Sensitized Sheep Red Cells Research Articles

Purification and properties of anti- trypanosoma cruzi antibodies isolated from patients with chronic Chagas' disease

Three purified human IgG antibodies against Trypanosoma cruzi (anti-F, anti-Mc and anti-Cs) showed different reactivities in vitro. While all of them agglutinated specifically sensitized sheep red cells the complement-fixing capacity of anti-Fc and anti-Cs was higher than that of anti-Mc antibody. This one also showed a minimal precipitating activity. When the ability to neutralize mice bloodstream trypomastigotes was analyzed, the results obtained indicated that anti-F antibody was the most effective.

Inhibition of whole blood antibody dependent cellular cytotoxicity by heat aggregated human IgG

Inhibition of the whole blood antibody dependent cellular cytotoxicity (ADCC) of a lymphoblastoid cell line by heat aggregated human IgG (HAI) is described. Optimal sensitising antibody and effector cell concentrations were established to permit the detection of 0.1 μg/ml HAI. Conditions which avoid the ADCC inhibitory effects of normal human sera were defined. Twelve paired normal human sera were stored at −70°C for prolonged (> 3 years) and shorter (<6 months) periods of time. Sensitised sheep red cells were used to determine complement depletion in serum dilutions heated at 40°C, 45°C, 50°C and 56°C. Concentrations of human IgG (1 000–0.1 μg/ml) were prepared in foetal calf serum before heating at 63°C for 30 min to aggregate the IgG. ADCC inhibitory activity, frequently characteristic of normal human serum was minimised (<10%) when sera were stored at −70°C and heated to 50°C for 30 min to inactivate serum complement. This assay provides an economical, reproducible and sensitive test for the detection of circulating IgG complexes.

A simple method for detecting complement-fixation by autologous platelets in autoimmune thrombocytopenic purpura.

A new modification of the microtitre complement fixation test, (CFT), is described for the detection of platelet-bound antibodies (PBA). The test was positive in 12 out of 16 patients, (75%), with active autoimmune thrombocytopenic purpura (AITP). It was negative in four patients who were in remission of AITP when tested, in 10 patients with non-immune thrombocytopenia and in 51 normal blood donors. This is a semi-quantitative method in which suspensions of the patients' own platelets consume complement and therefore prevent the lysis of sensitised sheep red cells (SRBC). Sera from some of these cases were also tested for serum anti-platelet antibody (SPA) and immune complexes. The possible mechanisms and the relevance of positive results are discussed.

Quantitative determination of circulating immune complexes by inhibition of the hemolytic activity of polyclonal IgM rheumatoid factor

A practical and sensitive method for detection and quantification of soluble complement-fixing immune complexes in sera of patients with various disease states has been developed. The assay is based on inhibition of complement-dependent sheep red cell hemolysis mediated by polyclonal IgM rheumatoid factor. Aggregated human IgG was used as an in vitro model of C-fixing human immune complexes and was quantified by its ability to inhibit hemolysis of sensitized sheep red cells by isolated IgM rheumatoid factor. The limit of sensitivity of this assay was 1–3 μg/ml. Fixation of complement in competition with isolated IgM rheumatoid factor, resulting in inhibition of hemolysis of sensitized sheep red cells, was used for detection and quantification of immune complexes in human sera. IgM rheumatoid factor was incubated with sensitised sheep red cells followed by addition of test sera; guinea pig complement was added; and the amount of IgM rheumatoid factor mediated hemolysis was determined spectrophotometrically and referred to a standard curve of inhibition of hemolysis by increasing amounts of aggregated human gamma-globulin. Good discrimination between sero-positive rheumatoid arthritis and systemic lupus erythematosus patients compared with normal and hospitalized subjects was found.

Protein-A-Hämagglutinationstest, eine zuverlässige Methode zur schnellen Identifizierung von S. aureus

369 staphylococcal strains isolated from clinical material were examined for tubeagglutination with sensitized sheep red cells in a standardized assay to study its reliability for routine identification of S. aureus. Colonies isolated from blood agar plates correlated in 99.5% with the (optimized) coagulase reaction. The test is easily to perform and results can be read after 2 hours, whereas the reference methods coagulase, hyaluronidase and deoxyribonuclease took as much as 24 h. The reliability of these tests is discussed.

