were treated with DNase to generate small, DNase-resistant IC, some of the IC incorporated C3b, but did not bind to RBC. In order to examine C3b incorporation and RBC binding by IC of specific sizes, the DNase treated IC were fractionated by sucrose density gradient (SDG) ultracentrifugation. Small IC containing one, two, three or four IgG molecules per fragment of 125I-ds DNA were identified by autoradiography after electrophoresis of the SDG fractions on 3-12% linear polyacrylamide gradient gels. The SDG fractions were tested for C3b incorporation and RBC binding ability. There was neither C3b incorporation nor RBC binding activity in fractions which corresponded to 9-11S (containing IC with one IgG/DNA). Fractions which corresponded to 12-22S (containing IC with up to four IgG/DNA fragment) demonstrated increased C3b incorporation with increased size, but did not show significant RBC binding activity. Fractions with IC containing four or more IgGs (22-24S) incorporated C3b and bound to RBC at approximately the same level. It is concluded that DNase digested IC which contain three-four IgG/DNA fragment are large enough to activate complement and incorporate C3b, but are too small to bind to RBC CR1. These IC could therefore escape rapid clearance from the circulation via the erythrocyte CR1 clearance mechanism. Such IC could persist in the circulation and potentially elicit pathogenic effects in patients with systemic lupus erythematosus.     

Infection of C3HeB/FeJ mice with the docile strain of lymphocytic choriomeningitis virus induces autoantibodies specific for erythrocyte Band 3.

C3HeB/FeJ mice infected with the docile strain of lymphocytic choriomeningitis virus (LCMV-d) develop a persistent infection with a transient haemolytic anaemia. Immunoglobulin can be eluted from the red blood cells (RBC) of these mice but it cannot be detected on the RBC by a conventional antiglobulin test. The present study demonstrates that RBC from such mice bear erythrocyte autoantibodies which are predominantly of the IgG2a subclass, with lower levels of autoantibodies of the IgG1, IgG2b and IgG3 subclasses. To identify the target antigen the autoantibodies were eluted from the RBC of LCMV-infected mice. The eluted autoantibody bound to intact normal RBC and precipitated a 105000 MW component that corresponds to murine Band 3 protein. A monoclonal antibody derived from mice infected with LCMV-d also precipitated mouse Band 3, and reacted specifically by enzyme-linked immunosorbent assay against a purified preparation of Band 3. This study has shown that in C3H mice infected with LCMV-d which develop autoimmune haemolytic anaemia, the target autoantigen is erythrocyte membrane Band 3.     

Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes.

Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The alternative complement pathway control protein H binds to immune complexes and serves their detection

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.     

Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus

C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.     

Anti-I antibody in normal human newborn infants

A cold agglutinin with anti-I specificity is present in a high percentage of cord sera. Evidence is produced that this antibody, like the `naturally-occurring' anti-I present in adult serum, is not a γG-globulin; thus the antibody, both in cord serum and in adult serum, is inactivated by treatment with 2-mercaptoethanol, is not eluted with γG-globulin using DEAE-cellulose chromatography and is recovered in the fast sedimenting fractions after ultracentrifugation. Furthermore, the antibody does not disappear from the circulation of newborn infants as do γG antibodies which cross the human placenta.

These findings indicate that the antibody in cord serum is a γM-globulin and that its production often begins before birth.

     

Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell

We have studied the interaction between C3 and natural human and rabbit anti-MTX IgM and hyperimmune IgG antibody bound to red cells to which MTX was covalently coupled. IgM Ab molecules bound to the cell surface were measured by their interaction with rabbit anti-human mu-chain IgG Ab: the bound anti-mu-chain or anti-MTX IgG was quantitated with 125I-labelled PA. C3 uptake by EMTX AC142 complexes from purified preparation of C3 was detected by the interaction of bound C3 with rabbit anti-C3 IgG Ab; the bound IgG was then measured with radiolabelled PA. In some experiments the uptake of C3 by the EMTX AC142 complexes was measured by using 125I-labelled C3. To find out if C3 was bound to Ab molecules, anti-MTX Abs were eluted from the cells by excess fluid-phase MTX. After dialysis the eluted anti-MTX IgM or IgG Ab was than reattached to fresh EMTX. All cells were then analyzed for Ab and C3 content by a radioimmunoassay. It was found that when anti-MTX IgM or IgG was eluted from EMTX AC423 complexes no C3 was removed from the cells and that eluted IgM or IgG did not carry with them either C3 antigen or 125I-labelled C3. It was concluded that, during the activation of the classical C-pathway at the red cell surface, no C3 was bound to IgM (rabbit or human) or IgG (rabbit) antibody molecules.     

