Analyte detection with DNA-labeled antibodies and polymerase chain reaction

We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.     

Stable and monodisperse lipoplex formulations for gene delivery

A stable single vial lipoplex formulation has been developed that can be stored frozen without losing either biological activity or physical stability. This formulation was identified by systematically controlling several formulation variables and without introducing either stabilizers or surfactants. Analytical assays were used to unambiguously characterize the formulations. The critical formulation parameters were: (1) the size of the cationic liposomes; (2) the rate and method of DNA and cationic liposome mixing; and (3) the ionic strength of the suspending vehicle. The mixing conditions were precisely controlled by using a novel, specially designed continuous flow pumping system in which the DNA and liposome solutions were mixed at the junction of a T-connector. Homogenous cationic liposome preparations were prepared by extrusion in two different size ranges of either 400 or 100 nm. Extruded liposomes produced more monodisperse and physically stable lipoplex formulations than unextruded liposomes, but the formulations prepared with 100 nm liposomes were less active in in vitro transfection assays than either the 400 nm or unextruded liposomes. Low ionic strength and 5% sorbitol were required for the lipoplex formulations to survive freezing and thawing. A frozen lipoplex formulation stored for more than a year maintained its biological activity. These results have broad implications for the pharmaceutical development of lipoplex formulations for gene delivery.     

Affinity liposomes in vivo: factors influencing target accumulation

To make universal and efficient liposome-based drug carriers, liposomes should be able to recognize and bind other targets beyond their natural targets, the cells of the reticuloendothial system. To make liposomes targeted, numerous methods to couple active substances, primarily, monoclonal antibodies, to the liposome surface have been developed. Resulting immunoliposomes (or affinity liposomes) demonstrate good targeting to cells and organs both in vitro and in vivo. However, the short circulation time of immunoliposomes prevented them from accumulating in targets with diminished blood flow or low antigen concentration. Long-circulating liposomes were prepared by coupling soluble and flexible polymers, such as polyethylene glycol, to the liposome surface. The mechanism of liposome steric protection with flexible polymers is based on the formation of dense "conformational cloud' by a grafted polymer over the liposome surface, and might be analyzed in terms of a statistical model of polymer solutions. By co-immobilization of specific antibodies and protecting polymers on the liposome surface, liposomes can be prepared combining both targetability and prolonged circulation in vivo. A biological model (experimental myocardial infarction in rabbit) was used to estimate the relative importance of different factors (liposome size and coating with protective polymer and/or specific antibody) for effective accumulation of liposomes in the target. Statistical analysis demonstrated that different types of liposomes have to be used in order to reach maximum absolute delivery of liposomes to the target, or maximum target-to-non-target ratio (relative delivery). Therefore, different liposomes should be used as carriers of diagnostic and therapeutic agents.     

Enzyme immunoassays for a new angiotensin-converting enzyme inhibitor, zabicipril, and its active metabolite in human plasma: application to pharmacokinetic studies

Zabicipril (S 9650) is a new angiotensin-converting enzyme inhibitor whose hydrolysis in vivo produces the pharmacologically active metabolite zabiciprilat (S 10211). Two competitive enzyme immunoassays specific for either zabicipril or zabiciprilat have been developed using acetylcholinesterase (E.C. 3.1.1.7) as label. Antibodies were raised in rabbits after immunization with lysil derivatives of zabicipril or zabiciprilat coupled with bovine serum albumin. Assays were performed in 96-well microtiter plates coated with a monoclonal antibody raised against rabbit immunoglobulin G, thus ensuring rapid separation of free and bound fractions of the tracer. The analysis does not require any extraction step. In the case of the assay of zabiciprilat, interference generated by endogenous angiotensin-converting enzyme (ACE) was eliminated by the addition of perindoprilat, another ACE inhibitor. Perindoprilat was not recognized by the antibodies (cross-reactivity < 0.01%) and did not affect assay efficiency. The specificity of the assays was checked by high-performance liquid chromatography of human plasma samples obtained after oral administration of 2 mg of zabicipril. No metabolites or endogenous substances were detected. The mean reproducibility was 15% for the assay of zabicipril and 19% for the assay of zabiciprilat. The quantification limits were 1.2 ng/ml for the zabicipril assay and 0.8 ng/ml for the zabiciprilat assay. These assays are therefore suitable for pharmacokinetic studies and drug monitoring in clinical studies.     

NMR characterization of a single-cysteine mutant of Escherichia coli thioredoxin and a covalent thioredoxin-peptide complex

Cationic liposomes are used as the carriers of polyanionic genes for combating against hereditary diseases in gene therapy. Studies directed to careful biophysical characterizations of the cationic liposomes commonly used in gene delivery have just begun. Herein, we report on a novel liposomal exo-surface bound indazolization reaction of an amphiphilic arenediazonium salt as evidence for the existence of remarkably alkaline exo-surface of cationic liposomes commonly used in gene transfection. Our results demonstrate that formation of 5-hexadecyl-7-methylindazole in thermal indazolization of 2,6-dimethyl-4-hexadecylbenzenediazonium tetrafluoroborate bound to liposome surface is a strong indication for the existence of significantly high exo-surface pH for cationic liposomes commonly used in gene delivery. The present method can be used in determining the relative exo-surface basicities of various cationic liposomes used in gene transfection and subsequently to find any possible correlation between the transfection efficiencies of these liposomes and their exo-surface basicities.     