BPO-Specific, Complement-Dependant Cell-Lysis of Differently Sensitized Sheep Red Cells: Evaluation of Haptenic Groups and their Influence on IgM- and IgG-induced Lysis

Sheep erythrocytes were coated with bencylpenicilloyl-(BPO)groups. Different incubation periods resulted in erythrocyte preparations with different hapten density. Complement dependent lysis induced by IgM or IgG antibodies was studied with the cell preparations. The calculation of hapten density on the erythrocyte surface was not possible by direct measurement of coupled radioactive BPO, since more than 90% of radioactive material was found in the soluble supernatant after osmotic cell lysis and less than 10% was fixed to the cellular membrane. Measurement of membrane bound immunologically relevant BPO-groups was achieved, therefore, by comparison of the inhibitory capacity of the test cells with that of a standard cell preparation. The latter consisted of tannic acid treated erythrocytes coated with protein complexed radioactive BPO. Surface hapten density of the different target cell preparations varied between 1.9 × 105 and 4.8 × 105 BPO-groups per cell depending on the time of incubation. Complement dependent antibody mediated cell lysis was significantly reduced by reduction of haptenic sites per target cell, IgG induced lysis being much more affected than hemolysis induced by IgM antibodies. Statistical calculations led to the conclusion that 18,000 protein islets per cell bearing 4 or more BPO-groups are not sufficient for hemolysis induced by IgG antibodies. 48,000 protein islets with this hapten density are necessary for «optimal« sensitization. IgG antibodies must be apparently bound to the cell surface in bivalent form.

Immunochemical behaviour of sheep non-precipitating antibodies isolated by immunoadsorption

A comparative study has been made of the precipitating and non-precipitating sheep antibodies in the γ 1 and γ 2 immunoglobulin fractions. The γ 1 precipitating and non-precipitating antibodies possess heterocytotropic activity and are able to fix complement, while those of γ 2 possess homocytotropic activity and do not fix complement. It is only the γ 1 precipitating antibody that originate a reverse Arthus phenomenon. All precipitating and non-precipitating antibodies are capable of agglutinating specifically sensitized sheep red cells. The non-precipitating antibodies purified by immunoadsorption are bivalent and make their appearance throughout immunization until representing 10–15% of the total antibodies. While the K 0 values of γ 1 and γ 2 non-precipitating antibodies are lower than those corresponding to precipitating antibodies, such values increase progressively and are never sufficiently low to consider them responsoble for the non-formation of insoluble complexes with the antigen on the part of non-precipitating antibodies.

Decreased antigenicity of mouse blastocysts after activation for implantation from experimental delay.

The antigenicity of mouse blastocysts in experimentally delayed implantation and after activation from delay by estradiol administration were determined by two different methods. CBA blastocysts were incubated in C57BL/6J anti-CBA serum, and the amount of antibodies bound to the blastocysts was traced by -125-I-conjugated sheep antimouse gamma-globulin (isotope antiglobulin technique) and sensitized sheep red cells (mixed haemadsorption technique). With both techniques it was possible to demonstrate that mouse blastocysts in experimental delay of implantation possess alloantigens, and that this antigen expression has decreased markedly 14 hr after activation for implantation. It is suggested that this phenomenon may be of significance for noncognition of the allogeneic conceptus during pregnancy.

