Liposome Immune Lysis Assay Research Articles

A liposome immune lysis assay for enrofloxacin in carp and chicken muscle

A homogenous complement-mediated liposome immune lysis assay (LILA) was developed for the determination of enrofloxacin (ENRO) in carp and chicken muscle. ENRO was covalently coupled to DPPE, and then immobilized onto the surface of liposomes by reverse-phase evaporation method. The performed liposome would be specifically lysed by the sequential additions of anti-ENRO monoclonal antibody (MAb) and guinea pig complement. Through a competitive assay format, the performed liposome can be used to detect ENRO in a range of 5.0–20 ng mL −1 in assay buffer. The limit of detection of ENRO in carp and chicken muscle was 1 ng g −1 and the limit of quantification was 2 ng g −1. Recoveries ranged from 58.3% to 65.2% for carp and 55.6–63.8% for chicken muscle at spiked levels of 2–8 ng g −1, with intra-assay and inter-assay variations 5.6–12.3% and 7.1–19.2%, respectively.

Analysis of neutral glycosphingolipids from Trypanosoma brucei

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M–H) − ions from m/ z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M–H) − ions from m/ z 944–987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.

Development of Liposome Immune Lysis Assay for the Herbicide Atrazine

A homogeneous complement‐mediated liposome immune lysis assay (LILA) was developed for determination of the herbicide atrazine. To dispose the antigen on the surface of lipid bilayer the atrazine was conjugated to a dimirystoylphosphatidylethanolamine (DMPE) carrier. Calcein was compared with sulforhodamine 101 as a fluorophore label for entrapping into the antigen‐sensitized liposomes. The liposomes were incubated with rabbit anti‐atrazine antibodies in the presence of guinea pig complement. Formation of the antigen–antibody complexes on the liposomal surface initiated the lytic action of the complement. As free competing atrazine inhibited the lytic reaction, the amount of calcein released was inversely proportional to the atrazine content in the probe. Concentration and kinetic dependences of the immunoassay were characterized to reach its maximal sensitivity. The developed assay allows detecting atrazine in concentrations up to 0.13 ng mL−1 in the sample (0.04 ng mL−1 in the final reaction mixture). The named sensitivity is two orders higher than those for the microplate enzyme‐linked immunosorbent assay (ELISA) with the same antibodies which allows us to recommend LILA for environmental monitoring.

Liposome Immune Lysis Assay of Antibodies by Liposomes Encapsulating Coenzyme .BETA.-NAD+.

Liposome immune lysis assay (LILA), which depends on immune lysis of artificial phospholipid vesicles by the activation of complement, is a homogeneous immunoassay method and much better than ELISA in respects of simplicity and rapidity in measurements. Fluorescent agents have been used as markers encapsulated in liposomes, but they need an expensive apparatus for detection. In this work, we measure the concentration of polyclonal and monoclonal antibodies by LILA by use of antigen-bound liposomes encapsulating coenzyme β-NAD+. Conjugated redox reactions involving β-NAD+ can accumulate a colored material and attain a high sensitivity similar to fluorescent agents in detection of released marker. We also analyze the characteristics of immune lysis of liposomes in LILA depending on the equilibrium relationship between antigen and antibody. The marker release depends on the concentration of the antibodies, and the decrease in the concentration of liposomes raises the sensitivity. The marker release is proportional to the concentration of antigen-antibody complex formed on liposomes. Polyclonal antibody can sensitively be detected even at a lower average association constant than monoclonal antibody.

Antigenicity of the carbohydrate moiety of ganglioside GM3 having 3-O-acetyl ceramide.

To elucidate the effect of a modification of ceramide on antigenicity of the carbohydrate of ganglioside, the reactivity of O-acetyl GM3 having 3-O-acetyl ceramide, which has been characterized as a glioma-related ganglioside, with monoclonal antibody M2590 was examined in comparison to that of non-acetylated GM3, by means of quantitative enzyme-linked immunosorbent assay, TLC-immunostaining and liposome immune lysis assay. In all these assay systems, O-acetyl GM3 showed less activity than GM3 as follows: GM3 was detected till 0.1 nmol in TLC-immunostaining, whereas O-acetyl GM3 could not be detected even at 0.25 nmol; the GM3 reaction was approximately twofold that of O-acetyl GM3 at each diluted point in the enzyme-linked immunosorbent assay; and 20% of the liposomes containing GM3 were lysed at 6 mol%, while liposomes containing O-acetyl GM3 did not lyse at that concentration. The lesser antigenicity of the sugar moiety of O-acetyl GM3 could be ascribed to the presence of an acetyl group in the ceramide at the 3-position of sphingosine.

