Relative Sensitivity of Gel Diffusion, Complement Fixation, and Immunoelectroosmophoresis Tests for Detection of Hepatitis-Associated Antigen and Antibody

The immunoelectroosmophoresis (IEOP) test was compared with gel diffusion and complement fixation (CF) tests for sensitivity in detecting hepatitis-associated antigen (HAA) in the sera of hepatitis patients, for titration of HAA, and for detection of antibody to HAA. The IEOP test was found to be slightly more sensitive than either gel diffusion or CF tests for detection of antigen in the patients' sera. Titers of HAA demonstrated by IEOP were higher than those seen in gel diffusion tests but lower than CF titers. The gel diffusion test with an "enhancement" pattern was found to be more reliable than the other two procedures for detection of low levels of anti-HAA, due to the greater inhibitory effect of an antigen excess in the IEOP system and the possible masking of low levels of antibody by anticomplementary activity in the CF test system. Staining of immunoprecipitates in the IEOP test contributed little to the sensitivity of the test for detection of HAA.     

Glass slide models for immunocytochemistry and in situ hybridization

We have developed a model system based on the immobilization of microdroplets (0.1 l volumes) of antigens or of target (sense) oligonucleotides onto aminoalkylsilane-coated glass slides. Oligopeptide antigens need to be vapour-fixed in order to achieve efficient immobilization, while oligonucleotides do not require fixation. Protein antigens, exemplified by rabbit immunoglobulin, may be subjected to liquid fixation. The glass slide models are optically translucent and useful both for in situ hybridization and immunocytochemistry. The slides are compatible with detection systems of both light and fluorescence microscopy and permit measurement of staining intensities by microfluorimetry or computerized microdensitometry. The model systems can be used for comparisons of method sensitivities, for characterizing antibody and probe sensitivities and cross-reactivities, and as internal standards or quality controls for immunocytochemistry and in situ hybridization.     

Binding of complement by complexes between antibodies to nucleic acid constituents and short oligodeoxyribonucleotides

Oligodeoxyribonucleotides act as inhibitors of the complement fixation caused by complexes between antibodies to defined oligodeoxyribonucleotides and denatured DNA. At concentrations higher than 50 micrograms oligodeoxyribonucleotide/ml complement fixation occurred in the absence of antigen. The extent of complement binding depends on the specificity of the antibodies as well as on the composition of the oligodeoxyribonucleotides. Complement fixation is observed most strongly with antisera to oligodeoxyribonucleotides and to denatured DNA, which belong predominantly to the IgM class. With two LE-sera, containing antibodies to denatured and to native DNA, no complement fixation was found. It is supposed that specific interactions of the oligodeoxyribonucleotides with amino acid residues closely neighbored to the antibody combining site lead to conformational changes in the antibody molecules and to an activation of the complement binding site.     

Comparison of the Reliability and Sensitivity of Three Serological Procedures in Detecting Antibody to Yersinia pestis (Pasteurella pestis)

Three serological procedures, the agar-gel precipitin inhibition, the complement fixation, and the indirect hemagglutination tests, were used to detect and measure antibody to Yersinia pestis in the sera from 383 individuals. Although all three tests were useful in detecting plague antibody, the most reliable and sensitive test procedure was indirect hemagglutination.     

Micro Indirect Hemagglutination Test for Cytomegalovirus

In an effort to obtain the flexibility and ease of performance of a rapid, serological test for detection of cytomegalovirus antibody, the indirect hemagglutination (IHA) technique was investigated by using a microserological system. Antigens were prepared from tissue cultures of infected human fibroblasts. The specificity of the cytomegalovirus antibody response detected by the IHA test correlated well with the standard neutralization test. The IHA method was more sensitive than the complement fixation test in detecting antibody in congenitally infected newborns. There appeared to be some heterologous antibody response with Herpesvirus hominis or varicella virus infections. The IHA test pattern was found to be very stable with excellent persistence of agglutination.     

Microprecipitation test for the detection of adenovirus antibody

A microprecipitation test (MPT) for the detection of adenovirus antibody has been developed. The new procedure combines precipitation of virus particles with specific antibody, separation of unreacted components from the resulting electroneutral virus-antibody complexes by electrophoresis, and detection of these complexes with a protein stain. Type-specific antibody was detected in rabbit antisera but, under similar conditions, antibody in convalescent human sera reacted with adnovirus antigens of types 4 and 7. Paired sera from 57 patients with suspected adenovirus infection were examined for significant rises in antibody activity by the microprecipitation test and by complement fixation.     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. V. Residual Complement Activity

Complement-fixation tests of three different antigen-bovine antibody systems, two antibacterial and one antiviral, were set up with or without normal bovine serum supplement. At the end of the fixation period all mixtures were tested for whole complement activity and for first, second, third and fourth complement components using the conventional, crude reagents R1, R2, R3 and R4. The increased fixation in mixtures containing the bovine serum supplement mainly reflected a greater decrease in second and fourth component activity than in the antigen-bovine antibody mixtures with non-supplemented complement. The decline in first component activity was relatively less. The results of tests for residual third component activity were not consistent.     

