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Quantitative assessment of hemadsorption by myxoviruses: virus hemadsorption assay.

The standardization and quantitative evaluation of an assay for myxoviruses, based on the enumeration of individual infected clone 1-5C-4 cells manifesting hemadsorption within 24 h of infection, are described. Hemadsorption was detectable earlier than immunofluorescence in infected cells or hemagglutinins in culture medium. The relationship between virus concentration and cells exhibiting hemadsorption was linear. The assay was highly precise, sensitive, and reproducible.

Animals↗

Quantitative assessment of hemadsorption by myxoviruses: anti-immunoglobulin G hemadsorption-inhibition test.

A quantitative hemadsorption-inhibition test was developed to estimate myxovirus serum antibodies within 24 h by determining the serum dilution inhibiting hemadsorption in 50% of the infected cells. The test depends on the interactions of virus-infected cell monolayers with antiviral serum and of the resultant complexes with antiimmunoglobulin G serum. The incorporation of species-specific anti-immunoglobulin G serum into the test significantly increased sensitivity.

Animals↗

Titration of Newcastle disease virus and its neutralizing antibodies in microplates by a modified hemadsorption and hemadsorption inhibition method.

Using the microtiter system, titration of Newcastle disease virus infectivity and neutralizing antibodies was carried out in chicken embryo fibroblasts grown in "U" or flat-bottomed plates. Infectivity was detected by a combined hemadsorption-hemagglutination method. Inhibition of that reaction indicated the presence of neutralizing antibodies. A 24-h microneutralization test was developed and compared to the plaque neutralization and microhemagglutination inhibition test. Reproducibility of the microneutralization test was statistically analyzed.

Animals↗

Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host.

Animals↗

Metabolic requirements for the development of hemadsorption activity and virus formation in BHK-21 cells infected with Newcastle disease virus.

Differential effect of various metabolic inhibitors on the development of hemadsorption activity and virus formation in cells infected with Newcastle disease virus (NDV) was investigated. It was found that, in BHK-21 cells infected with NDV, cycloheximide did not prevent the development of hemadsorption activity, whereas protein synthesis and virus formation by the cell were rapidly inhibited by the drug. When the drug was added to the culture at 4.5 h after infection or later, hemadsorption activity of the cell continued to develop normally for about 1 h. Similar increase in hemadsorption activity was found in cells which were treated with anti-NDV serum (to neutralize their hemadsorption activity) and then washed and incubated with cycloheximide. However, when cells were treated with the drug early in the infection (1.5 or 3.0 h), they did not show any detectable hemadsorption reaction throughout the infection. In contrast to cycloheximide, iodoacetate added to the culture together with sodium azide inhibited completely both the development of hemadsorption activity and the formation of progeny virus. These results suggest that the change of cell surface to become hemadsorptive may depend upon the energy generating system but not upon de novo synthesis of protein, whereas production of infectious virus may require continuous synthesis of protein.

Animals↗

A novel amino acid position in hemagglutinin glycoprotein of measles virus is responsible for hemadsorption and CD46 binding.

Three recent isolates of measles virus Fu, IMA, and SMD obtained by using B95a cells did not exhibit hemadsorption with African green monkey red blood cells (AGM-RBC). After long-term passage in Vero cells, these Vero cell-adapted strains derived from three isolates obtained the activity to agglutinate AGM-RBC. The primary sequences of the hemagglutinin (H protein) and fusion glycoproteins (F protein) from these two types of viruses were compared and revealed that several important amino acid residues in the H protein do not converge. After adaptation, Fu strain has an Asn to Tyr substitution at position 481 and IMA strain has two substitutions--an Asp to Asn at position 14 and a Ser to Gly at position 546, SMD strain also has a Ser to Gly substitution at position 546. Since the sequences of the F protein were identical between both types of viruses, the hemadsorption alteration from negative to positive might be the result of these substitutions. Site-directed mutagenesis of the H genes were performed to confirm that the substitution of Ser --> Gly at position 546 and Asn --> Tyr at position 481 in the H protein were responsible for hemadsorption alteration. Anti-CD46 monoclonal antibody (M75 and M160) study made clear that these two substitutions also governed the MV H protein's interaction with CD46 receptor. Our results showed that two important amino acid residues in MV H protein govern the binding to CD46 receptor and hemadsorption. In this paper, we reported a novel amino acid residue at position 546 in MV H protein, which was critical for hemadsorption and CD46 binding.

Amino Acid Substitution↗

Hemadsorption and virulence are separable properties of Mycoplasma pneumoniae.

A selective enrichment technique was used to isolate a hemadsorption-positive revertant of a hemadsorption-negative mutant strain of Mycoplasma pneumoniae. This hemadsorption-positive revertant was shown to have simultaneously regained both the ability to attach to neuraminidase-sensitive receptors on the tracheal ring respiratory epithelium in vitro and the ability to synthesize three virulent-strain-specific proteins which were not synthesized by the hemadsorption-negative mutant. Despite the persistence of the revertant in hamster lung tissue for 9 to 12 weeks postinfection, no cytopathology was observed. Intranasal inoculation of the revertant provided limited protection against a challenge dose of virulent M. pneumoniae.

