Frosco M, Fahed C, Chase T, Macmillan J D
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903.
Infect Immun. 1992 Mar;60(3):735-41. doi: 10.1128/iai.60.3.735-741.1992.
In preparing monoclonal antibodies to the elastase from Aspergillus fumigatus, we found that the enzyme was weakly immunogenic in BALB/c mice. Antiserum titers were only 1:1,000 to 1:5,000, and hybridomas secreted nonspecific immunoglobulin M (IgM). Denaturing the elastase in 0.5% sodium dodecyl sulfate at 80 degrees C for 10 min prior to injection increased titers of antiserum against the nondenatured (native) enzyme 10-fold. Of eight hybridomas selected following immunization with the denatured enzyme, seven produced IgG reactive with the native enzyme and one produced nonspecific IgM. The nondenatured immunogen tested again yielded mainly IgM producers. Immunoblots and enzyme-linked immunosorbent assay showed that the IgG monoclonal antibodies were reactive with both the denatured and nondenatured fungal elastases; none cross-reacted with human neutrophil elastase, porcine pancreatic elastase, or Pseudomonas elastase. Elastase-specific polyclonal antibody produced in mice inhibited elastase activity beginning at a molar ratio (antibody to elastase) of 4:1, and activity was completely inhibited at 14.5:1. Some individual monoclonal antibodies partially inhibited elastase, but certain pairs, at a molar ratio of each antibody to elastase of 5.4:1, acted synergistically to inhibit the activity completely.
在制备针对烟曲霉弹性蛋白酶的单克隆抗体时，我们发现该酶在BALB/c小鼠中免疫原性较弱。抗血清效价仅为1:1000至1:5000，并且杂交瘤分泌非特异性免疫球蛋白M（IgM）。在注射前于80℃下用0.5%十二烷基硫酸钠将弹性蛋白酶变性10分钟，可使针对未变性（天然）酶的抗血清效价提高10倍。在用变性酶免疫后选择的8个杂交瘤中，7个产生了与天然酶反应的IgG，1个产生了非特异性IgM。再次检测未变性的免疫原主要产生IgM的细胞。免疫印迹和酶联免疫吸附测定表明，IgG单克隆抗体与变性和未变性的真菌弹性蛋白酶均有反应；无一与人类中性粒细胞弹性蛋白酶、猪胰弹性蛋白酶或铜绿假单胞菌弹性蛋白酶发生交叉反应。小鼠产生的弹性蛋白酶特异性多克隆抗体从摩尔比（抗体与弹性蛋白酶）为4:1时开始抑制弹性蛋白酶活性，在14.5:1时活性被完全抑制。一些单个的单克隆抗体部分抑制弹性蛋白酶，但某些抗体对，在每种抗体与弹性蛋白酶的摩尔比为5.4:1时，协同作用完全抑制活性。
