Preparation Of Immunoliposomes And Their Application In Laboratory Diagnosis

Objective:To study the methods of preparing immunoliposomes anduse them to establish liposome immune lysis assay and enzyme-linkedimmunoabsorbent assay in clinical diagnosis tests. Methods:The liposomes containing horseradish peroxidase wereprepared by reverse phase evaporation after comparing different methodsand enzymes. Optimal techniques were chosen with single factor reviewand orthogonal design. Liposomes and rabbit anti-human IgG wereconjugated by glutaraldehyde. The optimal conjugation conditions wereexplored and liposomes not only encapsulated enzyme but also hadimmune activity were prepared, and human IgG was tested using liposomeimmune lysis assay. The enzyme-linked immunoabsorbent assay withimmunoliposomes marker was established and antibody to hepatitis C viruswas detected. Results:Liposomes prepared by reverse phase evaporation had perfectshape and high bioactivity of horseradish peroxidase. The optimal reactionconditions to prepare liposomes were determined. Phospholid mixturescontaining egg yolk lecithin(30 mg),cholesterol(15 mg) andphosphatidylethanolamine(5 mg) were dissolved in 15 ml of organicsolvent containing chloroform and aether(volume ratio of 1:1).The organicsolvent was removed at 37℃ using a rotary evaporator at reduced pressureand the dried layer film was then redissolved in 15 ml of organic solventand 5 ml of phosphate buffered saline containing 3 mg of horseradishperoxidase. After ultrasonic emulsification for 5 minutes, the organic phasewas removed under reduced pressure at 37℃ until the mixture phaseschanged from gelatin state to liquid state. Adding phosphate buffered salineto 5 ml, horseradish peroxidase liposomes were prepared after ultrasonictreatment. Using 25% glutaraldehyde and 2mg/ml of rabbit anti-human IgG,liposomes and antibodies were conjugated. The method was efficient,convenient and practical. The bound efficiency of protein inimmunoliposomes was 27.5%.The limit of detecting human IgG withliposome immune lysis assay was 5μg/ml.The coefficient of variation wasacceptable. Establishing enzyme-linked immunoabsorbent assay withimmunoliposomes marker for hepatitis virus C, the positive results could begotten after positive samples were diluted in 1:500. In a clinical trial with30 serum samples, results of this assay were completely in agreement withthose detected by commercial ELISA kit . Conclusion: Immunoliposomes with bioactivity of enzyme andantibody are prepared through reasonable methods. It has practicalimportance in clinical diagnosis to establish liposome immune lysis assayand enzyme-linked immunoabsorbent assay using immunoliposomes...