The use of micropeptide maps and monoclonal antibodies for antigenic variation analysis of influenza A viruses isolated in China from 1972 to 1983

125I-labelled haemagglutinin (HA) micropeptide mapping and haemagglutinin inhibition (HAI) tests with monoclonal antibodies were used in analysing antigenic variation of six strains of influenza A virus isolated from 1972 to 1983 in China. The results from micropeptide mapping generally coincided with those of sequence analysis. 125I-labelled HA micropeptide mapping is a simple, reproducible and practical method for surveillance of influenza, especially in combination with HAI test using monoclonal antibodies.     

Liquid-phase and solid-phase radioimmunoassay with herpes simlex virus type 1 nucleocapsids

Liquid-phase radioimmunoassay (LPRIA) and solid-phase radioimmunoassay (SPRIA) are described utilizing either 125I-labelled or immobilized nucleocapsids (NC) of herpes simplex virus type 1 (HSV-1). These techniques appeared sensitive and specific for quantification of HSV-NC antigens and corresponding antibodies.     

The use of 125I-labelled protein A for the detection of humoral immunity to gross murine leukaemia virus

A solid-phase radioimmunoassay utilising bind of 125I-labelled protein A to antibodies bound to virus adsorbed onto microtitre plates was shown to be suitable for detection of humoral immunity to Gross murine leukaemia virus (MuLV). The specificity of the reaction was shown by the fact that only homologous or closely related viruses effectively inhibited binding of antibodies to adsorbed virus. With this method a low level of spontaneous humoral immunity was demonstrated in sera from AKR/Crc mice, a strain with high concentrations of endogenous virus, whereas little or no anti-viral activity was found inCBA/H-T6Crc, a subline that does not appear to express MuLV.     

Evaluation of a Sindbis virus vector displaying an immunoglobulin-binding domain: antibody-dependent infection of neurons in living mice

Viral vectors that genetically incorporate an immunoglobulin-binding domain on their surfaces provide many advantages because of the availability of a spectrum of antibodies that allow the selection of a wide range of target cells. However, the specificity and the effectiveness of this system have not been evaluated in the field of neuroscience. We investigated the effectiveness and specificity of a recombinant Sindbis virus displaying an antibody-binding domain of bacterial protein A (ZZ Sindbis). We found that the ZZ Sindbis virus vector specifically infected hippocampal neurons in an antibody-specific manner in living mice, although the efficiency of the gene transduction was not high. However, the ZZ Sindbis virus vector that did not display any specific antibodies continued to exhibit intrinsic tropism toward Bergmann glial cells in the cerebellum. These data indicate that the antibody-displaying viral vectors are potentially useful for delivering a gene of interest to a specific subset of neurons in the central nervous system with the help of neuron type-specific antibodies.     

Prevalence of sindbis virus neutralizing antibodies among Swedish passerines indicates that thrushes are the main amplifying hosts

The Sindbis virus uses birds as vertebrate hosts in the summer amplification cycle, and the virus is transmitted by ornithophilic Culex species. Previous field and experimental studies have shown that mainly passerine birds are involved in the amplification. To delineate the pattern of Sindbis virus infections among passerines, we collected and sampled birds for blood at five study sites located in northern, central, and southern Sweden. All study sites were lowland forested wetlands and humid forests. The blood samples were assayed for Sindbis neutralizing antibodies, and we tested if the prevalence of Sindbis antibodies varied in relation to bird characteristics (i.e., species, body-mass, sex, and age), and environmental factors (i.e., year, month, and location). We found that Sindbis virus infections occurred in almost all passerine species sampled, but that the infection prevalence was unequally distributed among species. The fieldfare, the redwing, and the songthrush each had significantly higher prevalence than the average for all species. Large passerine species had higher infection prevalence than small species. The infection was less prevalent in hatching-year birds than in older birds during June and July, but not in August. Males and females had the same infection prevalence. The prevalence of Sindbis antibodies was higher in central than in southern Sweden, which coincided with a higher proportion of fieldfare-redwing-songthrush samples in the central region of the country. Thus, it is possible that regional and annual variations in the prevalence of Sindbis antibodies in Swedish passerine species depend on the number of fieldfares, redwings, and songthrushes available for feeding by vector mosquitoes.     