献血者献血前后红细胞免疫功能和T细胞亚群水平的变化

目的：探讨了献血者献血前后红细胞免疫功能和T细胞亚群水平的变化。方法：分别应用红细胞酵母花环法和单克隆抗体法,对40例献血者进行了献血前后红细胞免疫功能和T细胞亚群水平的测定。结果：献血后外周血中RBC-C3b、CD3、CD4、CD8水平均显著性提高（P〈0.01）。CD4/CD8比值变化无统计学意义（P〉0.05）。结论：献血能增强献血者红细胞的C3b水平,T细胞亚群比例升高,使机体进入新的免疫平衡状态。     

Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3.

Phagocytosis of Cryptococcus neoformans is markedly influenced by the presence of a polysaccharide capsule. We examined activation and binding of C3 fragments to eight isolates of C. neoformans. All isolates were shown to have capsules by light and electron microscopy. These strains differed in susceptibility to phagocytosis by neutrophils. Yeast cells were opsonized by incubation in normal human serum. Five strains were resistant to ingestion, two strains showed intermediate levels of resistance to ingestion, and one strain was quite sensitive to phagocytosis. Yeast cells opsonized with heat-inactivated serum (56 degrees C for 30 min) neither attached nor were ingested by neutrophils. A quantitative estimate of the amount of C3 bound to the yeast cells was determined by use of normal human serum containing 125I-labeled C3. The results showed approximately 5 X 10(6) to 10 X 10(6) C3 molecules per yeast cell regardless of whether the yeast cells were sensitive or resistant to phagocytosis. Bound C3 was eluted from the yeast cells by treatment with 0.1 M NH2OH (pH 10), and the eluted fragments were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Results of this analysis showed that little of the C3 was in the form of C3b, and there was substantial decay to iC3b, the inactive decay product of C3b. This pattern of decay was similar with all strains. Immunoelectron microscopy was used to assess the ultrastructural location of the C3 fragments bound to the yeast cells. C3 fragments were bound to the perimeter of the capsule regardless of whether the isolate was sensitive or resistant to phagocytosis. Thus, phagocytosis-sensitive and phagocytosis-resistant isolates were similar with regard to the amount, molecular form, and ultrastructural location of C3 fragments bound to the cryptococcal capsule. These results further indicate that activation of the complement cascade is necessary but not sufficient for phagocytosis of the yeast cell.     

Serum and Plasma Fibronectin Binds to Complement Reacted Immune Complexes Primarily via C1q

The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after incubation of the solid-phase IC with serum or plasma at conditions where Clq was also bound, whereas only minor amounts of fibronectin bound to the solid phase IC via C3b. Purified fibronectin showed a dose-dependent binding to solid-phase IC pretreated with Clq or fibronectin-depleted serum, confirming that the binding of fibronectin to IC largely occurred via Clq. Significantly smaller amounts of fibronectin were bound to solid-phase IC incubated with plasma instead of serum, despite the higher fibronectin concentration in plasma. This difference was found not to be due to a fibrinogen-fibronectin interaction in plasma. Binding of fibronectin to preformed fluid phase IC incubated with serum was demonstrated by SDS-PAGE analysis of PEG-precipitated IC.     

Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes

An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.     

Advances in immunoserology tests in clinical medicine: autoantibodies, immune complexes and DNA analysis

I reviewed first the history of detection for autoantibodies and the methods to detect the circulating immune complexes. Then I presented several unusual tests for detecting autoantibodies which I had experienced. They are agglutination tests in agarose gel using the particulate antigens of human tissues or lipid antigens and the mixed agglutination test using the cultured cells. The characteristics of rheumatoid factor(RF) were analyzed by the following methods: 1) solid-phase radioimmunoassay for IgG-RF using the formalinized sheep RBC, 2) mixed agglutination test for IgG-RF on the slide glass smeared with the sensitized sheep RBC, 3) hemolysis in agarose gel for competitive reaction of RF with complement to the IgG hemolysin and 4) ELISA for detecting the complement-fixation of the monoclonal IgM-RF. The IgG-Fc and C3b receptors on the tissue sections of the mammalian aorta were detected by an adhesion of the IgG-sensitized or C3b-bound RBC. DNA-analysis studies using the molecular biology techniques which were done in the Department of Internal Medicine II, Fukushima Medical University were presented. 1) Nucleic acid sequences of the cloned DNA polypeptide fragments in the serum of patients with systemic lupus erythematosus. 2) SSCP analysis for clonality of Vb repertoires in T cell receptors in the patients with rheumatoid arthritis, primary biliary cirrhosis or CREST syndrome. 3) Detection of mRNA of TNFa and Fas ligand in the mononuclear cells(FICL-PCR) in the synovial fluids of RA patients.