Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).     

Improvements in the detection of pea seed-borne mosaic virus by ELISA

The detection by ELISA of pea seed-borne mosaic virus (PSbMV) in pea leaves and seeds was improved by the addition of cellulase or Triton X-100 to the extraction fluid, probably because the additives aided the release of virus particles from host materials. With leaf extracts the additives were most effective at 0.1%. In initial tests cellulase was used with macerozyme, but the latter enzyme was then shown to decrease the effectiveness of cellulase. Triton X-100 was as effective as cellulase and the absorbance values obtained in ELISA of infected leaf extracts, diluted to 1/10 in extraction fluid containing the additive, were about six times greater than those of infected extracts diluted in normal extraction fluid. Five named isolates of PSbMV, in addition to the homologous isolate, were readily detected in infected leaves extracted in fluid containing Triton X-100. In tests on seeds and seedlings of seven infected seed lots of pea cv. Waverex, using Triton X-100 in the extraction fluid, PSbMV was detected in five times as many seeds as seedlings, probably mainly because in many infected seeds the virus was in the testa and not in the embryo. About 9% of infected seedlings were without recognisable symptoms 4 wk after emergence.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K=10¹⁰M^–1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of ³H-folate labelled protein (>=130 and 100kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.     

Identification of a mucosal form of enteropeptidase in triton X-100 extracts of porcine duodenal mucosa

Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.     

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Association of a photoreceptor-specific tetraspanin protein,ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.     

Modulation of the Access of Exogenous NAD(P)H to the Alternative Pathway in Potato Tuber Callus Mitochondria with Triton X-100

Alternative oxidase activity in potato tuber (Solanum tuberosum L. cv Bintje) callus mitochondria with exogenous NAD(P)H as substrate is inhibited by low concentrations of the detergent Triton X-100. Alternative oxidase activity with succinate or malate as substrate is not affected by these low concentrations of Triton X-100. Cytochrome pathway activity was not influenced under these conditions, neither with endogenous nor with exogenous substrate. Washing of Triton X-100-treated mitochondria did partially restore both uninhibited and CN-resistant NADH oxidation, indicating that under these conditions Triton X-100 does not permanently remove major components from the mitochondrial membrane. Apparently, it is possible to manipulate mitochondria in such a way that the access of exogenous NADH to the alternative pathway is blocked while access to the cytochrome pathway is uninhibited. It is suggested that membrane conditions have a regulatory function (possibly via influencing the diffusion path) in the oxidation of exogenous NADH via the alternative pathway.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The K(m) of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.     

Identification and characterization of a Triton X-100 replacement for virus inactivation

Triton X-100 detergent treatment is a robust enveloped virus inactivation unit operation included in biopharmaceutical manufacturing processes. However, the European Commission officially placed Triton X-100 on the Annex XIV authorization list in 2017 because a degradation product of Triton X-100, 4-(1,1,3,3-tetramethylbutyl) phenol (also known as 4-tert-octylphenol), is considered to have harmful endocrine disrupting activities. As a result, the use of Triton X-100 in the European Economic Area (EEA) would not be allowed unless an ECHA issued authorization was granted after the sunset date of January 4, 2021. This has prompted biopharmaceutical manufacturers to search for novel, environment-friendly alternative detergents for enveloped virus inactivation. In this study, we report the identification of such a novel detergent, Simulsol SL 11W. Simulsol SL 11W is an undecyl glycoside surfactant produced from glucose and C11 fatty alcohol. We report here that Simulsol SL 11W was able to effectively inactive enveloped viruses, such as xenotropic murine leukemia virus (XMuLV) and pseudorabies virus (PRV). By using XMuLV as a representative enveloped virus, the influence of various parameters on the effectiveness of virus inactivation was evaluated. Virus inactivation by Simulsol SL 11W was effective across different clarified bioreactor harvests at broad concentrations, pH, and temperature ranges. Simulsol SL 11W concentration, temperature of inactivation, and treatment time were identified as critical process parameters for virus inactivation. Removal of Simulsol SL 11W was readily achieved by Protein A chromatography and product quality was not affected by detergent treatment. Taken together, these results have shown the potential of Simulsol SL 11W as a desirable alternative to Triton X-100 for enveloped virus inactivation that could be readily implemented into biopharmaceutical manufacturing processes.     

Insights into virus inactivation by polysorbate 80 in the absence of solvent

Triton X-100 has long been used either alone or in combination with solvent to inactivate enveloped viruses in biopharmaceutical manufacturing. However, European Chemicals Agency (ECHA) officially placed Triton X-100 on the Annex XIV authorization list in 2017 because 4-(1,1,3,3-tetramethylbutyl) phenol, a degradation product of Triton X-100, is of harmful endocrine disrupting activities. As a result, any use of Triton X-100 in the European Economic Area would require an ECHA issued authorization after the sunset date of January 4, 2021. In search of possible replacements for Triton X-100, we discovered that polysorbate 80 (PS80) in absence of any solvents was able to effectively inactive enveloped viruses such as xenotropic murine leukemia virus and pseudorabies virus with comparable efficacy as measured by log reduction factors. Interestingly, PS80 did not show any virucidal activities in phosphate buffered saline (PBS) while achieving robust virus inactivation in cell-free Chinese hamster ovary (CHO) bioreactor harvests. This intriguing observation led us to speculate that virus inactivation by PS80 involved components in the cell-free CHO bioreactor harvests that were absent in PBS. Specifically, we hypothesized that esterase and/or lipases in the cell-free bioreactor harvests hydrolyzed PS80 to yield oleic acid, a known potent virucidal agent, which in turn inactivated viruses. This theory was confirmed using purified recombinant lysosomal phospholipase A2 isomer (rLPLA2) in PBS. Subsequent characterization work has indicated that virus inactivation by PS80 is effective and robust within temperature and concentration ranges comparable to those of Triton X-100. Similar to Triton X-100, virus inactivation by PS80 is dually dependent on treatment time and temperature. Unlike Triton X-100, PS80 inactivation does not correlate with concentrations in a simple manner. Additionally, we have demonstrated that PS20 exhibits similar virus inactivation activities as PS80. Based on the findings described in the current work, we believe that PS80 is potentially a viable replacement for Triton X-100 and can be used in manufacturing processes for wide spectrum of biopharmaceuticals to achieve desirable virus clearance. Finally, the advantages and disadvantages of using PS80 for virus inactivation are discussed in the contexts of GMP manufacturing.     