A Modified Crossed Radioimmunoelectrophoretic Method for Determination of Allergen-Specific IgG Antibodies

A crossed radioimmunoelectrophoretic (CRIE) method for detection of specific IgG antibodies in patients' sera against horse hair and dander was developed. The unacceptably high non-specific binding encountered when substituting 125I-labelled antihuman IgG for 125I-labelled antihuman IgE in an ordinary CRIE was eliminated by the combined use of 125I-labelled Protein A as detector, and F(ab')2-fragments of the allergen-specific rabbit antibodies. The low background binding thus obtained makes the method useful for detection of specific IgG in sera where the ratio between specific and non-specific IgG is low. Therefore the method should also be applicable to other antigen/allergen systems.     

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.     

Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response.

An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.     

A solid phase micro-radioimmunoassay to detect minute amounts of Ig class specific anti-viral antibody in a mouse model system.

A simple and rapid micro-radioimmunoassay was developed to detect and quantitate class specific mouse anti-Sendai virus antibodies. Two different 125I-labelled indicator systems were studied. After incubation of test serum with antigen one system used 125I-rabbit anti-mouse IgG (RIA 1) and the second employed rabbit anti-mouse IgG, IgA or IgM followed by 125I-sheep anti-rabbit immunoglobulin reagent (RIA 2). The RIA 2 method was adopted for routine use as it was more sensitive, gave better discrimination between sample and background counts and eliminated the need for several labelled rabbit anti-mouse Ig class specific antisera. The technique was found to be about 100 times more sensitive than conventional HI tests, specific, reliable and economical of reagents and time.     

Radioallergosorbent test (RAST) for measurement of IgG antibodies to Aspergillus fumigatus in sera of patients with different lung diseases

A radioallergosorbent test (RAST) for measuring human anti-Aspergillus fumigatus (Af) antibodies of the IgG class is described. The use of 125I-labelled animal antibodies against human IgG is compared with the use of 125I-labelled protein A. Under optimal conditions the radioactivity binding ratio between pooled patients' serum and pooled healthy persons' serum is 8-11.5. The immunoblotting technique was used to investigate so-called non-specific binding. The results obtained show that most if not all human sera contain anti-Af antibodies of the IgG type. The difference between pathological and normal immunological response to Af antigens seems to be in the antibody titres rather than in the presence or absence of antibodies to these antigens.     

The use of [125I]C1q subcomponent for the measurement of complement binding antibodies on cell surfaces.

An assay using 125I-labelled human C1q has been developed for the measurement of complement fixing antibodies bound to cell monolayers or cell suspensions. The method has been adapted for use either during or after sensitisation of the cells with antiserum, is simple to perform and does not require require prelabelling of the target cells.     

A cleavable biotinylating agent and its use in protein electroblots.

A direct method for the synthesis of N-biotinyl penicillamine is described. It has been shown to be a convenient biotinylating agent for antibodies which have been previously coupled with SPDP. The biotinylated antibodies can be used to detect antigens on protein electroblots using 125I-labelled streptavidin and radioautography. The biotin and its attached streptavidin and radiolabel can be removed under mild conditions and the blot reprobed with a different antibody using an identical protocol.     

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.     

Molecular properties of T-lymphoma immunoglobulin. III. Peptide composition of the light chain.

Structural studies of T-cell immunoglobulin light chains have been carried out in order to ascertain whether they possess a unique structure or if they resemble standard kappa or lambda isotypes. T-cell immunoglobulin was isolated from 125I-labelled culture medium of monoclonal, continuously cultured T-lymphoma cells, and the purified 125I-labelled light chains were subjected to either cleavage by cyanogen bromide or digestion with trypsin. These peptides were then resolved and compared with those derived from 125I- and 131I-labelled murine kappa and lambda chains in mixed label experiments, by means of polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers and ion exchange chromatography. The profiles obtained suggested that T-lymphoma immunoglobulin light chains are immunoglobulin polypeptides and most closely resemble kappa chains. These results support available antigenic and mRNA hybridization data, which also suggest that T cells bear kappa-like light chains.     