Chorea-acanthocytosis with polyclonal antibodies to ganglioside GM1

A patient with chorea-acanthocytosis presenting with axonal neuropathy showed an elevation in IgM polyclonal antibodies to the GM1 ganglioside, which were estimated by enzyme-linked immunosorbent assay and complement-mediated liposome immune lysis assay (LILA). This is the first demonstration of such antibodies in chorea-acanthocytosis. Anti-GM1 antibodies might have directly caused the axonal neuropathy by binding to GM1 or cross-reactive antigens in the nerves.

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transfrmation and bovine i-active glycolipid ( NeuAcα2→3Gal β1→ 4GlcNac β1 → 3Gal β1 → 4GlcNac β1 → 3Glc β1 → 1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

Peptide-coupled liposomes for homogeneous immunoassay of polyclonal antibodies against proteins

The concentration of anti-native insulin antibodies was measured by liposome immune lysis assay (LILA) using liposomes coupled with a peptide which corresponds to residues 19–30 of the B-chain of porcine insulin. Although native insulin was slightly soluble in the reaction solution used in binding the antigens to the liposomes, this peptide was readily soluble in the solution. Thus liposomes coupled with a sufficient amount of the antigen could be easily obtained. Antibodies against native insulin from several species bound to the peptide on the surface of the liposomes, and the antigen-antibody complexes activated the complement system. By using the peptide-coupled liposomes, the concentrations of antibodies against insulin from several species were measured by the LILA method without the limitation of the solubility of the antigens.

Immune lysis assay of antibodies by use of antigen-coupled liposomes

The concentrations of antibodies were measured by the use of immunoliposomes coupled with the corresponding antigens. The addition of complement under conditions of the existence of the antigen-antibody complex on the surface of the liposomes caused lysis of the liposomes. The degree of marker release from the liposomes was increased with the concentration of the antibodies, as well as the amount of antigen coupled to the liposomes, and was proportional to the amount of antigen-antibody complex formed. The liposomes made of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPhyPC) showed excellent stability and consequently could bind large amounts of antigen on the surface of the liposomes in comparison with liposomes made of ordinary lipids such as dipalmitoylphosphatidylcholine (DPPC). Although the characteristics of liposomal membranes consisting of DPhyPC in lysis by the classical pathway of complement were similar to those of DPPC liposomes, the DPhyPC liposomes showed higher resistance against non-specific lysis caused by the complement activity in serum samples, which may be effective to reduce false-positive errors in liposome immune lysis assay.

Analysis of neutral glycosphingolipids of Theileria sergenti piroplasms.

Neutral glycosphingolipids (GSLs) were isolated from a lipid extract of Theileria sergenti piroplasms and analyzed by thin-layer chromatography (TLC) and liposome immune lysis assay (LILA); two predominant GSLs, designated as N-1 and N-2 were separated on TLC. N-1 GSL showed the same mobility as lactosylceramide (LacCer) on the TLC plate. On the other hand, the mobility of N-2 GSL on the TLC plate was identical to that of galactosylparagloboside. In order to characterize the molecular species of neutral GSLs from T. sergenti, N-1 and N-2 GSLs were tested by LILA with antibodies against LacCer and galactosylparagloboside, respectively, N-1 GSL had reactivity to anti-LacCer antibody and N-2 reacted with the antibody to galactosylparagloboside. These results suggest that N-1 and N-2 GSLs are LacCer (Gal beta 1-4Glc beta 1-1Cer) and galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), respectively.

Comparison of liposome immune lysis assay (LILA) and enzyme-linked immunosorbent assay (ELISA) for detection of antibodies

Anti-α-chymotrypsinogen A antibody was assayed by both enzyme-linked immunosorbent assay (ELISA) and liposome immune lysis assay (LILA). The detection limit was slightly affected by the measurement conditions in ELISA; however, it was possible to control the detection limit and to achieve a lower level by adapting the measurement conditions in LILA. LILA is believed to offer a simple and highly sensitive method for measuring the concentration of antibody in serum.

Application of Liposome Immune Lysis Assay for Studies on Anti-Ganglioside Antibodies.

