Immunoassay has been extensively used as a diagnostic tool in biotechnology and biomedical field. Though there are so many methods exist, recently, open-sandwich immunoassay (OS-IA) for the non-competitive detection of haptens has been developed which is based on the phenomenon of increased association of two antibody variable domains (VH and VL) upon binding with antigens. In this study we established OS-IA for thyroxine (T4; 3, 3', 5, 5'-tetraiodo-thyronine), which is the most commonly measured thyroid hormone for diagnosis of thyroid function. Stimulation of thyroid gland by the pituitary hormone (TSH) causes the release of T4 in bloodstream. It exists either as a complex with carrier protein or as a free form in blood. Only the free portion (unbound) of T4 is responsible for the biological action.

So on the first chapter of PhD thesis I overviewed this area and made introduction to the following two topics: 1) Direct construction of open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4, and 2) Micro open-sandwich ELISA to rapidly evaluate thyroid hormone concentration from serum samples.

In Chapter 2, direct construction of open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4 is described. To obtain antibody fragments suitable for OS-IA swiftly by-passing time-consuming conventional hybridoma technology, direct acquisition of such genes from immunized mice by phage display technology was attempted. After obtaining antibody V region genes from the spleen of immunized mice, we successfully isolated anti-T4 antibodies using a Fab phage display system (pDong system) with an estimated diversity 1.5x105. To estimate the affinity of the two isolated clones, indirect competitive ELISA with immobilized T4-BSA was performed. For the both clones, unconjugated T4 completely inhibited the binding of Fab to T4-BSA in a dose-dependent manner. In both cases, the obtained IC50 was about 5-13 ng/mL, indicating their high antigen binding affinity. Also, the clones showed similar cross-reactivity to another thyroid hormone ,3,3',5-triiodothyronine (T3).

After the deletion of CH1 gene from pDong1 by SgrA1 digestion and self ligation, the resulting phagemids were used for the expression of VH-displaying phage (VH-phage) and free light chain (VL-CL) for OS- ELISA The culture supernatant containing VH-phage and L chain was added to the microplate immobilized with goat anti-human kappa chain antibody, and the bound phages were detected with anti-M13 HRP conjugate. As a result, D11 showed a clear antigen-dependent increase in signal, suggesting its suitability to OS-IA. According to the dose-response, a significant signal at 1 ng/mL of free T4 was detected. On the other hand, F11 showed higher signal even in the absence of antigen, but low signal in the absence of immobilized anti-light chain antibody, suggesting stronger VH/VL interaction of this clone than D11.

The results of phage OS ELISA indicated the superiority of D11 in OS-IA than F11. With the intent of clarifying this reason, we determined the nucleotide sequences of the two clones. To our surprise, from the deduced amino acid sequences of the VH/VL both clones share very similar primary structure even within three CDRs for both VH and VL. However, two residues (37 and 74) in VH and four located in VL (31, 94, 96 and 100) are different between the two, which make them distinctive. Among them, the residues H37, L96 and L100 are located at the VH-VL interface and could be attributable to the stronger VH / VL interaction in F11. Especially, the H37 of D11 is small Ala, while that of F11 is Val. This residue is also in the interchain interface, and potentially playing an important role in VH/VL interaction. However, preliminary experiment to exchange VH/VL sequences of D11 and F11 in pDong1 indicated that both VH and VL are important in deciding the VH/VL interaction strength in the absence of antigen.

To further confirm the T4-dependency in phage OS ELISA of clone D11, we attempted to reproduce the assay with purified fusion proteins. As in our previous studies, we used E. coli maltose binding protein (MBP) as a fusion partner for the ease of soluble expression the fusion proteins and also their immobilization/conjugation. Hence, the obtained purified proteins were either immobilized directly (MBP-VL) to the microplate, or used as a conjugate with horseradish peroxidase (HRP-MBP-VH) to probe its binding activity to the other variable region fragment (MBP-VL). The result indicated similar or superior antigen (free T4)-dependent association of purified HRP-MBP-VH conjugate to immobilized MBP-VL than VH-displaying phage, which successfully detected less than 0.1 ng/mL T4 in PBS solution. Furthermore, we tried to apply OS-ELISA to estimate T4 concentration in serum using the recombinant proteins. The estimated total T4 concentration using an ethanol-extracted pooled serum was 90 ng/mL (9μg/dL). The value was in good agreement with the normal adult range of 5-12μg/dL.

In the third chapter of this thesis, micro open sandwich ELISA to rapidly evaluate thyroid hormone concentration from serum was described. Since the patients suffering from thyroid diseases are increasing, rapid and sensitive assays are strongly desired. To answer such demand, here we tried to integrate the sensitive OS-IA into a microfluidics-based analytical system. The resultant system is expected to hold the promise of integrating an entire laboratory onto a single chip (i.e., lab-on-a-chip) equipped with a thermal lens microscope (TLM), requiring less reagents, sample volumes and short analysis time (20 min). The two variable domains (VH and VL) of the anti-T4 antibody D11 were expressed as fusion proteins with E. coli maltose binding protein (MBP) and MBP-VH HRP were used to perform the os(open-sandwich) ELISA in a portable micro-ELISA system (μELISA IMT-501). Greater than 99% of T4 is reversibly bound to plasma proteins, while approximately 0.03% of T4 exists free in the blood. To precipitate the plasma proteins, a brief ethanol extraction was carried out both the control (T4/T3 deficient, Sigma, Germany) and test sera (healthy human donor).