Adhesiveness↗

Hemadsorption immunosorbent technique for determination of mumps immunoglobulin M antibody.

We used hemadsorption immunosorbent technique (HIT) to detect mumps immunoglobulin M (IgM) antibody. IgM from human sera was adsorbed into anti-human IgM-coated wells in plates, and mumps-specific IgM was detected by adding mumps virus hemagglutinin and guinea pig erythrocytes consecutively. Specific IgM-positive sera showed hemadsorption, whereas negative sera showed hemagglutination. All 81 patients with current mumps infections tested showed mumps-specific IgM antibody, with titers ranging from 160 to 327,680. In most cases IgM antibody was already present on day 1 or 2 after the onset of illness. IgM antibody persisted for 6 to 10 weeks. Of 57 patients with acute respiratory illnesses caused by parainfluenza virus, 5 showed cross-reactions in the hemadsorption immunosorbent technique assay for mumps IgM. The hemadsorption immunosorbent technique assay is specific for the IgM class of antibody and avoids false-positive results due to rheumatoid factor. This test is an efficient and sensitive method for rapid and early diagnosis of mumps infections.

Animals↗

Detection of cell-bound immunoglobulins by a radioisotopic micro-mixed hemadsorption reaction with technetium-99m-labeled erythrocytes.

The mixed hemadsorption (MHA) reaction detects antibodies reactive with cell surface antigens by means of antiglobulin-coated indicator erythrocytes. We have developed a radioisotopic modification which employs sheep erythrocytes (SRBC) that have been prelabeled with technetium-99m (99mTc), a high specific acitivity metastable gamma-emitter of short half life. The 99mTc MHA reaction was performed on human and murine cells cultured in Micro-test II plated with six replicate wells per serum dilution. Antibody activity in species-specific xenoantisera and mono- and polyspecific alloantisera was detected in high titer. The sensitivity of 99mTc micro-mixed hemadsorption was 2 times that of the visual assessment of mixed hemadsorption, 100 to 200 times that of the 125-I-mixed antiglobulin reaction and 500 to 1000 times more sensitive than indirect immunofluorescence. The assay system was applied successfully to confirm the species of origin of a panel of previously karyotyped human and mouse cell lines. Our results indicate that the 99mTc micro-mixed hemadsorption method is a rapid, sensitive, quantitative test for the detection of cell surface antigens and membrane reactive antibodies.

Animals↗

Hemadsorption-negative plaque test: new assay for rubella virus revealing a unique interference.

A simple and rapid plaque procedure has been developed for detecting and accurately assaying rubella virus in a noncytopathic virus-cell relationship. Plaque-formation is based on the development, in individual cells infected with rubella virus, of a unique type of intrinsic interference to infection with Newcastle disease virus. Rubella virus-infected cells challenged with Newcastle disease virus and tested for hemadsorption 15 hours later stand out as hemadsorption-negative areas. Individual living cells infected with rubella virus can be resolved under conditions allowing standard cloning procedures. In principle, the hemadsorption-negative plaque test can be used to search for a new class of noncytopathic, non-hemadsorbing viruses-those that induce an intrinsic interference to infection by any hemadsorbing virus.

Animals↗

Hemadsorption and enzyme-linked immunosorbent assay nitrocellulose replica methods for identification of colonization factor antigen (CFA)-positive Escherichia coli colonies and for isolation of CFA-negative mutants.

Methods were developed that allow demonstration of individual colonies carrying colonization factor antigen (CFA) I or CFA/II or E8775-type antigen in mixed bacterial cultures on solid media. These methods are based on mannose-resistant hemadsorption or CFA enzyme-linked immunosorbent assay (ELISA) on nitrocellulose replicas of the cultures allowing simultaneous analysis of up to 200 colonies per plate. The sensitivity and specificity of the CFA ELISA nitrocellulose replica method were 97 and 99%, respectively, for CFA/I-carrying colonies and 99 and 100% for CFA/II-positive colonies; corresponding figures for the quicker and simpler hemadsorption modification were somewhat lower. Both methods seem to be useful for studying excretion of CFA-carrying bacteria in feces, as indicated by studies in rabbits infected with enterotoxin-producing Escherichia coli in a nonligated-intestine model. By initially absorbing CFA-carrying bacteria on erythrocytes and then performing nitrocellulose replicas of agar colonies of the nonabsorbed bacteria, CFA-deficient mutants could be identified by the hemadsorption method, as well as by the CFA ELISA. Treatment of CFA-carrying bacteria with antiserum against CFA and complement also resulted in enrichment of spontaneous CFA-deficient mutants that could be identified by the replica methods. Several stable CFA-deficient mutants from enterotoxin-producing E. coli carrying CFA/I, CFA/II, or E8775 were isolated by these approaches.

Animals↗

Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains.