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.     

Contact-dependent, immunecomplex-mediated lysis of hapten-sensitized liposomes

Large unilamellar liposomes (d approximately 160 nm) composed of dioleoylphosphatidylethanolamine (DOPE) (80-90%), a negatively charged phospholipid stabilizer (10-20%), and a small amount (0.1-1%) of a haptenated lipid are unusually stable in divalent cation-free isotonic buffer at pH 7.4. The liposomes can be stored under this condition at 4 degrees C for at least 6 months without any detectable leakage of the entrapped fluorescent dye calcein. However, the liposomes undergo a rapid (1 h) aggregation and lysis reaction in the presence of free bivalent anti-hapten antibody. The liposome destabilization was immunospecific in that it did not occur with the normal IgG or in the presence of excess free hapten. Liposome lysis was always accompanied by liposome aggregation. Aggregation and lysis of the liposomes was completed in 5 min if the incubation temperature was raised to 70-80 degrees C. Replacing DOPE with dioleoylphosphatidylcholine in the liposomes did not abolish the liposome aggregation, but no liposome lysis was observed even at 80 degrees C. Since liposome aggregation appeared to be a necessary (but not sufficient) prerequisite for liposome lysis, we have named this new class of liposome "contact-sensitive liposomes." The immunodiagnostic potential of the contact-sensitive liposome was demonstrated with liposomes containing theophylline-DOPE. The aggregation and lysis of the liposomes induced by a monoclonal anti-theophylline antibody could be inhibited by free theophylline at concentrations of therapeutic significance. The observation could be the basis of a homogeneous assay for theophylline.     

Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin

Liver uptake of liposomes containing phosphatidylserine was studied in a single pass liver perfusion system and found to be serum dependent. The effectiveness of serum in mediating liposome uptake by the liver depends on liposomes size. Large liposomes appeared to be opsonized more efficiently and, therefore, taken up more by the liver than the smaller ones. The effects of liposomes size on liver uptake did not occur in the absence of serum. Treatment of serum at 56 degrees C for 30 min abolished the serum activity, suggesting the involvement of complement components. Inhibition of the hemolytic activity of complement through the alternative pathway by PS-containing liposomes suggests that components in this pathway are responsible for liposome opsonization. Liposomes containing phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate compete in different degrees for serum components which mediate the liver uptake of PS-containing liposomes. These results suggest that the opsonization of liposomes by serum opsonins are the determining factors for the recognition and clearance of liposomes by the RES. Complement components are most likely involved in this process. The results presented here are relevant to the use of liposomes as drug delivery vehicle in vivo and to the PS-mediated clearance of red blood cells from the blood circulation.     

Free-roaming and feral cats--their impact on wildlife and human beings

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.     

Identification of regulatory sequences of juvenile hormone-sensitive and -insensitive serum protein-encoding genes

Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate-containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress.     

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.     

Improved DNA: liposome complexes for increased systemic delivery and gene expression

To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.     

Postnatal changes of nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 7 and beta 2 subunits genes expression in rat brain

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.     

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid

The antiphospholipid antibodies (aPL) present in "antiphospholipid-protein syndrome and autoimmune disorders" are associated with thromboembolic episodes, such as venous and/or arterial thrombosis and fetal loss. Patients with antiphospholipid antibodies have, by definition, laboratory abnormalities in either coagulation assays or various solid phase immunoassays ELISA or radioimmunoassays (RIA). These assay systems were initially thought to detect antibodies against phospholipids. The problem was complicated when it was reported that phospholipid is not the sole antigen but only a part of it, the other contribution being due to b2-glycoprotein I (b2-GP I). More findings, demonstrate that the aPL are in fact anti-b2-GP I antibodies directed against a epitope which is expressed when b2-GP I is bound to anionic phospholipid or another suitable surface. Recent studies have demonstrated that antibodies related to lupus anticoagulant (LA) induce an anticoagulant activity in b2-GP I. Some of these LA require binding to phospholipid. However, not all LA require b2-GP I as a cofactor. Human prothrombin is an antigen for some LA IgG's. Finally, a subclassification of phospholipid-dependent coagulation test anticoagulants is described, there appear to be several subclasses of LA, and the clinical and laboratory criteria required to establish the diagnosis of antiphospholipid-protein syndrome is emphasised.     

