Liposome spin immunoassay: A new sensitive method for detecting lipid substances in aqueous media

Journal o f Immunological Methods, 9 (1975) 165--170

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© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

LIPOSOME SPIN IMMUNOASSAY: A NEW SENSITIVE METHOD FOR D E T E C T I N G LIPID SUBSTANCES IN AQUEOUS MEDIA

ROBERT WEI*, CARL R. ALVING**, ROBERTA L. RICHARDS**, and EDMUND S. COPELAND*** Electronucleonics, Inc, 368 Passaic Ave., Fairfield, N.J. 07006* and Departments o f Immunology * * and Biological Chemistry ** * Walter Reed A r m y Institute o f Research, Washington, DC 20012, U.S.A.

(Received 1 July 1975, accepted 10 July 1975) A new sensitive immunoassay procedure is described for quantitative detection of glycolipids and other lipids in aqueous media. As with other immunoassays, specific antiserum is first reacted with the free lipid hapten. The amount of antibody activity remaining is measured by assaying the release, in the presence of complement, of spin label marker from liposomes containing the same lipid hapten. Using this method, 2.6 pmol of aqueous Forssman hapten was detected, and the sensitivity could be increased further.

INTRODUCTION In recent years there has been great interest in the development of sensitive, specific, safe, convenient and cheap immunoassays for detecting small molecular weight haptens (reviewed by Haber and Poulsen, 1974). Radioimmunoassay (RIA) has the feature of great sensitivity. Disadvantages include the hazard, inconvenience and expense of radioactive materials. In addition, antigen--antibody complexes must be physically separated from unreacted labeled hapten or antibody. A new m e t h o d called 'spin immunoassay' (SIA), recently has been devised in which a spin-labeled analogue of the hapten is synthesized (Leute et al., 1972a,b; Wei and Almirez, 1975). The SIA m e t h o d does n o t require physical removal of antigen--antibody complexes, but, as yet, it does n o t compare with RIA in terms of sensitivity. Furt herm ore, when using each of the above m e t hods for measurements of lipids, additional problems might be e n c o u n t e r e d in obtaining reliable and reproducible immunological reactions with insoluble lipid antigen standards. In the present report, a new technique is described which is different from RIA or SIA. This m e t h o d utilizes the fact that a lipid hapten may be 'solubilized' in a lipid bilayer m em br ane matrix, called a 'liposome'. Liposomes were invented in 1965 (Bangham et al., 1965) and were adapted in 1968 for immunological applications by Kinsky and coworkers (H axby et al., 1968; Kinsky, 1972). The liposomes consist of concentric shells of lipid bilayers,

1.66 and these are separated by aqueous interspaces which contain a trapped marker. When the liposomes are 'immunologically complete', that is, when they contain an appropriate lipid hapten, they can react with specific antib o d y and, in the presence of complement, release the trapped marker (Haxby et al., 1968; Kinsky et al., 1969; Kinsky, 1972). A variety of trapped markers have been utilized for studying immune reactions of liposomes, including: glucose (Haxby et al., 1968), galactose (Knudson et al., 1971), chromate (Lachmann et al., 1970), a radiolabel (Hesketh et al., 1972), several enzymes (Kataoka et al., 1973), a fluorogenic substrate, umbelliferone phosphate (Six et al., 1974), and a spin label, tempocholine chloride (Humphries, 1974; Humphries and McConnell, 1974). In the procedure described here, specific antiserum was first reacted with an aqueous solution containing 'free' lipid hapten to be assayed. The degree of inhibition of antibody activity caused by this reaction was then measured by adding liposomes containing the same lipid hapten and also containing a spin label in the aqueous interspaces. Antibody activity which remained after reaction with the free hapten was measured by the degree of spin label release from the added liposomes in the presence of complement. MATERIALS AND METHODS

Preparation and handling o f liposomes The liposomes were prepared, as before (Kinsky et al., 1969; Kinsky, 1974; Alving et al., 1974c), using dipalmitoyllecithin, cholesterol, dicetyl phosphate and Forssman hapten in molar ratios of 2/1.5/0.22/0.01. The dried lipids were swollen in an isotonic veronal buffer solution which contained 0.1 M tempocholine bromide (Kornberg and McConnell, 1971). The swollen liposomes were dialyzed against 6 changes of 1000 vols of 0.15 M NaC1, for 30 rain each, to remove untrapped marker. Total trapped spin label marker was determined by dissolving an aliquot of liposomes in CHC13, evaporating the solvent and determining the amount of released marker after adding water.

Measurement of spin label release The method for measuring spin label release caused by immune damage to liposomes was essentially identical to the previously described methods which employed glucose release (Kinsky, 1974), with the exception that quantities of reactants employed were scaled down. In a total vol of 40 pl, 40 nl of liposomes were incubated with appropriate quantities of heated antiForssman serum (Alving et al., 1974c) and the immune reaction was started by adding 500 nl of fresh guinea pig serum, as a complement source. The reactants were added as dilutions in 0.15 M NaC1 containing 1 mM MgC12 • 6 H 2 0 and 0.15 mM CaC12. Spin label was detected with an E-4 EPR spectrometer (Varian, Palo Alto, Cal., USA).