We have used site-directed mutagenesis of the hemagglutinin (H) glycoprotein of measles virus (MV) to investigate the molecular basis for the phenotypic differences observed between MV vaccine strains and recently isolated wild-type MV strains. The former downregulate CD46, the putative cellular receptor of MV, are positive for hemadsorption, and are fusogenic in HeLa cells, whereas the latter are negative for these phenotypic markers. CD46 downregulation in particular, could have profound consequences for the immunopathology of MV infection, as this molecule protects the cell from complement lysis. Mutagenesis of two amino acids, valine and tyrosine at positions 451 and 481, respectively, in the H protein from the vaccine-like Hallé MV strain to their counterparts, glutamate and asparagine, in the H protein from the wild-type Ma93F MV strain (creating the V451E/Y481N double mutation) abrogated CD46 downregulation, HeLa cell fusion, and hemadsorption. The converse double mutagenesis of the Ma93F H protein (E451V/N481Y) transferred the CD46-downregulating, fusogenic, and hemadsorption functions to this protein. The data provide the first mapping study of the functional domains of MV H. The consequences of these results for MV vaccine design and the role of CD46 in MV infection are discussed.

Animals↗

Detection of antibody and complement complexed in vivo on membranes of human cancer cells by mixed hemadsorption techniques.

The mixed hemadsorption (MHA) techniques demonstrated antibody and complement fixed in vivo to the surface of human cancer cells. Tumors from 12 cancer patients and normal tissues from 5 cancer patients and 8 patients with cerebrovascular or cardiac diseases were collected from biopsy and autopsy for in vitro testing. Antiserum to human whole immunoglobulins and antiserum to human C3 were used in the MHA techniques. Positive MHA patterns were demonstrated on the surface of cancer cells by both methods. Positive reactions ranged from 12 to 32% in mixed hemadsorption for anitbody detection and from 10 to 34% in mixed hemadsorption for complement component 3 detection. Normal tissues obtained from cancer patients or from patients who died of causes other than cancer rarely exhibited distinct MHA reactivity. Collectively, the data suggest that most human cancers are antigenic in the autologous host and that tumor-associated antigens of cancer cells react in vivo with their humoral antibody to fix complement.

Antibodies, Neoplasm↗

[Use of the hemadsorption method to demonstrate the African swine fever virus in Angola].

Attempts were made to modify the hemadsorption method used in the demonstration of the African swine fever virus. Use was made of fourteen strains of the virus with known hemadsorption properties and infectious titers, which were replicated in leukocyte cultures of swine blood, in three variants: (a) at direct infection of the cultures without changing the medium; (b) at infecting with changing the medium and adding erythrocytes to the new medium; and (c) infecting the cultures with changing the medium and adding 0.2 cm3 2 per cent erythrocyte suspension to each test tube at the 16th hour following infection. It was shown that in its third variant the hemadsorption method yielded most clearly expressed results, and all suspective fields were eliminated, the result being recorded as early as the second hour after the addition of erythrocytes, i.e., at the 18th hour following the infecting of cultures. With the third modification of the method no phagocytosis of the present erythrocytes effected by the emerging macrophages, which is common for the other two variants, could take place.

African Swine Fever↗

Temperature-dependent rosette formation by mouse lymphoma cells as a result of viral hemadsorption.

Cells from several mouse lymphomas formed rosettes with nonsensitized foreign erythrocytes through C-type virus particles clustered on the cell surface in serum-free medium held at 4 degrees C. This type of rosetting was found most typically in a lymphoma induced by Rauscher leukemia virus in tissue culture (RD-12), but it also occurred in 23 of 61 spontaneous thymic lymphomas in AKR mice. Chemically or X-ray-induced leukemias and spontaneous reticulum cell sarcomas did not form rosettes. The nature of the rosette formation may be interpreted as viral hemadsorption, with a possible relationship to hemagglutination by murine leukemia viruses. The receptor on virus particles was trypsin sensitive and showed high affinity to serum inhibitors (RIF). Serum rosette-inhibiting activity was assessed by a quantitative rosette inhibition test; rosette inhibition proved widely distributed among species. Physicochemical properties of serum RIF and their function both in vivo and in vitro were described. Rosette formation with similar temperature requirements, previously reported in a mouse lymphoma carrying membrane-bound heterophile cold hemagglutinin, was readily distinguished from viral hemadsorption by its insensitivity to mouse serum RIF.

Animals↗

Identification of immunoglobulin class and subclass of mouse monoclonal antibodies to human cell surface antigens by mixed hemadsorption assay.

A microassay is described to determine immunoglobulin Ig class or IgG subclass of mouse monoclonal antibodies by mixed hemadsorption assay. Monoclonal antibody bound to adherent target cells is reacted with serial dilutions of a panel of class or subclass specific rabbit anti-mouse Ig antisera and binding of the latter is traced by anti-rabbit globulin-coated indicator erythrocytes. The class or subclass of the bound monoclonal antibody is revealed by preferential binding of the corresponding rabbit antibody. Unlike gel immunodiffusion analysis, the mixed hemadsorption assay may be performed with unconcentrated hybridoma culture supernatants.

Animals↗