Antigen-antibody Systems Research Articles

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.

Studies on the Interference between the Allergic Reactions Caused by Different Antigen-Antibody Systems

Interference between two different in vivo antigen-antibody reactions which occurred simultaneously was studied in guinea pigs. Animals were presensitized with an aqueous fraction (CP) extracted from cells of Corynebacterium equi, strain KO-85. Skin reactions of the Arthus type elicited by the CP fraction inhibited passive cutaneous anaphylaxis (PCA) induced by bovine serum albumin (BSA) and rabbit anti-BSA serum. PCA reactions of the CP-sensitized animals were also inhibited upon simultaneous challenges of BSA and the CP as evidence by reduction of PCA titer of the anti-BSA serum.

Isolation and partial chemical characterization of the three major yolk polypeptides from Drosophila melanogaster

The three major yolk polypeptides of Drosophila melanogaster have been isolated from yolk spheres of early embryos. Their molecular weights, as determined by SDS-polyacrylamide electrophoresis, are 44,000, 45,000, and 46,000. A number of approaches have been used to show that each of these yolk polypeptides are different. They have different isoelectric points, they have different digestion products upon peptide mapping by limited proteolysis, and they show three different antigen-antibody systems when each polypeptide is reacted with an antisera made to a mixture of all three. Both the digestion with chymotrypsin and the immunoelectrophoresis studies indicate similarities between two of the polypeptides while the third appears unique. This is the first example of multiple yolk polypeptides of similar molecular weight.

Assay of antibodies to caliciviruses by radioimmune precipitation using staphylococcal protein A as IgG adsorbent.

A radioimmune assay method designated St-RIP using a staphylococcal IgG adsorbent, which potentially has broad applications to viral (and nonviral) antigen-antibody systems, was applied to detection of calicivirus antibodies. Purified 125I-labeled virions of San Miguel sea lion virus serotypes 4 (SMSV-4) and 5 (SMSV 5) were incubated with sera; the immune complexes were reacted with an immunoadsorbent, formaldehyde-fixed staphylococci (Staphylococcus aureus protein A producer, strain Cowan I), and collected by centrifugation. Broad cross-reactivity was observed among serotypes of SMSV and vesicular exanthema of swine virus (VESV), but there was no reaction with antisera to six noncaliciviruses. Antibody production in a rabbit inoculated with SMSV-5 polypeptide was monitored by St-RIP assay; reactivity with intact SMSV-4 virion antigen was slightly less than, but closely paralleled, reactivity with SMSV-5 virion antigen. Applicability of the St-RIP test to serologic survey was demonstrated with pinniped, swine, and human (laboratory personnel) sera; numerous positive St-RIP reactions suggested the occurrence of widespread contacts with caliciviruses.

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals. Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi. Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection. Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.

Studies on the Interference between the Allergic Reactions Caused by Different Antigen‐Antibody Systems

Interference between two different in vivo antigen-antibody reactions which occurred simultaneously was studied in guinea pigs. Animals were presensitized with an aqueous fraction (

A micro-procedure for quantitative precipitin tests

•A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 μm pore size in a specially designed multisample apparatus, and (2) spectrophotometric determination at 210 nm of the amount of proteins in the antigen-antibody precipatates dissolved in 0.01 N HCl. At this wavelength, the =C=O group of the polypeptide chains constitutes the main chromophoric group.•In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 μg of antigen. The concentration range of protein solutions in 0.01 N HCl which is measured at low UV can be regulated by a factor of 8–10 by changing absorption between 200 and 230 nm.•Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5–6 h. The standard precipitin technique, by contrast, requires a 5–7 -day reaction period for completions.

Determination of the position of electroimmune precipitates of antigen—Antibody systems on their respective quantitative precipitation curves

Using corresponding monospecific antisera from the rabbit, the precipitation curves for albumin, alpha-1-antitrypsin, haptoglobin 1-1 and fibrinogen were produced. In parallel, four different dilutions of these antigens were examined by line immunoelectrophoresis, employing the same antisera. The distance of the resulting line immune precipitates from the start served to calculate the amount of antigen-antiserum reacting with each other. After converting these values to the amount of antiserum used for producing the precipitation curves, the ‘line antigen/antibody ratios’ were drawn into the appropriate precipitation curves. All electroimmune precipitates (in this case, the ’line immune precipitates’) were found to be located on the side of antigen excess of their corresponding precipitation curve.

Recent progress in the study of autoantibodies to nuclear antigens

Autoantibodies to nuclear antigens can now be classified according to their immunologic specificities. They include antibodies that react with DNA, deoxyribonucleoprotein, nuclear histones, and nuclear acidic protein antigens. It has been established that there are several antinuclear antibodies that react with nuclear acidic proteins, and these include antibodies to Sm antigen, nuclear ribonucleoprotein, and SS-A and SS-B antigens. It has also been established that certain systemic rheumatic diseases, such as systemic lupus erythematosus, Sjögren's syndrome, and scleroderma, are characterized by antibodies of some specificities and not of others. Thus, distinct profiles of antibodies to nuclear antigens may be present, and these may be used as diagnostic aids. Further characterization of these specific nuclear antigen-antibody systems may help in unraveling the etiology and pathogenetic mechanisms of these diseases.

Hepatitis B: current concepts

SummaryAfter a short description of the Hepatitis B virus (HBV>, its newly associated antigen-antibody systems are briefly introduced. The natural course of hepatitis is then presented with regard to the evolution of these serological markers, whose clinical significance is discussed. An’mmunercsponse responsible for the destruction °f the HBV infected hepatocytes is proposed and related to the various clinical pictures commonly encountered Virus transmission, epidemiology and prognose of hepatitis B are reviewed and also the Possibilities of prevention, i.e. passive (specific and non-specific gamma globulins) and active immunization.

Quantitative immunoelectrophoretic assay for murine oncornavirus p30: noncovalent facilitation by sodium dodecyl sulfate.

Treatment of Rauscher murine leukemia virus lysates with the anionic detergent sodium dodecyl sulfate (SDS) at concentrations between 0.2 to 2.0% SDS per mg of viral protein greatly increased the anodal electrophoretic mobility of p30, the major internal polypeptide. SDS treatment did not reduce p30 antigenicity or cause nonspecific precipitation of normal serum proteins during subsequent immunoanalysis. The increased anodal electrophoretic mobility allowed assay of Rauscher murine leukemia virus p30 by Laurell rocket immunoelectrophoresis. An SDS-facilitated rocket immunoelectrophoresis assay is described that was highly reproducible (coefficient of variability, less than 3.0%) and capable of detecting 125 ng of viral protein. To our knowledge, this is the first report of a quantitative immunoelectrophoretic assay for an oncornavirus antigen. Since SDS binding is a general property of proteins, this method of noncovalently altering electrophoretic mobility appears to be applicable to other antigen-antibody systems.

Immunochemical studies on the galactomannans isolated from mycelia and culture broths of three Hormodendrum strains

Six major antigenic galactomannans isolated from both mycelia and culture broths of three representative strains of three species of the genus Hormodendrum, H. pedrosoi IFO 6071, H. compactum IFO 6726, and H. dermatitidis IFO 6421, were investigated for their immunochemical properties by precipitin reaction and the passive cutaneous anaphylaxis test. We found that partial acid hydrolysis of the these galactomannans with 0.005 M sulfuric acid yielded free galactose and corresponding acid-resistant core moieties. In agar-gel double-diffusion reactions, no acid-resistant core moiety of galactomannan showed a corresponding precipitin line against homologous antiserum, indicating that the acid-labile galactofuranosyl residues were solely responsible for the precipitin activity of these galactomannans. All galactomannans and their core moieties gave sharp cross-precipitin lines against anti-Saccharomyces cerevisiae serum, specific for the alpha1 leads to 3- or alpha1 leads to 2-linked D-mannopyranosyl residue. Quantitative precipitin reactions of the same antigen-antibody systems showed that all acid-resistant core moieties were completely inactive in the homologous systems but were highly cross-reactive with anti-S. cerevisiae serum. Passive cutaneous anaphylaxis tests using guinea pigs sensitized with homologous antisera or with anti-S. cerevisiae serum provided results consistent with those obtained in the in vitro assay.

Influence of immunosuppressants on the establishment of Paragonimus miyazakii in albino rats.

The present investigation has shown that combined treatment with dexamethasone and prednisolone of with hydrocortisone and dexamethasone enhances the susceptibility of albino rats to Paragonimus miyazakii, presumably by suppressing the host's immune responses. In the rats given these combined treatments, the size and egg production of worms were markedly increased. Use of dexamethasone or prednisolone alone had relatively little influence on the growth and maturity of P. miyazakii. In serological analysis and agar double diffusion, considerable differences were recognized between the gamma-globulin fractions and antigen-antibody systems (precipitin bands) of the treated groups and the untreated group. The mechanism of action of these immunosuppressants in this system remains obscure.

Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Ib, and Ic.

The opsonization and phagocytosis of group B streptococci of types Ia, Ib, and Ic were studied in vitro by measuring the uptake of radioactivity by coverslip cultures of rabbit alevolar macrophages during incubation with radiolabeled, nonviable bacteria which had been exposed to rabbit serum. The uptake of counts per minute was quantitative, reproducible, and reversibly inhibited by cold, indicating that it was largely a measurement of phagocytic ingestion rather than of attachment of bacteria-immunoglobulin complexes to macrophage membranes. Moreover, suspended macrophages killed approximately 90% of viable streptococci in the presence of specific antiserum. The opsonic activity of immune serum was heat stable, and phagocytosis of streptococci was insignificant after incubation with normal serum and antiserum to some heterologous group B streptococci. By absorption studies, it was possible to identify the effect of antibodies to specific bacterial antigens. Phagocytosis of streptococci containing the corresponding antigens was maximal after opsonization with homologous or heterologous sera containing antibody to IaCHO, IbCHO, or Ibc protein. Phagocytosis of all three serotypes was intermediate when opsonization could be attributed to anti-IabcCHO. The opsonization of a specific group B streptococcus is complex and may involve two or more antigen-antibody systems.

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimized and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic sytem. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fact-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic ontamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzy,e markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of potential use in screening immune responses to N. gonorrhoeae.

An immune hemagglutination procedure with very high sensitivity applied to the detection of hepatitis B antibody and antigen and immune complex.

An immune agglutination procedure is described in which red cells are coated with antigen and agglutination is observed after a short incubation with antibody in reagent serum. Conversely, the cells may be coated with antibody and used to test for the presence of antigen. Treatment of the reagent serum with cobra venom factor results in inhibition, and there is a marked lo-s of sensitivity with cells pretreated with formalin or chromic chloride, suggesting that C3 and some type of reactive sites on the cells are required. The distinctive features of the new procedure are that incubation time is very short, sensitivity is several orders of magnitude higher than with other related tests, and, within some limitations, bovine (steer) red cells with bovine serum can be used as an alternative to human red cells with human serum. It is necessary to remove an inhibitor from the reagent serum, human or steer. While the mechanism of the procedure needs further elucidation, the findings reported here suggest that it ought to be sufficiently sensitive to detect hepatitus B antigen if present in virtually any blood sample. With appropriate modifications it should be useful for blood donor screening, diagnosis, and for studying many other antigen-antibody systems.

Serological studies of Actinomyces israelii by crossed immunoelectrophoresis: taxonomic and diagnostic applications.

Crossed immunoelectrophoresis (CIE) with intermediate gel was applied to the serological analysis of Actinomyces israelii to develop a test with high efficiency in the laboratory diagnosis of human actinomycosis and classification of A. israelii. Recently developed standard antigen-antibody systems for A. israelii by CIE were used as reference. The reference systems were based on standard preparations of cytoplasmic and whole cell-associated antigens of A. israelii and a standard immunoglobulin G pool purified from rabbit antisera to formalin-treated whole cells and cell lysates of A. israelii. The specificity of the standard antigens for A. israelii was evaluated in CIE studies by screening for antibodies to components of the antigens in rabbit antisera raised against related bacteria. The standard system for A. israelii based on cytoplasmic antigens formed species-specific precipitins whereas antisera raised against A. naeslundii and/or Propionibacterium acnes precipitated components of the other standard antigens. As a result of these analyses, the standard system for A. israelii based on 10 cytoplasmic antigens was used as reference for CIE studies to detect humoral antibodies to A. israelii in sera from nine patients with actinomycosis. All the sera from the patients formed at the time of diagnosis one or more precipitins in terms of the 10 reference precipitins. Up to five precipitins were found in single sera. Follow-up studies covering a period of one-half year after treatment showed a gradually decreased precipitin response in the course of time. In control sera from patients with newly diagnosed tuberculosis, nocardiosis, deep Candida infection, and aspergillosis, and in sera from healthy blood donors, no antibodies were detected with specificity for the reference antigens.