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Liposome spin immunoassay technique D i f f e r e n t quantities o f purified F o r s s m a n substance (Alving et al., 1 9 7 4 c ) ( 2 6 0 n m o l / m l in m e t h a n o l ) were diluted with 0.15 M NaC1. Diluted antigen solution (10 pl) was p r e i n c u b a t e d f o r 5 min with 1 pl o f a n t i s e r u m which had been diluted to 10 pl with 0.15 M NaC1. The liposomes and excess c o m p l e m e n t t h e n were a d d e d , as above, and spin label release was determined a f t e r at least a 5-min i n c u b a t i o n at r o o m t e m p e r a t u r e ( a p p r o x . 22 ° C). RESULTS

Immune damage to liposomes T h e kinetics o f spin label release in the presence o f d i f f e r e n t quantities o f a n t i s e r u m are illustrated in fig. 1. The results with spin label were virtually identical to those observed with glucose release u n d e r similar c o n d i t i o n s (Kinsky et al., 1 9 6 9 ) . As b e f o r e , separate e x p e r i m e n t s also d e m o n s t r a t e d an absolute r e q u i r e m e n t f o r fresh guinea pig c o m p l e m e n t .

Liposome spin immunoassay for Forssman glycolipid T h e spin label release caused b y the presence o f u n r e a c t e d a n t i b o d y after i n c u b a t i o n with d i f f e r e n t a m o u n t s o f free F o r s s m a n h a p t e n is s h o w n in fig. 2. T h e results o b t a i n e d , showing inhibition o f spin label release, were in

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Fig. 1. Antibody requirement and kinetics of spin label release. The data were corrected for baseline spin label observed in the absence of antibody or complement. Fig. 2. Inhibition by fluid phase hapten of spin label release. The data were corrected for baselin spin label observed in the absence of antibody or complement.

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the form of a sigmoid curve. The data of fig. 2 were transformed into a straight line (fig. 3) by using an adaptation of the Von Krogh equation for a sigmoid curve (Von Krogh, 1916; Mayer, 1961), namely: free antigen = K ( y /1 - - y ) l / , , or, log(free antigen) = log K + 1/n log(y/1 -- y) where y is the degree of spin label released compared to the plateau, as shown in fig. 2, and K is an 'inhibitory unit' of antigen, or AgIs 0, as explained below. DISCUSSION

The liposome spin immunoassay (LSIA) described here combines the advantages of homogeneous 'solubilization' of lipid hapten in a membrane, with the safety and convenience of spin labels, and sensitivity usually found only with radioimmunoassays. Because of spin-exchange (Humphries, 1974; Humphries and McConnell, 1974) the spin label was not detected readily when present in the aqueous interspaces of the liposomes. Upon release, however, the characteristic pattern was observed, as previously described. Because of this the liposome--antibody complex, which still contained some spin label, did not need to be physically removed from the solution containing released marker and, as with glucose release (Kinsky, 1974), the entire series of reactions and measurements could be conducted conveniently in a single reaction vessel.

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The degree of release of trapped liposomal marker is dependent on the number of antigen--antibody complexes which are formed on the membrane and which lead to complement activation (Kinsky et al., 1969). Unlike erythrocyte lysis, the release mechanism from liposomes occurs by a process of simple diffusion, and this is increased by additional 'hits' (Kinsky et al., 1969). Factors which may influence the degree of immunological reaction of a glycolipid, and hence the magnitude of release, include: length of the haptenic carbohydrate chain (Alving et al., 1974a); length (Graf and Rapport, 1974) and integrity (Kataoka et al., 1971b) of fatty acyl groups of the hapten molecule; concentration of the hapten in the liposome (Kinsky et al., 1969); fatty acyl chain length of liposomal phospholipids (Alving et al., 1974a,c), size of the liposomes (Six et al., 1974); and the specificity (Alving et al., 1974a) and affinity (Six et al., 1973; Alving et al., 1974c) of the antibody. It is clear, therefore, that various means could be used to alter the membrane, or the soluble reactants, to increase the sensitivity of the LSIA beyond the picomole levels described in this report. This could even be achieved simply by increasing the relative volume of the sample assayed. The liposome assay method described in this report requires that the lipid molecule be inserted properly into the liposomal membrane so that the haptenic group is available for antibody binding. Besides Forssman, other suitable (or potentially suitable) substances now include: galactocerebroside (Inoue et al., 1971; Alving et al., 1974a), ganglioside (Alving et al., 1974a; Nagai and Ohsawa, 1974), galactosyl diglycerides (Alving et al., 1974a), schistosomal lipids (Alving et al., 1974b), lipopolysaccharides (Kataoka et al., 1971a,b), cardiolipin (Inoue and Nojima, 1967) and phosphatidylinositol mannosides of mycobacteria (Pangborn, 1968). In addition, it is clear that many other molecules, even including a protein which could be attached to an appropriate lipid (Six et al., 1973), might serve as functional antigens in liposomes, thus greatly increasing the potential utility of the LSIA technique. It should be pointed out that the Von Krogh equation, used to convert the sigmoid curve of fig. 2 to the straight line of fig. 3, could also be used to calculate a free hapten 'inhibitory unit'. By analogy with CHs 0 units of complement lysis of erythrocytes (Mayer, 1961), AgI50 corresponds to the amount of free hapten required to inhibit 50% of the maximal marker release. This occurs when y / 1 - - y = 1(2.6 nmol in fig. 3). This calculation, using volume units, might be useful, therefore, in circumstances in which the antigen solution for the standard curve is not well characterized or purified.

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