A simple method to quantify staphylococcal protein A in the presence of human or animal IgG in various samples.

Immunoassays designed to measure low concentrations of staphylococcal protein A (SPA) that have been leached into antibody preparations intended for therapeutic use are subject to differing degrees of interference. Methods established to quantify SPA in murine antibody preparations are not accurate in the presence of human or humanized IgG. We report the development of an enzyme-linked immunosorbent assay (ELISA) for SPA with a detection limit of 7 pg/ml and the optimization of a method that permits complete dissociation of SPA-immunoglobulin-complexes. This assay is a modification of our heat-mediated dissociation (HD-SD) treatment with sodium dodecyl sulfate (SDS) and diethylenetriaminepentacetic acid (DTPA) for total immune-complex dissociation, in which the heat treatment has been prolonged and the diluent is characterized by increased protein content and buffering capacity. The diluent developed contains SDS, DTPA and bovine serum albumin dissolved in a 0.1 M phosphate buffer (pH 7.2). To validate the efficiency of this novel method, a series of samples have been assayed, including samples reconstituted in vitro, samples of purified antibodies, and plasma from patients. The described method has been shown to be generally efficient in quantitating all native and recombinant SPA in samples containing up to 50 mg/ml of human IgG. These data demonstrate the utility of this technique in determining SPA contamination of recombinant immunoglobulin therapeutic products.

A simple and robust method for the complete dissociation of HIV-1 p24 and other antigens from immune complexes in serum and plasma samples.

Accuracy of antigen determination in human plasma samples is often adversely affected by immune complex formation between antigens (e.g., HIV-1 p24 protein) and specific antibodies. In this study we describe an optimized method for complete immune complex dissociation (ICD) in plasma. This method is based on heat denaturation of antibodies and utilizes a defined solution of sodium dodecyl sulfate (SDS) and diethylenetriaminepentaacetic acid (DTPA) as diluent. The efficiency of this procedure for ICD was compared with those of published methods, employing heat denaturation alone and acidification. Plasma samples from patients participating in anti-retroviral treatments and samples reconstituted in vitro were treated and analyzed in parallel. HIV-1 p24 antigen was determined by quantitative enzyme-linked immunosorbent assay (ELISA). In 312 samples from 97 patients, antigenemia was found in 44.9% when measured directly and in 87.2% after this treatment. In a subset of 56 samples, 21.4% tested positive prior to treatment, while after either novel treatment, heat denaturation or acidification, these samples tested positive in 80.4%, 62.5% and 60.7%, respectively. In 94% of cases viral RNA was detected. This improved procedure for ICD provides a reliable and convenient method for complete and accurate p24 antigen detection in human plasma and is applicable to commercially available test kits.

Interferon alpha primes early proliferative response of bone marrow cells in vivo.

Using colony-forming assays, a number of previous studies established that interferon alpha (IFN-alpha) could cause bone marrow cell (BMC) suppression. In this study, the suppressive effect of IFN-alpha is however shown to be time-dependent, occurring only 7-8 days after transfer of BMC obtained from IFN-alpha-treated mice to growth factor-containing culture medium. In contrast, in the interval before suppression is observed, BMC obtained from IFN-alpha-treated mice initially proliferated more rapidly than BMC from placebo-treated mice. These findings suggest that IFN-alpha acts in vivo to prime the proliferative responses of BMC, a hitherto unexpected action which may have clinical relevance.

Acquisition of host cell-surface-derived molecules by HIV-1.

OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.

A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees.

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.

Detection of the trans activity of the plum pox virus NIa-like protease in infected plants.

The NIa-like protein of plum pox virus is a protease with high sequence specificity that is autocatalytically released from the viral polyprotein. In order to determine whether the protease is active in trans we constructed a fusion protein consisting of the C-terminal region of the plum pox virus polyprotein and the staphylococcal Protein A. The authentic protease recognition sequence Asn-Val-Val-Val-His-Gln-Ala occurs in the centre of this protein fusion. This protein was cleaved specifically by extracts of plum pox virus-infected plants due to the strong activity of the viral protease making it a useful tool for diagnostic purposes.