Serology of Rubella—Comparison of Fluorescent Antibody,Complement Fixation and Neutralization Tests for Diagnosis of Current Infections and Determination of Sero-immunity

Neutralization, complement fixation (CF) and indirect fluorescent antibody (FA) assays for rubella virus were compared for sensitivity in the serologic diagnosis of infection, for demonstrating antibody in the sera of infants with suspected rubella syndrome, and in the detection of antibody elicited by past infection (determination of immunity status). The combination of CF and FA tests was shown to be the most useful for serologic diagnosis of infection, largely eliminating the need for the slower and more cumbersome interference neutralization test.Neutralizing antibodies were found to appear rapidly in the course of infection, antibodies demonstrable by immunofluorescent staining appeared slightly later, and CF antibodies were rarely demonstrable in sera collected earlier than 14 days after onset of illness. Antibodies detected by all three techniques showed good correlation in infants with clinical evidence of rubella syndrome and corresponding maternal sera. The indirect FA technique compared favorably with the neutralization test for the detection of antibody elicited by past infection (determination of immunity status) and offered distinct advantages in ease of technical performance and more rapid results. In both current and past infections, FA titers tended to be higher than neutralizing antibody titers.     

Description and application of an immunological detection system for analyzing glycoproteins on blots

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Sugars are oxidized to obtain aldehyde groups, which then react with digoxigenin-succinyl--amido caproic acid hydrazide. A high-affinity antibody, conjugated to alkaline phosphatase, is used for the detection of the incorporated digoxigenin.This system allows the detection of nanogram-amounts of glycoproteins on blots, and it's specificity allows a clear distinction of a glycoprotein from a non-glycoprotein. In combination with endo- and exoglycosidases it is very useful for determining the type of carbohydrate linkage in a glycoprotein, and by varying the oxidation conditions, specific labeling of sialic acids and terminal galactoses can be achieved.     

Heated histoplasmin as a control for non-specificity in the complement fixation test for histoplasmosis

We present data in support of the use of a heated histoplasmin control for the complement fixation (CF) test for histoplasmosis to assist in the detection of the presence of h or m antibody.     

Detection of mouse hepatitis virus antibody by protein A-ELISA in 6 prevalent inbred strains or outbred stocks of mice.

Protein A was applied as a reagent for the secondary reaction in ELISA (protein A-ELISA). Mouse hepatitis virus antibody in 6 prevalent mouse strains or stocks reared in a MHV-contaminated room was effectively detected by protein A-ELISA, whereas significant strain differences in the antibody detection rate were demonstrated using the complement fixation test. C57BL/6 mice were particularly reactive in the protein A-ELISA test.     

A new fluorescent detection system for identifying variant hemoglobins after gel electrophoresis using immunobinding with monoclonal antibodies

This paper describes a low-resolution system for identifying variant hemoglobins with great sensitivity and specificity. After electrophoresis of the hemoglobin sample in a gel, fixation is used to entrap the hemoglobin. The gel is dried, incubated with a monoclonal antibody against the desired hemoglobin, then incubated with a second antibody against the first antibody which is conjugated with the enzyme beta-d-galactosidase. An enzyme overlay membrane containing a fluorogenic substrate is then placed on the gel surface, incubated, and removed, yielding an immunofluorescent print. The entire procedure takes only two hours, and by virtue of fluorescent detection gives sharper band resolution and greater sensitivity than conventional dye methods. The system clearly distinguishes SS sickle-cell hemoglobin from heterozygous and "S-like" hemoglobins. The technique therefore holds promise as a powerful probe for allelic variants.     

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.     

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.     

Immunofluorescence as an Aid in the Early Diagnosis of Trichinosis

The established serological tests for trichinosis are often negative during the period when laboratory investigation is most likely to be useful.Another serological test, the immunofluorescence test, appears to be more promising in this respect. The results were based on studies involving experimental animals and human patients. In two rabbits orally infected with Trichinella spiralis larvae, antibodies were demonstrable by immunofluorescence on the fourth day after infection, by complement fixation on the eighth and tenth days, and by the precipitin test on the thirteenth and twenty-eighth days, respectively. In three human cases the immunofluorescence antibody test was positive two weeks (the earliest blood samples available) after onset, while precipitin and complement fixation tests did not become positive until the end of the fourth week. The immunofluorescence test thus becomes positive at least two weeks earlier than the other two, a factor which undoubtedly increases its value in diagnosis.     

Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.     

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.     

Mononuclear phagocyte maturation: a cytotoxic monoclonal antibody reactive with postmonoblast stages

The rat IgM monoclonal antibody B23.1 was found to bind to mononuclear phagocytes that had matured beyond the monoblast stage. Macrophages from several anatomical sites, elicited by different means, as well as those cultured from bone marrow precursors, bound B23.1 antibody. The increase, with time, of B23.1-positive cells in the nonadherent fraction of cultured bone marrow paralleled that of immature mononuclear phagocytes as detected by esterase staining. Treatment of freshly explanted bone marrow cells with B23.1 and complement did not reduce the number of macrophage colony-forming cells (monoblasts) in that population. Treatment with B23.1 antibody alone did not alter the activation of macrophages for tumor cell killing; however, with added complement, B23.1 reduced activated macrophage-mediated cytotoxicity to background levels. In similar studies B23.1 and complement did not affect antibody production by B cells, the cytotoxic capacity of T cells, or NK cell-mediated lysis. These data indicate that antibody B23.1 is useful for either the detection or elimination of mouse mononuclear phagocytes from the promonocyte stage to that of the mature macrophage.