In the serum from patients with certain neurological diseases, antibodies against GM1 ganglioside are detected in high titers, suggesting that these antibodies may be involved in the pathological process. In this present study the liposome immune lysis assay was applied to determine anti-ganglioside antibodies in serum, and the results were compared with those of the commonly used enzyme-linked immunosorbent assay, where gangliosides are fixed on a solid surface. This liposome method is based on the measurement of a fluorescent dye released from liposomes by complement-mediated immune lysis. The value of the specific release of a fluorescent dye from liposomes by the serum from normal control individuals was used as a criterion to evaluate the antibody level. The values of the specific marker release from liposomes by anti-GM1 antibodies were scarcely correlated with the titers of IgM or IgG antibodies measured by the enzyme-linked immunosorbent assay, suggesting that the nature of anti-GM1 antibodies detected by the two assay methods may be different. The antibodies detected by the liposome immune lysis assay may represent those involved in membrane lysis, which occurs in the pathogenesis of some neurological diseases.

A novel liposome immune lysis assay (LILA) for determination of CRP antigen using two monoclonal antibodies recognizing different antigenic determinants.

We have already developed the liposome immune lysis assay (LILA) for the determination of C-reactive protein (CRP) by employing an inhibition method and a sandwich method. We herein report a new LILA system involving the use of monoclonal antibodies-bearing liposomes. We established five monoclonal antibodies to CRP antigen, AC-1, -2, -3, -4, -5 which had the capacity to activate complement and form antigen-antibody complex. Each of these antibodies was covalently coupled to carboxyfluorescein-entrapped multilamellar liposomes. When the liposomes were incubated with CRP antigen in the presence of guinea pig complement, CRP antigen-dependent liposome lysis was observed but the sensitivity was not great enough for practical use. On the other hand, when liposomes coupling two monoclonal antibodies (AC-1, AC-2) which recognized distinct CRP antigenic determinants were employed in the assay, the sensitivity increased compared with that using only one monoclonal antibody, and the detectable concentration range was 5-300 ng/ml. These results indicated that the combination of two or more monoclonal antibodies which recognize distinct CRP antigenic determinants is effective for increasing the sensitivity of the assay.

Homogeneous immunoassay of antibody by use of liposomes made of a model lipid of archaebacteria

Liposomes made of 1,2-di(3 RS,7 R,11 R-phytanyl)- sn-glycero-3-phosphocholine(DPhyPC), which was synthesized as one of the model lipids existing in archaebacterial halophiles, showed excellent stability. Because of this high stability, DPhyPC liposomes could be constituted high ratios (50%) of N-[4-( p-maleimidophenyl) butyryl] dipalmitoyl phosphatidylethanolamine (MPB-DPPE), and consequently could bind large amounts of antigen (α-chymotrypsinogen A) on the liposome surface in comparison with those made of ordinary lipids, such as dipalmitoylphosphatidylcholine (DPPC). Though the characteristics of the DPhyPC liposomal membranes in lysis by the classical complement pathway were similar to those of DPPC liposomes, a high sensitivity and a low detection limit in the liposome immune lysis assay (LILA) of antibodies were attained by binding large amounts of the antigen. Further, by coupling sufficient amounts of antigen, almost all the DPhyPC liposome surface was covered with the antigen, and such liposomes showed higher resistance against non-specific lysis caused by complement activity in serum samples, which may be effective in reducing positive-false errors in LILA.

Establishment of monoclonal antibodies specific for ganglioside GM1: detection of ganglioside GM1 in small cell lung carcinoma cell lines and tissues.

Three kinds of anti-GM1 monoclonal antibodies, AGM-1, -2, and -3, of the IgM class were produced by the immunization of BALB/c mice with ganglioside GM1 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides and fusion of the spleen cells with a mouse myeloma cell line. The specificities of the monoclonal antibodies obtained were elucidated through complement-dependent liposome immune lysis assay and enzyme immunostaining on thin-layer chromatograms. All of the monoclonal antibodies reacted only with ganglioside GM1, and structurally related glycosphingolipids, such as fucosyl-GM1, asialo-GM1, GM2, and GD1b, and the other gangliosides (GM3 and GD1a) tested showed no reactivity to the 3 monoclonal antibodies. These findings suggest that the monoclonal antibodies obtained may be specific for ganglioside GM1. These anti-GM1 monoclonal antibodies were used to define the expression of ganglioside GM1 on small cell lung carcinoma (SCLC) cell lines and tissues. In flow cytometric analysis and immunostaining studies, we observed that ganglioside GM1 was highly expressed on the SCLC cell lines. Results obtained with flow cytometry and immunohistochemistry agreed well with the immunochemical determination of ganglioside GM1 in lipid extracts of cell lines. Furthermore, expression of ganglioside GM1 in tumor tissues from patients with SCLC was ascertained by the immunohistochemical examination of acetone-fixed paraffin-embedded tissue sections. Ganglioside GM1 was detected in 5 of 19 SCLC tissues. These results suggest that ganglioside GM1 is expressed in SCLC cells.

