Addition Of Guinea Pig Complement Research Articles

Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein.

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.

Complement-dependent serum neutralization with virulent and a virulent Bucyrus strains of equine arteritis virus

Virulent and avirulent strains of Bucyrus equine arteritis virus (EAV) were used to raise antiserum in horses. Serum neutralization (SN) tests were performed with and without the addition of guinea pig complement. The inclusion of ten percent guinea pig serum in the virus suspension was sufficient for optimal enhancement of SN titres at any immune stages after immunization. Immune serum prepared against avirulent virus reacted only with homologous virus and there was no complement enhancement. Immune sera raised against live or inactivated virulent virus neutralized both virulent and avirulent virus. The reaction with virulent virus demonstrated complement enhancement. There was also moderate potentiation in the presence of complement when serum raised against inactivated virulent virus reacted with avirulent virus.

Antigenic and Functional Characterization Of Rinderpest Virus Envelope Proteins Using Monoclonal Antibodies

A total of 24 monoclonal antibodies (MAbs) against the haemagglutinin (H) and the fusion protein (F) of rinderpest virus (RPV) were used to characterize their antigenic structure and biological properties, and to analyse natural variation in the envelope proteins of morbilliviruses. The anti-H and anti-F MAbs defined seven and three distinct antigenic sites, respectively. The MAbs to six sites on H were able to neutralize the infectivity of RPV. The addition of guinea-pig complement or anti-mouse immunoglobulin increased the virus-neutralizing antibody titre of most of the anti-H MAbs, including those lacking neutralizing activity. One of the antigenic sites on H was conserved among morbilliviruses and the MAbs to this site had haemagglutination inhibition activity against measles virus (MV). The remaining sites were specific for RPV and varied antigenically between strains of RPV. The anti-F MAbs lacked neutralizing activity, but two of the five MAbs did show activity in the presence of complement or anti-mouse immunoglobulin. On the whole, the antigenic sites on F were conserved in some strains of MV, but not in canine distemper virus. All of the sites on the surface proteins were sensitive to SDS and, although those on F were not affected by 2-mercaptoethanol, five of the seven sites on H were destroyed by it. These results suggest that the epitopes on the envelope proteins are conformation-dependent.

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.

Killing ofLegionella pneumophila by human serum and iron-binding agents

The inhibitory effect of serum on the growth and survival ofLegionella pneumophila Bloomington 2 was investigated. When incubated in the presence of 20%–50% normal human serum for 10 h, viability was decreased by >99%. Heat-inactivated or <40% normal serum supplemented with 50 μM iron was not inhibitory. The addition of guinea pig complement to heat-inactivated serum resulted in killing of approximately 98% of the cells. Growth in buffered yeast extract broth was inhibited by the addition of ferric iron-binding compounds. Minimum bactericidal concentrations at 37°C were 10 μM apotransferrin, 35 μM 1,10-phenanthroline, and 50 μM deferoxamine. Addition of iron chelators to normal serum did not accelerate killing. Egg yolk-passaged “virulent” strains and agar-grown “avirulent” strains exhibited similar serum sensitivity. Results of this study indicate that complement and serum transferrin are antagonistic to the growth ofLegionella in serum.

Endoneurial injection of antisera to myelin antigens

When antisera to purified myelin antigens were injected into rat sciatic nerves, some produced significant demyelination, whereas others merely induced an inflammatory infiltrate. Extensive demyelination was produced by antisera to galactocerebroside and the peripheral nerve glycoprotein P0. Demyelination resulting from injections of antisera to ganglioside GM1, P2, myelin basic protein, sulfatide, and glucocerebroside did not exceed that produced by normal rabbit serum. Addition of guinea pig complement had no effect. It is of interest that the greatest demyelination followed injection of antisera to two molecules, galactocerebroside and P0, the main antigenic determinants of which present at the Schwann cell surface.