Transportation of cytotoxic liposomes to malignant cells using a carbohydrate determinant

A method of the synthesis of lipophilic glycoconjugates (vectors) on the basis of polyethyleneglycol-containing detergent was proposed. It has been shown by flow cytofluorometry that fluorescent labeled liposomes equipped with beta-galactosyl conjugate are bound human leukosis HL-60 cells more effectively than liposomes embedded with the beta-glucosyl conjugate or vector-free liposomes. A new lipid derivative of antitumor drug rubomycin (daunorubicin), N-(rac-1,2-dioleoylglycero-3-oxalyl)rubomycin (RubDG) has been synthesized. Liposomes loaded with RubDG and equipped with galactosyl vector showed higher cytotoxic activity in vitro against HL-60 cells than analogous unvectored liposomes or liposomes bearing glucosyl conjugate.     

A novel in situ method for the detection of deficient transglutaminase activity in the skin

Autosomal recessive congenital ichthyoses are disorders of epidermal cornification, but are clinically and etiologically heterogeneous. Some cases, known as lamellar ichthyosis, are caused by mutations in the TGM1 gene encoding transglutaminase 1, which result in markedly diminished or lost enzyme activity and/or protein. In some cases, this enzyme is present but there is little detectable activity, and in other clinically similar cases, transglutaminase 1 levels appear to be normal. Since conventional enzyme assays and mutational analyses are tedious, we developed a novel assay for the rapid screening of transglutaminase 1 activity using covalent incorporation of biotinylated substrate peptides into skin cryostat sections. Coupled with immunohistochemical assays using transglutaminase 1 antibodies, our method allows rapid identification of those cases caused by alterations in this enzyme.     

Lupus anticoagulant activity of some antiphospholipid antibodies against phospholipid bound beta 2 glycoprotein I

AIMS: To determine whether beta 2 glycoprotein I (beta 2GPI) dependent anticardiolipin (aCL) antibodies detected in solid phase enzyme linked immunosorbent assays can also have lupus anticoagulant activity. METHODS: Six anticardiolipin antibodies were affinity purified from patients with these antibodies and lupus anticoagulant activity in their plasma. RESULTS: The anticardiolipin antibodies bound only to anionic phospholipids in the presence of beta 2GPI and bound to beta 2GPI in the absence of phospholipids. Four out of six had lupus anticoagulant activity in the dilute Russell viper venom time test. CONCLUSIONS: The results show that some beta 2GPI dependent aCL are lupus anticoagulants. It is unclear why only some should have lupus anticoagulant activity while others do not.     

Effectiveness of liposomes possessing surface-linked recombinant B subunit of cholera toxin as an oral antigen delivery system

Liposomes appear to be a promising oral antigen delivery system for the development of vaccines against infectious diseases, although their uptake efficiency by Peyer's patches in the gut and the subsequent induction of mucosal immunoglobulin A (IgA) responses remain a major concern. Aiming at targeted delivery of liposomal immunogens, we have previously reported the conjugation via a thioether bond of the GM1 ganglioside-binding subunit of cholera toxin (CTB) to the liposomal outer surface. In the present study, we have investigated the effectiveness of liposomes containing the saliva-binding region (SBR) of Streptococcus mutans AgI/II adhesin and possessing surface-linked recombinant CTB (rCTB) in generating mucosal (salivary, vaginal, and intestinal) IgA as well as serum IgG responses to the parent molecule, AgI/II. Responses in mice given a single oral dose of the rCTB-conjugated liposomes were compared to those in mice given one of the following unconjugated liposome preparations: (i) empty liposomes, (ii) liposomes containing SBR, (iii) liposomes containing SBR and coadministered with rCTB, and (iv) liposomes containing SBR plus rCTB. Three weeks after the primary immunization, significantly higher levels of mucosal IgA and serum IgG antibodies to AgI/II were observed in the rCTB-conjugated group than in mice given the unconjugated liposome preparations, although the latter mice received a booster dose at week 9. The antibody responses in mice immunized with rCTB-conjugated liposomes persisted at high levels for at least 6 months, at which time (week 26) a recall immunization significantly augmented the responses. In general, mice given unconjugated liposome preparations required one or two booster immunizations to develop a substantial anti-AgI/II antibody response, which was more prominent in the group given coencapsulated SBR and rCTB. These data indicate that conjugation of rCTB to liposomes greatly enhances their effectiveness as an antigen delivery system. This oral immunization strategy should be applicable for the development of vaccines against oral, intestinal, or sexually transmitted diseases.     

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL)

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.     

Highly sensitive and stable phosphatidylserine liposome aggregation assay for annexins

Annexins are a gene family of Ca(2+)-dependent membrane binding proteins which interact specifically with acidic phospholipids. We describe here details of highly sensitive and precise assays for annexins I and V, utilizing turbidometric analysis of phosphatidylserine liposome aggregation. In the case of annexin I, the new assay is 7-fold more sensitive than the conventional chromaffin granule aggregation assay in terms of threshold for detection and the rate of increase of initial absorbance is 15-fold greater. Annexin V, which binds but does not aggregate liposomes, can be assayed on the basis of inhibition of Ca(2+)-dependent liposome aggregation. Further comparative advantages of the assay include lower expense and increased shelf life of the liposome reagent.