Characterization of antigen-coupled liposomes for homogenous immunoassay of polyclonal antibodies

Abstract The effects of the preparation conditions of antigen-coupled liposomes on their characteristics in liposome immune lysis assay (LILA) of specific antibodies were studied. The amount of antigen coupled to liposomes depended on the molar ratio of N-[4-( p -maleimidophenyl)butyryl] dipalmitoyl phosphatidylethanolamine (MPB-DPPE) constituting the liposomes as well as on the molar ratio of modified antigen to MPB-DPPE in the reaction mixture. In depended also on the molecular weight of the antigen. With increases in the amount of antigen coupled to liposomes higher sensitivity and a wider dynamic range were obtained. Though an increase in the average association constant between antigen and antibody lowered the detection limit, the wide dynamic range, which prevents false positive results in LILA, can be controlled mainly by the amount of coupled antigen.

The use of streptavidin-biotin interaction for preparation of reagents for complement-dependent liposome immunoassay of proteins: Detection of latrotoxin

We have developed liposome sensitization by a protein, latrotoxin (LT), using immobilization of biotinylated LT via streptavidin with biotinylated phosphatidylethanolamine contained in liposomes. The use of such liposomes in the complement-dependent homogeneous liposome immune lysis assay (LILA) has allowed us to detect in the test sample as little as 2 μg/ml of polyclonal and 50–100 ng/ml of monoclonal IgG and IgM antibodies to LT. LT concentration in solution was determined by inhibition of immune lysis by free LT. The sensitivity of the LT assay varied from 1 × 10 −9 to 5–50 × 10 −9 m when antiserum (polyclonal antibodies) and monoclonal antibodies to LT were correspondingly used. The results show that a streptavidin-biotin spacer can be used to immobilize protein antigens on liposomes for a subsequent application in LILA. The suggested technique greatly simplifies the sensitization procedure and extends the applicability of the LILA.

Aberrant expression of GM1 on lymph node cells of MRL/Mp-lpr/lpr mice: influences on the autoreactivities of anti-asialo GM1 antibodies.

Although changes in surface carbohydrate expression of abnormally expanded MRL/Mp-lpr/lpr (MRL/lpr) lymph node (LN) cells have previously been described, the composition and function of glycolipids present on these cells as well as the spectrum of specificity of anti-carbohydrate antibodies reactive with these cells remains obscure. Analysis of antibodies to a panel of 22 carbohydrate structures using a liposome immune lysis assay (LILA) showed that, except for anti-asialo GM2 (GA2) antibodies, marked reduction of antiglycolipid antibody levels was observed in sera from 4-mo-old MRL/lpr mice compared with these from MRL/Mp(-)+/+ (MRL/+) mice. Absorption experiments revealed that both anti-asialo GM1 (GA1) and globoside antibodies had binding capacity to MRL/lpr LN cells. To elucidate the glycolipid profiles of MRL/lpr LN cells, glycolipids were extracted from LN cells of both MRL/lpr and MRL/+ mice and analysed. A 30-fold elevation of GM1 was found in MRL/lpr LN cells compared with MRL/+ LN cells. From the results of LILA using GA1/GM1 mixed liposomes, aberrantly expressed GM1 inhibited the classical complement pathway but did not interfere with the binding of anti-GA1 antibodies to liposomal GA1. These findings suggest that a drastic GM1 increase on MRL/lpr LN cells would inhibit the action of anti-GA1 antibodies and complement on the cell surface. This may explain the escape of these cells from an activated self directed immune response.

Liposome immune lysis assay (LILA) for gelonin

A complement-mediated liposome immune lysis assay using entrapped calcein was developed for a plant toxin gelonin. Gelonin was covalently coupled to DPPE, and then adsorbed on to the surface of liposomes. Such antigen-bearing liposomes when incubated with anti-gelonin antibody in the presence of guinea pig complement undergo lysis. The detection range is from 3 ng to 60 ng. The method was used to monitor isolation of gelonin by affinity chromatography. It was observed that a minor peak in addition to the major one comes with gelonin, shared common epitopes/epitope with gelonin immunological reaction. This was further confirmed by SDS-gel electrophoresis indicating the former being an isoform of gelonin. A comparative study of the immunocross-reactivity of ricin and A chain with anti-gelonin antibody was carried out. It was found that while ricin A chain cross-reacted extensively with gelonin antibody and intact ricin elicited little or no cross-reactivity. It is suggested that the present LILA may be employed for the detection and quantitation of ricin A chain by this LILA method.

Production of Monoclonal Antibodies Specific for Ganglioside GD31

Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3.