Immunologic comparison of the conformations of apolipoprotein B. Investigation of methodologies for the reconstitution of delipidated and denatured apolipoprotein B with nonionic surfactants

Immunologic probes have been used to examine the conformation of apolipoprotein B (apo-B) as it exists within native low density lipoprotein (LDL) after lipid displacement with Triton X-100 and after denaturation with guanidine hydrochloride organic solvent delipidation and reconstitution with Triton X-100. Antigenic expression was assayed in two systems: by using either Triton X-100 or bovine serum albumin to maintain protein solubility. Apo-B delipidated by lipid displacement using Triton X-100 was virtually identical to LDL-apo-B in both systems, as assayed by polyclonal antisera prepared in rabbits against either antigen. Thus the native antigenic sites are preserved, although the displacement of the lipid core of LDL drastically alters the physical properties of the particle. Apo-B delipidated by solvent extraction in guanidine was reconstituted with Triton X-100 by several methods, and the products were examined immunologically. One method yielded a product that resembled apo-B as delipidated with Triton X-100, although full reconstitution could not be achieved. Nevertheless, Triton promoted refolding of apo-B to reform partial native structure as judged immunologically. By using both physical and immunologic methods for assessing structure, it is clearly evident that the perceptions of the conformational states of reconstituted apo-B can be very different, and multiple criteria need to be used to assess lipoprotein reconstitution.     

Triton X-100 can alter the temporal sequence of the light-driven proton pump of archaerhodopsin 4

We report that Triton X-100 can alter the temporal sequence of the light-induced proton uptake and release of archaerhodopsin 4 (AR4), a proton pumping protein in a species of Halobacteria from a Tibetan salt lake. Under physiological conditions, AR4 isolated from the bacterium exhibits a reversed temporal order of proton release and uptake compared to what is observed for bacteriorhodopsin (BR). However, in the presence of Triton X-100 early proton release was observed in AR4 at neutral pH by us. Further, this temporal order for light-driven proton release and uptake for AR4 was found to be recovered after the removal of Triton X-100 by Biobeads. This phenomenon of detergent-induced alteration of the order of proton release and uptake has not yet been reported in any other retinal-containing membrane protein such as BR. Our findings indicate that the function of AR4 is influenced by its self-assembled state, and meanwhile imply some subtle protein-lipid interactions or protein-protein interactions in adjusting the proton pumping behavior of AR4.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Effects of detergents on the West Nile virus protease activity

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at ?0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC₅₀ value of 5.7 ± 2.5 μM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.     

Further evidence for an active site polypeptide of galactosyl transferase

In a previous report it was shown that galactosyl transferase activity after blotting from acrylamide gel was present in a molecular weight range of less than 14 kDa, in Triton X-100 (1). Molecular sieve chromatography on Superose 12, in the presence of Triton X-100, gave the same result. The low molecular weight activity peak was eluted together with peptides as a part of the covalent structure of the enzyme or as absolutely requires effectors. Peptide mapping showed a new poly-lysine-like peptide and a new hydrophobic peptide in this low molecular weight activity peak as effectors of the enzyme inside its hydrophobic environment.     

Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate((R)) has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6log after 30min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.     

磷脂酰肌醇－4－激酶的的免疫分析

在纯化猪肝微粒体磷脂酰肌醇－４－激酶（ＰＩ－４－Ｋ）的基础上,制备该酶的免疫血清、提纯ＩｇＧ,并建立了ＰＩ４－Ｋ的ＥＬＩＳＡ,进行不同组织中ＰＩ４－Ｋ的免疫沉淀分析和免疫定量。结果证明：猪肝微粒体ＰＩ４－Ｋ的抗血清或抗体ＩｇＧ可沉淀猪肾和猪肺的微粒体膜、猪肝细胞膜和人中性粒细胞细胞膜ＴｒｉｔｏｎＸ－１００增溶液（ＴＸＳＭ）中的ＰＩ４－Ｋ,表明不同组织、不同亚细胞器和不同种属的ＰＩ４－Ｋ有相似的抗原性。猪肝、肾、肺、胃、小肠和大肠ＴＸＳＭ中ＰＩ４－Ｋ的活力和酶蛋白含量相仿,急性中性粒细胞白血病患者的中性粒细胞膜上ＰＩ４－Ｋ的含量和活力都增加３．５倍左右．加上ＰＩ４－Ｋ抗体沉淀各组织ＰＩ４－Ｋ活力和酶蛋白的百分率基本相同,支持文献报道组织中微粒体或细胞膜中的ＰＩＫ主要是ＰＩ４－Ｋ,而免疫性不同的ＰＩ３－Ｋ含量极少的观点．