Phagocytosis of virulent and avirulent leptospires by guinea-pig and human polymorphonuclear leukocytes in vitro

Chemiluminescence (CL) and electron microscopy were used to study the phagocytosis of both virulent and avirulent strains of Leptospira interrogans serovar copenhageni by guinea-pig and human polymorphonuclear leukocytes (PMN). A significant CL response was observed when guinea-pig PMN were incubated with virulent leptospires in the presence but not in the absence of specific immune serum. This response was markedly enhanced by the addition of guinea-pig complement. Phagocytosis was confirmed by the observation of intracellular leptospires in guinea-pig PMN by electron microscopy. The phagocytosis of avirulent leptospires by guinea-pig PMN and of both virulent and avirulent leptospires by human PMN required the presence of both specific immune serum and complement. Thus the ability of leptospires to resist phagocytosis by PMN in the absence of immune serum does not appear to be a major determinant of virulence.

Mouse immunoglobulin antibodies require complement for neutralization of mouse retroviruses.

The addition of guinea pig complement was found to enhance the neutralizing capacity of mouse antibodies directed against the endogenous ecotropic murine leukemia viruses. The same immune sera, when tested without complement, had low or negligible neutralizing capacities, regardless of whether freshly harvested, unfrozen virus was used to preserve virus infectivity. Antibodies in high titers were found in sera from NFS congenic mice carrying the mouse leukemia virus inducing locus Akv-2. These mouse antibodies were type specific and failed to neutralize either Friend or Moloney leukemia virus. The mouse serum immunoglobulin fraction containing the complement-dependent antibodies was tentatively identified as immunoglobulin M.

Inactivation or elimination of potentially trypanolytic, complement-activating immune complexes by pathogenic trypanosomes.

Trypanosoma brucei subsp. brucei and T. congolense incubated in homologous antibody at 0 degrees C for 30 min were lysed by subsequent addition of guinea pig complement. Trypanosomes incubated in antibody at 37 degrees C before complement treatment remained intact. Parasites bearing adsorbed antibody also remained intact when incubated at 37 degrees C before complement treatment. The proportion of cells which survived complement treatment decreased with increasing antibody concentration. Parasites which survived complement treatment continued to express antigens which could bind complement-activating antibody, but did not bear complement-activating immune complexes. Virtually all cells in T. brucei populations exposed to antibody at 37 degrees C, but only 10(-3) to 10(-5) of the cells in populations exposed to antibody at 0 degrees C before complement treatment, remained infective for X-irradiated mice. Only 10(-6) to 10(-7) of T. brucei populations exposed to antibody at 0 or 37 degrees C before complement treatment infected mice immunized with homologous antigens. Serotype analysis of substrains and of T. brucei populations isolated from mice infected with antibody and complement-treated parasites suggested that variant-specific antigens participated in trypanolysis and that T. brucei which survived complement treatment could undergo antigenic variation. Mechanisms by which trypanosomes may inactivate or eliminate surface immune complexes and the possible significance of this phenomenon in trypanosomiasis are discussed.

Further Investigations of Live Respiratory Syncytial Virus Vaccine Administered Parenterally

SummaryFurther clinical tests were carried out of a live attenuated respiratory syncytial virus vaccine administered parenterally. Neutralization of respiratory syncytial virus by homologous human antibody was found to be a complement-dependent event and addition of guinea pig complement greatly increased antibody titers and the sensitivity of the test. Ninety-seven percent of 116 initially seronegative children developed antibody following vaccination and there was no evidence for contagious spread of the infection. Clinical reactions, if any, were mild and inconsequential. A small portion of initially seropositive persons appeared to develop antibody by booster effect and a portion of initially seronegative and seropositive persons developed detectable nasal antibody. Long-term follow-up failed to reveal any increase in severe respiratory disease in initially seronegative persons who were vaccinated. A very substantial reservoir of seronegative persons was found in the 7 to 47 month age group (35%) reve...

