Antigens In Cultured Cells Research Articles

La protéine immunodominante de <em>Cowdria ruminantium</em> Cr32 conservée dans le genre <em>Ehrlichia</em>

Serological tests for cowdriosis are hampered by cross-reacting antibodies from animals suspected to be infected with Ehrlichia species. We have monitored infections with Ehrlichia bovis, E. ovina, E. canis and E. phagocytophila in experimental animals by competitive ELISA, Western blotting and immunofluorescence using Cowdria-infected endothelial cell culture antigens. Cross-reactions due to Ehrlichia antibodies could be attributed to the recognition of epitopes on the immunodominant Cr32 Cowdria protein. This was especially true for E. canis and E. ovina, much less for E. bovis, but not at all for E. phagocytophila. In addition, strong cross-reactivity between Cowdria and antibodies to E. Chaffeenis were demonstrated. These findings are in agreement with the phylogenetic relationships, recently reported by van Vliet et al. in 1992, between Cowdria and other members of the tribe Ehrlichieae, which showed Cowdria to be closely related to E. canis and also to E. chaffeensis. Although the tests used in this study remain valuable tools under laboratory conditions, their specificity requires improvement. It is suggested to study recombinant Cowdria antigens for the development of second generation serological tests for cowdriosis.

Molecular specificity of two commercial enzyme linked immunosorbent assays for human immunodeficiency virus antigens.

Only "fair" agreement has been shown between the Abbott and DuPont enzyme linked immunosorbent assays when used for the detection of human immunodeficiency virus (HIV) antigen in serum samples from asymptomatic HIV antibody positive homosexual men. To investigate the discrepancies between the two ELISA results, further experiments were performed. The rabbit detector antibody solutions of both tests were western blotted and showed that the DuPont test was specific for p24; the Abbott detector antibody had bands for p18, p41-43, gp120 as well as p24. By using dilutions of a known amount of HIV antigen, the Abbott test could detect 20 pg/ml p24; the DuPont test could detect 30 pg/ml p24. The DuPont test was also more sensitive than the Abbott test at detecting a synthetic 104mer peptide of p24. Within the 104mer sequence two regions (294-318, 334-348 amino acids) inhibited the binding of the DuPont detector antibody, but no blocking was observed with the Abbott antibody. Although the Abbott test was slightly more sensitive at detecting HIV protein than the DuPont test, the major difference between the tests was in the molecular specificity, in that the Abbott test detected proteins other than p24. This may not be important for detecting antigen in cell culture, but it may affect the detection of antigenaemia in patients' sera.

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

Summary An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could detect SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was detected by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of detection of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was detected in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.

Rapid detection of cytomegalovirus DNA in urine samples with a dot blot hybridization immunoenzymatic assay

A dot blot hybridization immunoenzymatic assay for the rapid detection of cytomegalovirus DNA in urine samples was developed by using a digoxigenin-labeled probe which was immunoenzymatically visualized by antidigoxigenin Fab fragments labeled with alkaline phosphatase. A total of 516 urine samples from different groups of subjects were analyzed, and the hybridization assay was able to yield results within 24 h. The results obtained were compared with results for detection of cytomegalovirus antigens in infected cell cultures.

Early detection of cytomegalovirus in cell culture by a new monoclonal antibody, CCH2

A CMV monoclonal antibody, CCH2, produced in this laboratory was evaluated for rapid detection of CMV. Two staining procedures, immunofluorescence and an immunoenzymatic technique using biotin-streptavidin peroxidase, were compared. The CCH2 monoclonal antibody was used to demonstrate early CMV antigen in cell culture 24 h after inoculation of 598 urine samples from kidney transplanted patients by indirect immunofluorescence in comparison with virus isolation. One hundred and sixty of the specimens were stained additionally by an immunoenzymatic technique and the results were compared. CMV was isolated from 170 out of 598 specimens within 6 weeks. Early CMV antigen was demonstrated in 114 of these specimens by immunofluorescence giving a sensitivity of 67% and a specificity of 95%. In the comparison with the immunoenzymatic staining procedure the results for all three tests agreed for 81% (130/160) of the specimens. After resolving discordant results into true positives and true negatives, the sensitivity was 87, 85 and 70%, respectively for virus isolation, immunoenzymatic staining and immunofluorescence and the specificity 100, 96 and 99%. The CCH2 monoclonal antibody proved to be useful for rapid detection of CMV in urine specimens and using immunoenzymatic staining with biotin-streptavidin a sensitivity comparable to that of virus isolation was found.