Antiidiotypic antibodies as probes for the Sindbis virus receptor

Rabbit polyclonal antiidiotypic antibodies were made to mouse monoclonal antibodies that neutralize the infectivity of Sindbis virus. One of the antiidiotypic antisera obtained has properties characteristic of an antireceptor antiserum. It binds to the surface of chicken cells as shown by immunofluorescence and partially blocks virus binding to these cells as determined by binding of radiolabeled virus or by a plaque reduction assay. It also immunoprecipitates a protein with a molecular weight of 63,000 from chicken cells. From the fact that the antiserum will only partially block virus uptake, and that it does not block uptake of a variant of Sindbis virus resistant to the monoclonal antibody used to produce the antiidiotypic antiserum, we propose that at least two distinguishable receptors can be used by Sindbis virus to enter chicken cells. Furthermore, the receptors used by Sindbis to enter BHK cells appear to be different from those on chicken cells, at least in part, in that the antiidiotypic antiserum does not recognize the BHK counterpart of the chicken cell receptor. We suggest that the alphaviruses use a number of distinguishable receptors which differ depending on the host and the tissue. In chicken cells the 63,000 molecular weight protein may be one of them. The diversity of such multiple receptors could account for the very wide host range of the alphaviruses, which infect mosquitoes, birds, and mammals.     

Haemorrhagic manifestations with Sindbis infection. Case report

Sindbis infection in man occurs rarely in Australia. Most recorded cases are either asymptomatic or result in a fever sometimes accompanied by a macular or vesicular rash. This case is of particular interest because of the severe haemorrhagic vesicular rash and the repeated recurrence of symptoms over a 5 month period together with the persistence of IgM antibodies to Sindbis virus.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

A radioimmunoassay detecting the bovine leukaemia virus transmembrane protein gp30 and anti-gp30 antibodies in the serum of cattle

By means of SDS PAGE we isolated from virus-infected foetal lamb kidney (FLK) cells a relatively homogenous envelope transmembrane protein gp30 of bovine leukaemia virus (BLV). As shown by a partial sequence analysis of the N-terminus of this protein, our gp30 preparation contained only traces (less than 5%) of p24 gag protein: Rabbit anti-gp30 serum did not cross react with the BLV proteins gp51, p12, p15(1), p15(2), and p10 but reacted weakly with the p24 polypeptide. 125I-labelled gp30 (chloramine-T) was precipitated with the serum of BLV-infected cattle. Nonlabelled preparation of gp30 competitively inhibited the reaction of 125I-labelled gp30 with the natural antibodies. We investigated 193 cattle sera by liquid phase radioimmunoassays (RIA) using 125I-gp30, gp51 and p24 antigens. Sixteen noninfected cattle sera were negative in all tests. The 177 serum samples of BLV-infected animals were examined to the diagnostic value of the three tests. Of these, 175 were positive in gp51 RIA, 172 in p24 RIA and 164 in gp30 RIA. In all three tests, 159 sera were positive while 18 sera, mostly coming from animals with normal leukocyte counts, were positive only either with gp51 or p24, or were double positive with either gp51/p24 or gp51/gp30. We conclude that the gp51 RIA is superior to both the gp30 and the p24 RIA and that the gp30 RIA will be useful for investigating the role of gp30 in virus pathogenicity.     

Radioimmunoassay for the measurement of mass and avidity of anti-nerve growth factor antibodies

A radioimmunoassay is described for measuring on a weight basis the total amount of humoral antibodies to the purified protein nerve growth factor (NGF). The technique is based on the primary interaction of the antibodies with ¹²⁵I-labelled NGF added in large molar excess. The resultant immune complexes are precipitated by ammonium sulfate at 37% saturation, while unbound ¹²⁵I-labelled NGF is removed with the supernatant. The assay permits measurement of antibodies in a range of sensitivity of 0.1–1.0 ng antibody protein per ml. It is specific and reproducible and permits estimates of the antibody avidity.     

Measurement of antibodies to herpesvirus types 1 and 2 in human sera by microradioimmunoassay.

The binding of 125I-labelled anti-human antibodies against the fc IgG fragment to unlabelled antiviral immunoglobulins in the surface of infected cells was used to quantitate antibodies against herpes simplex virus type (HSV-1) and type 2 (HSV-2) in sera from patients with cervix carcinoma. The microradioimmunoassay technique (micro-RIA) proved to be 5-10 times more sensitive than the microneutralization test. Antibody titres determined by micro-RIA correlated with neutralizing antibody titres to both HSV-1 and HSV-2. The relative antibody titres to HSV-1 and HSV-2, as determined by micro-RIA, could be used to distinguish persons previously infected with HSV-2 by means of II/I indices.