“REJECTION” OF HEART PERFUSED IN VITRO BY ALLOTRANSPLANTATION SERUM

SUMMARY In vitro “rejection” of heart perfused by allogeneic sera was studied. Following ligation of pulmonary veins and both venae cavae, the perfusing fluid entered the heart through the aorta, crossed the coronary circulation, and left the heart through the pulmonary artery. The heart action was recorded by means of an electrogram. Rabbit hearts, perfused by rabbit transplantation sera containing corresponding alloantibodies, stopped beating within 7 to 35 min. During the perfusion a great proportion, up to 90%, of antibodies were bound by the heart, as estimated by titration of the perfusing serum by means of mixed agglutination with cell cultures. In addition to antibody, complement played a role in this damage to the heart as evidenced by the inactivity of sera heated for 30 min at 56 C. Rat hearts were also “rejected” by alloantibody-containing transplantation sera, but addition of guinea pig complement to the perfusing rat serum was needed to obtain clear-cut results. Rabbit and rat hearts perfused by normal allogeneic sera showed good function for several hours. These experiments showed that secondary factors such as deposition of polymorphonuclear leukocytes, thrombocytes, or fibrin are not essential for antibody-mediated organ rejection.

In vitro lysis of tumor cells by antibody and complement: detection of antibody with low cytotoxic reactivity.

The factors affecting the in vitro lysis of PARA-7 tumor cells mediated by antibody and complement were examined with the use of the 51Cr-release assay. The addition of immune syngeneic hamster antisera (HA) to 51Cr-labeled PARA-7 target cells 30 minutes before the addition of guinea pig complement (GPC) failed to produce significant levels of lysis. Similarly, the addition of rabbit anti-PARA-7 serum (RAS) 30 minutes prior to the addition of GPC resulted in low levels of lysis. Longer incubations in the presence of RAS, but not RAS and GPC, increased levels of lysis. Extended incubation in the presence of RAS increased the amount of antibody bound to the target cells. This increase did not appear to be due to the expression of additional antigen, because the increase occurred at 4 degrees C and was not sensitive to inhibitors of protein synthesis. The inability to obtain increased levels of lysis by extended incubation in the presence of both RAS and GPC appeared to be due to the inhibitory effects of GPC on the interaction of antibody and antigen. Similar inhibitory effects could be produced by inactivated GPC and various other substances. When extended incubations in the presence of antiserum were used for the reexamination of the cytolytic activity of syngeneic immune HA, significant lysis was detectable.

Effect of Concanavalin a on the Killing of Tumor Cells by Antibody and Complement

Abstract Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with α-d-methylglucopyranoside or by incubation at 37°C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.

Effect of interaction between hapten-specific cells preselected for different receptor affinities and carrier-primed cells on antibody avidity.

Our previous studies,in vivo (1) and in vitro (2), have shown that carrier priming enhances antibody response and avidity. Under optimal conditions for these carrier effects to occur, C3HeB/FeJ and (C57BI/10×DBA/2)F1 mice either carrier-primed with HRBC or -unprimed were given the conjugate TNP-HRBC. Thereafter, spleen cells from the carrier-primed or -unprimed mice were daily pooled and assayed by the hemolytic plaque technique with TNP-SRBC to determine the number of direct PFC anti-TNP. Avidity of antibodies secreted by PFC was estimated from inhibition of PFC by soluble TNP-BSA (35 mols TNP/mol BSA). Aliquots of TNP-BSA dissolved in PBS at different concentrations and aliquots of spleen cells were plated in agar containing TNP-SRBC. The number of direct PFC developed after addition of guinea pig complement was decreased by TNP-BSA. Percent inhibitions of PFC were transformed to probits which appeared linearly related to the log dose of inhibitor. The reciprocal of the dose of inhibitor that suppressed 50% of the PFC was taken as an estimate of avidity: the higher the antibody avidity, the lower the inhibitor dose and the higher its reciprocal. The dose of inhibitor was referred to TNP-lysyl residues and calculated from spectrophotometric measurements of the TNP-BSA solution at λ max 348 nm by assuming 15,400 as the molar extinction coefficient for the TNP-lysyl residue. In each PFC inhibition assay, spleen cells were exposed to amounts of TNP-lysyl residues ranging from 10−5 to 10−1 mg.