Detection of human cytomegalovirus early and late antigen and DNA production in cell culture and the effects of dimethyl sulfoxide, dexamethasone, and DNA inhibitors on early antigen induction

Recently a conventional method for the laboratory diagnosis of human cytomegalovirus (HCMV) infection was improved by using centrifugation culture to enhance viral adsorption and by detecting HCMV early antigen and DNA. Comparison of the sensitivity of three rapid methods using commercial diagnostic reagents for the detection of HCMV early antigen (EA), late antigen (LA), and DNA was quantitatively evaluated in centrifugation cultures of human fibroblast cells (MRC-5) infected with HCMV. HCMV-EA was first detected 4 hours after infection, and the number of antigen-positive cells increased rapidly thereafter. Using biotinylated DNA probe, viral DNA was first detected 12 hours postinfection; the number of DNA-positive cells increased slowly. HCMV-LA was first seen 48 hours postinfection, and the number of LA-positive cells also increased thereafter. Thus detection of HCMV-EA was the most rapid and sensitive method for HCMV diagnosis. Several chemical compounds have been used to enhance HCMV replication. The effect of dimethyl sulfoxide (DMSO), dexamethasone (DEX), 5-bromo-2-deoxyuridine (BrdU), 5-fluoro-deoxyuridine (FdU), and cytosine arabinoside (Ara-C) on HCMV-EA induction was evaluated in centrifugation cultures of MRC-5 cells infected with HCMV. Infected cells treated with 1% DMSO alone or with DMSO plus DEX (10(-5) M) have been shown to increase the number of HCMV-EA-positive cells three- to fivefold over the untreated control cultures. The enhancing effects of Ara-C, BrdU, and BrdU plus FdU were demonstrated only occasionally.

Le Test De lL Coagglutination: Une Technique Simple Et Rapide Pour Le Diagnostic De La Variole Caprine

The coagglutination test was standardised using Staphylococcus aureus strain Cowan I (containing Protein A) coated with anti-goat pox serum for the detection of goat pox antigen of infected goat skin or kid kidney cell culture antigen. Agglutination was observed within 10 seconds in virus dilutions up to 10(-6). As the test is easy to perform it can be used for rapid diagnosis of goat pox.

Demonstration of Bovine Viral Diarrhoea Virus Antigens in Cell Cultures and in Paraffin-embedded Tissue Sections by the Peroxidase-antiperoxidase (PAP) Technique Using Monoclonal Antibodies

The diagnosis of both bovine viral diarrhoea (BVD) and mucosal disease (MD) is usually made on the basis of characteristic clinical and pathological findings. The definitive etiological diagnosis by virus isolation is time consuming, expensive and elusive. Isolation of the virus in cell cultures is rather difficult since it has no characteristic cytopathic effect (CPE). Furthermore, many strains have no CPE at all. Due to these uncertainties, virus isolation trials are generally supported by additional tests (Radostits & Littlejohns 1988).

Use of immunogold preembedding technique to detect hepatitis A viral antigen in infected cells

Localization of virus and viral antigen in cell cultures infected with a rapidly replicating isolate of strain HM-175 of hepatitis A virus (HAV; pHM-175) was accomplished by using immunogold probes. Cells infected under one-step growth curve conditions were prefixed with 2% paraformaldehyde and 0.1-0.001% saponin at appropriate times postinfection for detection of maximum virus and viral antigen. An indirect labeling technique was employed using monoclonal antibody to HAV followed by 5 nm gold-antimouse IgG conjugate. Cells were then fixed by standard electron microscopy techniques and thin sectioned. This prefixation technique allowed penetration of the immunogold probes and moderate preservation of ultrastructure. Within infected cell cytoplasm, numerous antigenic sites were labeled with six to 200 gold particles. Two types of cells were infected with HAV and somewhat different results were obtained with the two cell types. In BS-C-1 cells, where a cytopathic effect (CPE) was not observed, myelin figures were immunogold labeled or frequently were located near immunogold-labeled sites. Vesicles containing viruslike particles (14-22 nm) were also observed. A significant observation in infected FRhK-4 cells was the presence of multivesicular bodies labeled with immunogold. Microfilaments were commonly seen near the multivesicular bodies. Our results demonstrate that the choice of prefixation method for immunogold labeling should be empirically determined for the cell type and condition.

Detection of bluetongue virus antigens in Culicoides variipennis by enzyme immunoassay

A solid-phase enzyme immunoassay (EIA) was developed to detect bluetongue (BT) virus antigens in infected cell cultures and in suspensions of infected Culicoides variipennis midges. The technique was equally sensitive for detecting the five U.S. BT virus serotypes (2, 10, 11, 13, and 17) in cell cultures. EIA reliably detected about 3.8 log10 median tissue culture infective doses per ml of BT virus in infected cell culture lysates. The EIA readily detected virus antigens in pools of midges infected with BT serotypes 2, 10, 11, 13, and 17 and contained 2.3 to 4.8 log10 median tissue culture infective doses per ml of BT virus. The technique was sensitive enough to detect a single infected midge in a pool with 99 noninfected midges. The EIA may be a sensitive and rapid alternative to virus isolation for surveillance of BT viruses in vector populations.