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures.

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10(7) cells per ml were obtained. The described system is scaleable.

The complete nucleotide sequence of plum pox virus RNA.

The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.

Expression of the plum pox virus coat protein region in Escherichia coli.

A cDNA complementary to the 3' end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.

Early screening for anti-plum pox virus monoclonal antibodies with different epitope specificities by means of gold-labelled immunosorbent electron microscopy.

The technique of gold-labelled immunosorbent electron microscopy for the initial screening of monoclonal antibodies 10 days after cell fusion from 96-well culture plates is described. The technique is used to identify clones that secrete antibodies binding on the surface of the virion or to viral subunits, and compared to ELISA and Western blotting. High sensitivity was demonstrated.

Construction of a large scale membrane reactor system with different compartments for cells, medium and product.

A flat membrane reactor system has been designed where different membranes separate cells from medium and cells from product, respectively. By the use of this system the product can be enriched and partially purified within the reactor. Like other membrane systems perfect protection from mechanical stress of surface adherent as well as suspension type-cells is achieved. The scale up of the design is possible in a wide range. The prototype construction corresponds to a conventional reactor of appr. 300 l and contains a membrane area of 25 m2. Beside the three chamber operation mode it is possible to operate the system as a two chamber reactor. With slightly modified gaskets the reactor resembles a new type of tube reactor, where the cells can be refed on their way through the 50 m long tube.

Entrapment of animal cells for production of monoclonal antibodies and other biomolecules.

Animal cell technology is attracting considerable interest because of the capacity of animal cell cultures to synthesize or transform complex compounds such as virus vaccines, immunochemicals, hormones or enzymes. For the growth of surface-dependent cells, microcarrier technology is gaining importance. Here, we have attempted to immobilize surface-independent cells, normally grown in suspension, by entrapping them in polymer microbeads. Such entrapment should give increased stability to the normally fragile animal cells, allow for high cell densities to be achieved within the beads and make such preparations suitable for continuous operation. At the same time, the need for separation of the desired product from the cells is obviated. With the model systems studied, we showed that hybridoma, as well as other cell lines entrapped in agarose microbeads, remained viable. Both immunoglobulins and lymphokines were exported through the microbeads into the medium for 1-3 weeks, at levels corresponding well to those produced with free cells.

Entrapment of animal cells for the production of biomolecules such as monoclonal antibodies.

An important problem in the production of monoclonal antibodies is the large-scale cultivation of hybridoma cells in vitro. Fragility of cells and suboptimal in vitro cultivation methods have led to poor results in larger scale production up to now. To lower the mechanical stress on the cells we tried to entrap the cells into microspheres made of polymer material. In addition to other materials, agarose as embedding medium was investigated and results with hybridoma and other, non anchorage-dependent cell lines are given. The conclusion of the results is that encapsulation of living cells is possible and entrapped cells remain viable and continue to produce the desired substance for at least several weeks. The substances are secreted through the polymer matrix. Handling of microspheres is shown to be easy and simple fermentation apparatus may be used for the production on a reliable technical scale. Some problems remain unsolved, such as the determination of viable cell count within the microspheres and cultivation in columns which seems to be the simplest form of continuous production process.

Experience with testing for mycoplasma with five different methods.

Five methods for mycoplasma-content detection in cell-cultures are established in this laboratory. Results with about 20 different cell lines continuously grown in this laboratory in some determinations indicate that four methods give good correspondence. A microbiological method gave contrary results in many cases. It is possible that new infections will grow on mycoplasma broth but the older infections are adapted to cell-culture and give negative results on artificial medium. New low-level infections cannot be detected by the other methods which do not include any efficient enrichment step. The most convenient method in our opinion is a DNA-staining method according to Chen because this is a very quick, inexpensive and easily performable process. In practice it seems necessary to check cell cultures in two ways, firstly with a quick method and additionally with the microbiological test to ensure the detection of new low-level infections.