Viral induced immunity to syngeneic Rauscher murine leukemia cells, in the absence of allogeneic inhibition.

SummaryBALB/c mice were immunized with Rauscher murine leukemia virus. Both formalin-treated virulent virus, and a live, attenuated strain of the virus were used as antigens. Immune serums and immune spleen cells harvested 64 days later caused lethal damage to the virus transformed malignant cell. The effectiveness of immune serum was removed by heat inactivation and was restored by the addition of guinea pig complement. Either X-irradiation or freezing and thawing eliminated the competence of the immunocytes. Normal spleen cells agglutinated to target cells by phytohemagglutinin were not capable of causing lethal damage to the malignant cells. Although allogeneic inhibition has been suggested as the possible mechanism underlying tumor immunity and surveillance against neoplasia in syngeneic and autochthonous systems, it does not appear to play a significant role in the immunity to transplantation of malignant cells that follows immunization with killed or attenuated murine leukemia virus.

FLOCCULATION OF UNSENSITIZED BENTONITE PARTICLES BY CANCER SERUM.

Discussion and summaryThe high degree of flocculation of unsensitized particles of bentonite by cancer sera and not by normal or by sera from other disease conditions, may be of help in developing a test for cancer diagnosis. Heating of the sera at 50°C for 30 minutes is required to establish the difference in reactivity between cancer and other sera. The factors affected by such heat treatment are not yet clear.Although the flocculation reaction occurs in cancer sera in a gradient and at high titers, no other indication could be found to show that this may be an antigen-antibody reaction, as could have been expected if the bentonite particles became coated with either excess antigen or antibody present in the sera. Also, addition of guinea pig complement did not restore the reaction in sera heated to 56OC for 30 minutes. Since in vivo antigen-antibody interaction has been considered to take place in some acute or chronic diseases such as systemic lupus erythematosus and glomerulonephritis, this theory sh...

Passive transfer of cold urticaria

1. 1. A case of marked cold allergy manifested by urticarial reactions of the skin to contact with ice for 10 seconds is described. The reaction was not affected by ordinary doses of antihistamine drugs. 2. 2. The patient was allergic to pollens and horse danders, but apparently free of other diseases, and the serum did not contain cold agglutins, cold hemolysins, or cryoglobulins. The electrophoretic pattern of the serum was normal. 3. 3. Injection of the serum intracutaneously into normal skin produced passive local sensitization demonstrable by an urticarial reaction to contact with ice. The passive sensitization was exhausted by one reaction to cold. Stimulation of the skin of the patient with ice did not affect the sensitizing power of the serum. 4. 4. The skin-sensitizing activity of the serum was only slightly affected by heating one-half hour at 56° C. but was destroyed by heating 4 hours at this temperature. Addition of guinea pig complement to the heated serum did not restore activity. 5. 5. The sensitizing material in the serum was adsorbed by excised skin cells at 4° C., but not by Type O red cells. 6. 6. Attempts to demonstrate reversed passive transfer and to isolate an antigen liberated from normal skin by cold which would react with the serum were unsuccessful. 7. 7. Four fractions were separated from the serum by electrophoresis, containing respectively (1) albumin, (2) albumin and alpha globulin, (3) beta and gamma globulin, and (4) gamma globulin. None of these fractions passively sensitized normal skin to cold and addition of complement did not render them active. Mixtures of all 4 fractions or of the second and third fractions sensitized normal skin, suggesting that the presence of more than one protein component was necessary to produce sensitization.