Glutaraldehyde fixed-antigen sensitized stable cells in indirect hemagglutination test for rapid diagnosis of hydatid disease, trichinosis and amoebiasis

Glutaraldehyde fixed and antigen sensitized sheep red cells (SRBC) refrigerated or lyophilized in phosphate buffered saline, pH 7·2 (PBS) or in PBS containing 3% normal rabbit serum (PBS-NRS) were stable for more than 6 months in IHA test against sera from patients with hydatid disease, trichinoisis and amoebiasis. The final concentration of protein in hydatid, trichinella and amoebic antigens used for sensitization of SRBC was 0·43, 0·75 and 1·2 mg/ml, respectively. The lyophilized cells in general showed a slightly higher sensitivity but within acceptable limits. By contrast the sensitized cells refrigerated in PBS consistently showed a uniform reactivity and IHA titres were reproducible even 6 months after sensitization of the cells. No demonstrable change was observed in the sensitivity of the test when the PBS-refrigerated cells sensitized 6 months before were exposed to room temperature for 4 days. The following are the main improvements on existing serological tests: (a) a more rapid and reproducible test for sero-epidemiological investigations in areas with minimum laboratory facilities, (b) sensitized cells could be shipped to various laboratories without losing the sensitivity of the test, (c) reduction in the cost of personnel and reagents where these tests are needed infrequently.

Capacity of group A, B, C, D, and G streptococci to agglutinate sensitized sheep red cells.

The reactivity of streptococci with sheep red cells, sensitized with a subagglutinating dose of rabbit anti‐sheep red cell antibodies was studied; agglutination of sensitized and not non‐sensitized sheep red cells by streptococci was demonstrated in isolates belonging to groups A, B, C, D and G. The frequencies of isolates which had the capacity to agglutinate were 97, 88 and 100 per cent in group A, C and G, but only 47 and 33 in group B and D. The agglutination of sensitized sheep red cells by streptococci diminished after treatment of the streptococci with protease from Streptomyces griseus, but was not affected by pepsin or trypsin treatment. Heating the streptococci at 60°C for 30 minutes and repeated subculturing did not suppress their agglutinating activity. Besides, various streptococcal extracts agglutinated sensitized sheep red cells. The presence of an immunoglobulin G reactivity in streptococci might cause certain difficulties in the serological grouping and typing of streptococci.

A Surface Component in Group A, C, and G Streptococci with Non-Immune Reactivity for Immunoglobulin G

Abstract Fifty-seven strains of streptococci belonging to groups A, C, and G were tested for γ-globulin Fc-reactivity with sensitized sheep red cells. Twenty-six per cent of the strains were positive. Forty-four per cent of the strains produced a soluble factor with similar γ-globulin reactivity. With a radioimmunologic assay to detect streptococcal reactivity, human IgG was found to be positive, whereas human IgA, IgM, IgD, and IgE were negative. The reactivity was detected in heavy chain preparations but not in light chains, F(ab′)2 fragments nor in Fc fragments. Experiments using isolated myeloma globulins representing the four IgG subclasses indicated that streptococci might react not only with IgG1-, IgG2-, and IgG4-like protein A but also with IgG3 molecules. One of the two IgG3 myeloma globulins studied did react with a group A streptococcus and both myeloma globulins did react with a group G streptococcus. Serum samples from 14 selected animal species also indicated differences between the γ-globulin reactivity of protein A and streptococci. Marsupials as well as the Rhea bird were non-reactive with streptococci. The results obtained with eleven other species indicated that the group A and G streptococcal reactivities were similar but differed significantly from staphylococcal protein A reactivity.

SENSITIZED SHEEP RED CELLS AS A REACTANT FOR STAPHYLOCOCCUS AUREUS PROTEIN A

Staphylococci with cell‐bound protein A reacted with sensitized sheep red cells in a simple slide reaction with agglutination. Extracellular protein A proved capable of preventing the sensitized sheep red cells from reacting with cultures of Staph, aureus strains containing protein A. This confirms the reactivity of protein A with free Fc‐fragments of IgG on the surface of the sensitized sheep red cells as the cause of haemagglutination. The choice of culture medium proved important for demonstrating protein A. Peptone‐free media proved clearly unsuitable. 341 routinely isolated strains of Staph. aureus were examined for agglutination of sensitized sheep red cells in the slide test and for tube‐agglutination of sensitized sheep red cells with broth culture filtrate. 88.3 per cent of the strains were positive if examined on slides, while 6.7 per cent of the strains contained no cell‐bound or free protein A demonstrable by either method. Among the methicillin resistant strains, 50 per cent showed no agglutination on slide. Antibiotic‐sensitive strains belonging to phage group II tended to have weaker protein A reactivity than other groups.