Enzyme immunoassay for detection of human immunodeficiency virus antigens in cell cultures

A sensitive and specific avidin-biotin enzyme immunoassay that uses immunoreagents from naturally infected individuals was formulated for the detection of human immunodeficiency virus (HIV) antigens. A total of 500 cell culture samples from 111 cultures were tested in this assay and in a reverse transcriptase (RT) assay. Of 353 samples that were positive in the immunoassay, 174 were positive and 179 were negative in the RT assay. The specificity of the immunoassay results was supported by the failure of samples to react with nonimmune serum, by the ability of an anti-HIV type 1 monoclonal antibody to block the reactivity of selected samples, and by the appearance of RT activity in samples drawn from some cultures after a longer period of cultivation. HIV antigens were detected in 174 of 176 RT-positive samples (sensitivity, 98.9%). A comparison of the kinetics of antigen production and RT activity revealed that detectable antigen levels frequently preceded the appearance of RT activity. Thus, 50% of virus-containing cultures were identified within 9 days by immunoassay compared with 14 days by RT assay. In addition, RT activity was often detected intermittently in cultures sampled on several days, whereas antigen levels did not decline after initial appearance. Enzyme immunoassays for HIV antigen detection that use easily obtained reagents and simple technologies could facilitate laboratory and clinical research which requires cultivation of viruses.

A monoclonal antibody that binds to the surface of photoreceptors

A monoclonal antibody (MAB), 7H11, that binds to the surface of photoreceptors has been produced. Using immunoaffinity procedures, proteins with molecular weights of 53, 48 and 42 kDa were found to bind a MAB 7H11 column, although the 48- and 42-kDa proteins may be proteolytic breakdown products of the 53 kDa protein. Electron microscopic-immunohistochemical studies show that the antigen is concentrated on the external surface of inner and outer segments of photoreceptors. It is likely that the 7H11 antigen is a component of the interphotoreceptor matrix and, therefore, may contribute to some aspect of photoreceptor-pigment epithelial interactions. However, the expression of the 7H11 antigen in cell culture suggests that it is expressed independently of direct cell-cell interactions with either pigment epithelial cells, Müller cells or other photoreceptors.

Rapid diagnosis of cytomegalovirus infection by immunofluorescent detection of early antigen in cell culture.

The conventional culture method for the isolation of cytomegalovirus was used in parallel with the rapid method of early-antigen detection to test 485 specimens that had been collected from a total of 208 patients over a period of 11 months. Statistically, the rapid immunofluorescent method was significantly better than was the conventional test for the detection of cytomegalovirus infections. The rapid method is technically simple, sensitive and specific, and is also inexpensive when a suitable polyclonal antiserum is available. This test merits use in virology laboratories that service large hospitals.

Dot immunoperoxidase assay using monoclonal antibody for direct detection of cytomegalovirus in urine samples.

A rapid, simple dot immunoperoxidase assay for the direct detection of cytomegalovirus in clinical urine samples was developed. The assay was performed on nitrocellulose paper dotted with urine pellets free of cellular debris. Cytomegalovirus was detected with a monoclonal antibody to the capsid antigen, and the complex was visualized by immunoperoxidase staining. Positive reactions appeared as well-defined dark blue spots. Of the 87 urine samples examined, 10 proved positive in the dot immunoperoxidase assay, and 77 proved negative. The results agreed completely with the detection of cytomegalovirus-induced antigens in cell cultures inoculated with clinical specimens.

Expression and modulation of tumor-associated antigens in human glioma cell cultures

Expression and modulation of tumor-associated antigens in human glioma cell cultures

Tomorrow's hepatitis B vaccines: Arie Zuckerman discusses hepatitis B vaccines of the future and the impact of AIDS on immunization

Recombinant hepatitis B vaccines produced in yeast and in mammalian cells are the first of future vaccines against this important public health problem. Hybrid virus vaccines and the use of baculoviruses for expression of hepatitis B antigens in insect cell cultures will follow shortly and chemically synthetic vaccines are under development. The impact of AIDS on future immunization programmes should be considered.

Regulated Synthesis of Low-Molecular-Weight Antigens in Keratinocyte Cell Cultures

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low Ca++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [35S]methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32PO4, the 11K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocytes suggests that these proteins are important in keratinocyte proliferation and differentiation.

Early detection of cytomegalovirus in cell culture by a monoclonal antibody

A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytophatogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at −70°C were reinoculated. CMV was detected in 8 31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods.

Epidermal cell populations and antigen expression in cultures of human neonatal epidermis.

The persistence of Langerhans cells, melanocytes and ABH red cell antigens in human epidermal cell cultures derived from neonatal foreskin explants has been investigated. Langerhans cells were recognised via the detection of ATPase activity and by immunohistochemical staining for T6 and HLA-DR antigens. Melanocytes were detected by the dihydroxyphenylalanine (DOPA) technique; whilst the presence of red cell antigens was confirmed by immunohistochemical tests using monoclonal anti-A, anti-B, and anti-H antibodies. Langerhans cells were not detected in the cultured epidermis, and few melanocytes were found more than 0.2 mm from the periphery of the explants. However, both Langerhans cells and melanocytes were found to persist for up to 4 weeks in the explants themselves. Red cell antigens, consistent with the blood group of the donor infant, were consistently detected on the surface of the differentiating keratinocytes throughout their in vitro lifespan. The significance of these findings is discussed.

Neutralization test for bk virus: plaque reduction detected by immunoperoxidase staining

We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects.