The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

Microtiter Tests for Detecting Antibody in Bovine Serum to Parainfluenza 3 Virus, Infectious Bovine Rhinotracheitis Virus, and Bovine Virus Diarrhea Virus

Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

Rat adrenal dopamine-beta-hydroxylase: purification and immunologic characteristics

Dopamine-beta-hydroxylase (DBH) was purified from rat adrenal medulla by a series of steps including sedimentation of membranes, extraction with n-butanol, ammonium sulfate fractionation, gel chromatography and ion-exchange chromatography. Disk gel electrophoresis revealed two protein bands, both of which were active. Antiserum was prepared against homogeneously purified bovine adrenal and rat adrenal DBH; Ouchterlony immunodiffusion, enzyme neutralization and complement fixation tests demonstrated that the respective homologous antisera were monospecific and of high titer. Antiserum to bovine DBH was only 2- to 3-fold more potent than pre-immune serum in inhibition of rat DBH activity. Complement fixation tests demonstrate that antiserum to bovine DBH has a 25,000-fold lower immunoreactivity with rat DBH than with bovine DBH.     

A comparative study of detection methods for Aleutian disease viral antibody.

Four methods of detecting and quantitating mink antibody against Aleutian disease (AD) virus were compared. Counterelectrophoresis, modified, counterelectrophoresis, immunofluorescence, and complement fixation were performed blindly on 274 serum samples. All four methods were reliably specific for AD antibody. Immunofluorescence was less reproducible than the other systems. Immunofluorescence complement fixation were 4- to 8-fold more sensitive than regular or modified counterelectrophoresis, but were limited by background staining and anti-complementary activity, respectively, when used to detect small amounts of antibody in undiluted sera.     

Antibody Response of Animals to Mycoplasma pneumoniae Measured by Latex Agglutination

The standardization, application, and usefulness of latex agglutination for Mycoplasma pneumoniae antibody titration were investigated and compared with tetrazolium reduction inhibition and complement fixation. The sera of guinea pigs and monkeys reacted in a specific fashion, whereas rabbit serum required pretreatment to eliminate its nonspecific agglutinin. Human serum given such pretreatment still contained a nonspecific agglutinin. The latex agglutination procedure compared favorably with complement fixation and metabolic inhibition tests for evaluating vaccine antigenicity.     

Inactivated-concentrated virus antigen for indirect complement fixation test of foot-and-mouth disease.

To the culture fluids of BHK-21 cells infected with each of types O, A, and Asia 1 of foot-and-mouth disease virus was added acetylethyleneimine to 0.05% (v/v). The mixtures were incubated at 37 degrees C for 24 hours. To them were then added polyethylene glycol 6000 to 10% (w/v), and the mixtures concentrated to one-tenth of the initial volume. The resulting inactivated-concentrated virus antigens showed a complement fixation (CF) titer ranging from 12 to 24. The recovery rate of CF activity was 40 approximately 60%. This activity of each antigen was maintained at 4 degrees C or -70 degrees C for 6 months at least. Experimentally infected cattle were examined for the development of antibody by the aid of the indirect complement fixation (ICF) test with those antigens. As a result, ICF antibody began to be detected 3 approximately 5 days after inoculation. Its titer reached a maximum 10 approximately 14 days after inoculation and decreased gradually thereafter. It was detected even 232 days after inoculation. There was a tendency for the development of ICF antibody to be parallel with that of neutralizing antibody. It was suggested that ICF antibody might be type-specific. In conclusion, the antigens prepared had such high activity that they could be used for the determination of antibody by ICF. In addition, they were of great practical value because of their sufficient keeping quality and safety.     

An evaluation of elution techniques in the study of immune complex glomerulonephritis.

Antigen and antibody from glomerular immune complex deposits in rabbits with experimental bovine serum albumin-(BSA) induced chronic serum sickness (CSS) were quantitated in elutes from kidneys in which a portion of the antigen and antibody had been radiolabeled. The largest quantities of 125I BSA eluted with 1 M roprionic acid at pH 2.7 (86%) and 0.1 M borate buffer at pH 11.25 (80%). However, these buffers yielded less functional anti-BSA antibody than 0.02 M citrate buffer at pH 3.2 (344 mug/g kidney). Citrate buffer-eluted anti-BSA antibody was reactive in immunodiffusion, immunofluorescence, and radiolabeled BSA binding test systems, but complement fixation was impaired relative to chaotropic ion-eluted antibody. It was found that up to 75% of the eluted antibody was lost to further study by recombination with eluted BSA. This could be prevented by fractionation of the dissociated eluate before neutralization. IgG fractionated eluates were successfully fluorescein conjugated or radiolabeled for use as reagents. Elution of cryostat sections of CSS kidney was also studied; BSA, IgG, and complement (C3) eluted in parallel, and sub-microgram quantities of anti-BSA antibody were recovered.     

Demonstrations of the production of specific antibodies to poly(I).poly(C) in rabbits

The rabbit antiserum against poly(I).poly(C) purified by hydroxyapatite column chromatography contained three distinct antibodies. They were fractionated into three antibody populations by a series of precipitations (with poly(A).poly(U), poly(I), and poly(I).poly(C)) and their specificities were examined by quantitative complement fixation, double diffusion tests and radioimmunoassay. The first population was common to the double helical structure of double-stranded RNAs. The second was specific for poly(I) and the third was specific for poly(I).poly(C). These studies demonstrated that specific antibodies exclusively reactive with poly(I).poly(C) existed in the rabbit antiserum against poly(I).poly(C).     

Components of human complement system. Testing and isolation of C4

A modified reagent for testing the hemolytic activity of human complement component C4 has been obtained. Reagent R4 was obtained by treatment of human blood serum pools with 0.075 M solution of hydrazine hydrate. This reagent was found to be rich in the serum fraction obtained by chromatography on DEAE-cellulose DE-52 and containing an active complement C3 component. To test the sensitivity and specificity of the reagent, component C4 was subjected to purification. This procedure resulted in a hemolytically active, electrophoretically and immunoelectrophoretically homogeneous component C4. DEAE-cellulose DE-52, DEAE-Sephacel, Ultragel AcA-34 and, again, DEAE-Sephacel were used consecutively as purification agents. The activity yield of component C4 with regard to the initial serum level was 20%.     

Antibodies elicited by defined oligodeoxyribonucleotide sequences.

Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.     

Interactions of Standard Antibody with Australia Antigens in Au Ag-Ab Radioimmunoassay

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.     

Effect of the Purification of Virus Antigens on the Production of Specific Complement-Fixing Antibodies

The effect of viral purification procedures on the antibody response of guinea pigs to immunization with reovirus type 2 and echovirus type 19 was investigated. Three grades of antigens were employed: (i) infectious monkey kidney tissue culture fluid (TCF), (ii) virus sedimented in the ultracentrifuge and suspended in phosphate-buffered saline, and (iii) virus purified by centrifugation in CsCl density gradients. The antibody response of the guinea pigs was studied by the hemagglutination inhibition, complement fixation, and serum neutralization tests. Only sera produced from virus purified by CsCl density gradients reacted specifically with homologous antigen in the complement fixation test. Sera from animals receiving tissue culture fluid virus or sedimented virus cross-reacted with heterologous antigens such as tissue culture fluid from uninfected monkey kidney cells. All sera, however, reacted specifically in hemagglutination inhibition and serum neutralization tests. Sera from intranasally infected animals (reovirus type 2), even though reacting specifically in the complement fixation test, had much lower titers than sera from animals inoculated intramuscularly.     

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH₅₀ is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH₅₀ tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH₅₀ level will be zero; if one or more components of the classical pathway are decreased, the CH₅₀ will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.Download video file.^{(81M, mp4)}     

Experimental infection of domestic animals with R. prowazeki and R. canada]

The authors infected lambs with R. prowazeki and R. canada to ascertain their possible role in the natural infection of the animals. The lambs were infected subcutaneously with increasing doses; rickettsiemia was recorded with the aid of tests on guinea pigs and Ixodidae and Argasidae ticks fed on the lambs. Dynamics of antibody formation was ascertained in the infected animals in the agglutination reaction and in the complement fixation test. The antigenic affinity of R. canada and rickettsia of the typhoid group and the presence of common antigenic determinants with the Proteus OX19 was confirmed. The absence of any clinical manifestations, the character of antibody formation, impossibility of inducing the generalized infection and of the isolation of the causative agent from the blood pointed to the low susceptibility of lambs to R. prowazeki and R. canada; thus a possibility of circulation of the causative agents of typhius among the domestic animals scarcely probable.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

Antileptospiral Activity of Serum II. Leptospiral Virulence Factor

A definite relationship exists between the resistance of leptospires to the antibody-complement system and virulence. Leptospires capable of producing either lethal or renal infections in hamsters or guinea pigs were resistant to the leptospiricidal action of antibody and complement. Avirulent leptospires, in contrast to the virulent organisms, were rapidly immobilized and killed by these serum substances. The change of a virulent culture to the avirulent state as a result of growth in culture media was accompanied by the loss of resistance to antibody and complement. Virulent leptospires were phenotypically altered when grown in the presence of the purine analogue, 8-azaguanine. The cells became sensitive to antibody and complement without a corresponding decrease in virulence. The basis for a leptospiral virulence factor, the ability to multiply in vivo, appears to reside in their capacity to resist the leptospiricidal activity of the host antibody-complement system. The immune leptospiricidal assay provides a simple and rapid method of determining the virulence of a culture.     

A Comparative Examination of Swine Sera for Antibody To Aujeszky Virus with the Conventional and a Modified Virus-Serum Neutralization Test and a Modified Direct Complement Fixation Test

Sera of pigs from élite breeding herds, of boars and sows collected at slaughter-houses, and of pigs from herds known to be infected, were examined for antibody to Aujeszky virus. The conventional and a modified virus-neutralizing antibody (VNA) test and a modified direct complement fixation (CF) test were employed. In simultaneous titrations of positive sera the modified VNA test gave titers approx. 4 log2 units above the titers obtained by the conventional test. The conventional VNA test was found insufficiently sensitive. Unspecific neutralization in the modified VNA test was infrequent in serum dilution 1/2 and rare in dilution 1/4. The GF tests on sera of slaughter sows and animals from known infected herds showed a remarkable consistency with the VNA tests. Inconsistent results were obtained with but few sera. Abt. 5 % of the sera could not be examined because of complement fixation with control antigen.