Serological studies of actionomyces israelii by crossed immunoelectrophoresis: standard antigen-antibody system for A. israelii.

Standard preparations of crude cytoplasmic and whole cell-associated antigen mixtures of Actinomyces israelii were analyzed by crossed immunoelectrophoresis (CIE), with a standard polyvalent antiserum comprising purified and concentrated immunoglobulin G antibodies to formolized whole cells of A. israelii serotypes 1 and 2. The standard antigens provided four antigen-antibody systems for A. israelii. The immunoprecipitation patterns of the system were compared, and the immunochemical characteristics of individual precipitates were analyzed. Each system contained specific precipitates, but also one or two precipitates which were immunochemically identical to precipitates of the other systems. The standard system for A. israelii based on cytoplasmic antigens was best reproducible and revealed the highest number of immunoprecipitates. These precipitates possessed immunochemical properties which made them suitable for CIE studies. The cytoplasmic antigen mixture of A. israelii was, therefore, adopted as the most suitable for further development of a crossed immunoelectrophoretic system for A. israelii. In subsequent assays the cytoplasmic antigen mixture was raised in rabbit against cell lysates of A. israelii, serotypes 1 and 2. A standard antigen-antibody system for A. israelii was obtained which revealed an immunoprecipitation pattern of 10 distinguishable precipitates. The resolving power and separation by CIE of this standard system for A. israelii was compared with that of crossed immunoelectrofocusing. The results suggest that these methods supplement each other. Crossed immunoelectrofocusing appeared to be a useful tool for separation of specific components of the protein-antigen complex of A. israelii for analytic serology. The CIE in conjunction with a standard reference antigen-antibody system for A. israelii based on cytoplasmic antigens offers great potentialities in diagnostic A. israelii serology.

Differences in the sensitivity of tumor cells and normal lymphocytes toward lysis by alloantibodies and guinea pig or rabbit complement.

Antisera produced against sarcoma I (SaI) cells in C57BL/6 and CBA mice tested for their cytotoxic activity against normal A lymphocytes and SaI tumor cells, using guinea pig or rabbit complement. Whereas high levels of cytotoxic activity against normal A lymphocytes were revealed with both antisera using either complement, only rabbit complement produced significant lysis of tumor cells under the standard conditions of the cytotoxic test (30 min, 37 C). However, upon prolonged incubation, guinea pig complement also produced high levels of lysis of SaI cells with the C57BL anti-SaI, but not with the CBA anti-SaI serum. Because these two antisera contain antibodies directed mainly against the H-2K and H-2D specificities, respectively, these results seemed to suggest that SaI cells are lysed in the presence of guinea pig complement with anti-H-2K antibodies but not with anti-H-2D antibodies. This conclusion was further supported by the use of antisera of restricted specificity prepared by selective absorption of the C57BL anti-SaI serum with CBA or DBA/2 cells. However, a C57BL anti-A serum which did not contain antibodies capable of lysing SaI target cells on prolonged incubation with guinea pig complement did not inhibit their subsequent lysis with the C57BL anti-SaI serum. These findings might suggest that under these modified conditions, mainly tumor-specific antigen-antibody systems are revealed with guinea pig complement, whereas with rabbit complement, under the standard conditions of the cytotoxic test, all antigen-antibody systems are detected.

Antibody to hepatitis B core antigen.

Two distinct antigen-antibody systems are associated with the hepatitis B virus (HBV): hepatitis B surface antigen (HBs Ag) and antibody (anti-HBs) and the more recently described hepatitis B core antigen (HBc Ag) and antibody (anti-HBc). Testing of serial serum samples from patients with type B hepatitis demonstrates the regular occurrence of anti-HBc during the course of this disease. In general, highest titers of anti-HBc are seen with prolonged circulation of HBs Ag as in the chronic carrier state. Titers of anti-HBc begin to fall with recovery from HBV infection and anti-HBc appears to be shorter lived than anti-HBs. As such, anti-HBc testing is important in documenting the occurrence of infection with HBV and is of great value in epidemiologic studies and in evaluating the safety and efficacy of hepatitis B immune globulin and HBV vaccines.