對應人類血紅素的IgY抗體生產、純化與分析研究

Abstract

「大腸癌」為當前世界各國最盛行的癌症之一。定期的糞便潛血檢查可及早發現大腸癌徵兆，因而有助於降低其發生率與死亡率。本研究之目的即針對免疫糞便潛血檢驗試劑中的核心抗體，進行以雞隻為實驗動物的生產平台測試。 在測試內容上，我們一方面評估抗原劑量、佐劑、及激活注射對雞隻體液免疫反應的影響，另一方面也監測血清及蛋黃中抗體效價的變化情形，進而歸納出抗體生產的適當條件。在抗體純化部分，我們除了製備抗原管柱之外，也發展出效化清理抗原管柱的步驟；以該管柱吸附純化的專一性抗體，無論純度與活性均明顯優於市售抗體，並且不會辨識血紅素以外的蛋白質。此外，我們也對抗體應用的敏感度及是否受食源血紅素及上消化道出血的干擾進行初期評估。總體而言，以雞隻生產對應人類血紅素的抗體，不僅花費成本較低廉、操作較為方便、產量較高、過程更較符合動物福祉法規，所生產的IgY抗體也有良好的作用專一性與敏感度，因此具有取代當前業界使用的兔產抗體的潛力。

Colorectal cancer (CRC) is one of the most prevalent cancers in the world. Routine fecal occult blood tests (FOBT) help detect CRC in its early stages and thus decrease CRC mortality and incidence. The aim of this study is to establish a production platform, using chickens as experimental animals, for the core component of anti-human hemoglobin antibody (anti-hHb Ab) used in the immunochemical FOBT (I-FOBT). To that end, we have examined the effects of antigen dose, adjuvant and booster injection on chicken’s humoral immune response and monitored changes of antibody titers in the sera and yolksfrom that the optimal parameters for anti-hHb Ab production can be deduced. As for antibody purification, we have prepared affinity column with antigen-coupled beads and developed an effective cleaning procedure for that column. The affinity-purified sample of anti-hHb IgY does not cross-react with non-hemoglobin proteins and exhibits superior purity and reactivity to antigen than that of commercially available. In addition, preliminary assessments have been performed to evaluate the sensitivity of using anti-hHb IgY in a biochemical assay and to scrutinize the plausible interferences exerted by animal blood-containing food or by upper gastrointestinal tract bleeding. In conclusion, the use of chickens for anti-hHb Ab production brings many advantages over the conventional mammal hosts, including lower cost, higher yield, easier handling process and greater compliance to animal welfare regulations. Since the genarated anti-hHb IgY reveals great sensitivity and specificity in biochemical assays, they can be considered as an excellent alternative for the rabbit anti-hHb IgG currently used in the commercial I-FOBT kits.