Libération d'un composant du complément par des leucocytes humains in vitro

Human blood leucocytes were shown to liberate active C1q in vitro. An agarose gel containing sensitized sheep red cells and a serum depleted of C1q has been used to detect C1q activity. A population of mononucleated cells which display such activity could be isolated by centrifugation on an albumin gradient.

Inhibition of complement by a substance isolated from human erythrocytes—II: Studies on the site and mechanism of action

Extracts from human E stromata were found to be capable of causing inhibition of the complement mediated lysis of antibody sensitized sheep red cells. The partially purified inhibitor (I-H) could apparently bind spontaneously to sheep E, but another form of the inhibitor which was obtained by butanol extraction of human E stromata at a low ionic strength (I-L) would not bind. The latter material was employed in experiments designed to elucidate the site of inhibitory action. The low ionic strength form of the inhibitor was found to cause inactivation of shepp EAbC′la, 4, 2a, EAbC′la, 4, 2a, 3, and EAbC′la, 4, 2a, 3, 5 compelement component intermediate complexes. Indications were that the inhibition resulted from an increase in the rate of SAbC′la, 4, 2a decay to the SAbC′la, 4 state. Guinea pig C′3 was also observed to accelerate SAbC′la, 4, 2a decay.

Aspects of the Immune Hemolytic Reaction

Summary Sheep serum contains inhibitors that prevent the lysis of sensitized sheep red cells by guinea pig, pig or human C′, but do not interfere with the sensitization of the cells by rabbit antibody. The presence of a relatively large proportion of sheep serum during the formation of EAC′1,4,2 by guinea pig C′ results in slight loss of C′1 and C′2 activities on these cells and no detectable change in C′4 activity. The inhibitors affect primarily the C′3 step. Sheep serum, however, lyses sensitized human erythrocytes, and human serum inhibits the lysis of type A human erythrocytes by sheep C′. These results suggest a reason why a given C′ may be less active in lysing sensitized homologous red blood cells.

Studies on the antigenic properties of complement. I. Demonstration of agglutinating antibodies against guinea pig complement fixed on sensitized sheep erythrocytes.

Evidence is presented to show that guinea pig complement fixed on sensitized sheep red cells acts as a specific agglutinogen. Agglutinating antibodies that react with cell-fixed complement can be produced by immunizing rabbits with a complex of stromata-amboceptor-complement or with guinea pig serum globulin. These agglutinins can be removed by precipitation with guinea pig serum. They are, therefore, distinct from immunoconglutinins.

Concerning the Mechanism of Complement Action

Summary C′1 and TAMe esterase activity can be eluted from sensitized sheep red cells containing C′1 activity by ethylenediaminetetraacetic acid. On dialyzing the supernatant from cells so treated against a low ionic strength buffer, both activities can be recovered. The C′1 activity eluted from red cells in this fashion can be detected with case by a two-step procedure of adding the “eluate” to sensitized cells, washing and then adding an R1. If an R1, sensitized cells and the “eluate” are added together little or no evidence for C′1 activity is obtained. The properties of the enzyme when removed from the cell are the same so far as studied as when the enzyme is attached to the cell. When sensitized cells are treated with the eluate not only does C′1 activity attach itself to the cells but esterase activity as well. Allowing the eluate to stand in the cold at pH 3.3, affects its TAMe esterase activity little of at all; however, the C′1 activity and the ability of the esterase to attach to sensitized cells is destroyed and, as kinetic studies showed, destroyed to the same extent. In explanation of these latter observations the hypothesis was evoked that the C′1 molecule contains two distinct sites, a binding site and an enzymatic site, and both are required in order for the molecule to manifest C′1 activity.

SERUM FACTOR IN RHEUMATOID ARTHRITIS AGGLUTINATING SENSITISED SHEEP RED CELLS

SERUM FACTOR IN RHEUMATOID ARTHRITIS AGGLUTINATING SENSITISED SHEEP RED CELLS