Using the glass microfluidic chip equipped with a dam structure to keep microbeads for the reaction, first we optimized the measurement conditions for antigen T4 in PBS. MBP-VL in PBS was mixed with polystyrene beads for overnight, which was added with a blocking agent in PBS (PBS-IB) to block the remaining surface. The detection limit was influenced by the incubation/washing conditions and the blocking agent concentration in these reactions. The reactions of HRP-labeled MBP-VH, T4 and MBP-VL beads were also carried out in the presence of various concentrations of PBS-IB, and final concentration of 5% gave the highest signal/background (S/B) ratio. Hence we used these conditions in the following microchip experiments. Finally, HRP-MBP-VH at various concentrations was introduced into the microfluidic chip at various flow rates and the resulting S/B ratios were evaluated. Consequently, it was found that the reaction with 0.5μg/mL HRP-MBP-VH at 2 μl/min gave the best result and we successfully detected less than 1 ng/ml of free T4. The total serum concentration is a specific and sensitive index of thyroid function. Based on the established condition, we estimated the total T4 concentration of a healthy individual using ethanol extracted sample sera. The obtained value was 80 ng/ml (= 8μg/dl), which coincided well with the normal adult range for the total T4 in serum of 5-12μg/dl.

In the forth chapter of the thesis, conclusion and perspective are described. This would be the first micro open-sandwich ELISA constructed with antibody fragments directly selected from immunized mice. This system will be applied to the sensitive detection of many diagnostic markers.

本論文は,最近進展の著しい抗体工学的手法を用いて低分子化合物,特に甲状腺疾患の診断に重要なホルモンの一種チロキシン(T4, Mw:776.87)の高感度かつ迅速な検出を目指した研究について述べたものであり,4章より構成されている。

第1章は序論であり,本研究の意義を明確にするために、抗体工学に関する既往の研究を中心に、研究の背景が述べられている。

第2章では,本論文の主題となる抗原濃度に依存した抗体の2個の可変領域VH-VL間の相互作用変化を利用した免疫測定法であるオープンサンドイッチ法(OS法)による,T4検出系の構築に関して述べられている。従来,免疫測定法としては分子量1000以上の蛋白質を検出するためには主に2種類の抗体を用いるサンドイッチ法が,それ以下の低分子は競合法によって検出が行われてきた。しかし競合法にはその検出感度に抗体の解離定数によって定まる限界が存在し,さらに必要な感度と測定濃度域を得るための条件検討に相当の手間を要するという問題があった。これに対しOS法は,低分子であってもこれを非競合的に検出でき,高感度と広い測定域を得られる特長が知られていた。しかしその反面,これまでは測定系構築のためには確立された抗体産生細胞(ハイブリドーマ)から遺伝子をクローニングするための手間と時間がかかり,さらに得られた抗体断片が必ずしもOS法に最適な性質を持つとは限らないという問題があった。

そこで本章で筆者は,確立されたハイブリドーマでなく抗原コンジュゲートで免疫したマウスの脾臓細胞を材料とし,ここから抗体VH/VL cDNAを増幅しFab断片のファージ提示ライブラリを作製し,抗原に結合する抗体断片提示ファージを選択(パニング)することで迅速に所期の抗体断片を得ることを試みた。免疫原ならびに選択用分子としてそれぞれスカシ貝ヘモシアニンおよびウシ血清アルブミンに結合させたT4分子を用い,新規なファージ提示ベクターを用いて3回にわたるライブラリのパニングを行った結果,2個の特異的クローンが得られ,それらは競合ELISAで5~13 ng/mL (7~19 nM)という低いIC50を示すこと,そしてそのうち1個はVH断片提示ファージを用いたOS免疫測定によりOS法に適した性質を持っていたことが述べられている。また塩基配列から推定されるアミノ酸配列から,測定に適したクローンと適さないクローンとの差についての考察がされ,VH, VL両者の配列が重要であるとの結論が得られた。さらに得られた適性クローンを大腸菌マルトース結合蛋白質との融合蛋白質として発現させ,マイクロプレート上でのOS酵素免疫測定(OS-ELISA)系を構築したところ,0.1 ng/mL以下の検出限界LODが得られ,これは競合ELISAによるLOD(>1 ng/mL)を顕著に下回った。さらに血清からエタノール抽出した全T4濃度の検出結果と,類縁体T3に対する十分な選択性も示されており,本手法により臨床診断に応用可能な検出系が構築されたことが示されている。

第3章では,前章で得られたOS法に適した抗体断片を用い,これをマイクロ流路に基づく迅速ELISAシステムの検出素子として用いた結果について述べられている。近年確立されたマイクロ流路を用いたELISAシステムは,従来のELISA系よりはるかに少ないサンプル量と短い反応時間での抗原検出を可能にすることが明らかにされている。そこで,OS-ELISAにおいても同様の効果がみられるかどうか,検討された。反応条件最適化の結果,まずPBS中でマイクロプレートを用いて得られたLODを下回るLODを,少ないサンプル量(3 μL < 50 μL)と短い反応時間(20 min < 4 h)で得ることができ,さらにほぼ同等の結果を血清抽出サンプルにおいても得られたことが述べられている。以上の結果より,マイクロELISAとOS法の組み合わせにより,従来より迅速かつ高感度な甲状腺疾患の臨床診断が可能になったことが述べられている。

第4章では研究全体の総括と,今後の展望が述べられている。

以上本論文において筆者は,ファージ提示法による免疫動物からの低分子認識抗体遺伝子のクローニングとスクリーニング,さらにマイクロ流路・検出技術との融合という二つの側面で,抗体工学的手法を応用した高性能な低分子免疫測定系構築法を確立できたと言える。その成果は汎用分子認識素子である抗体の蛋白質工学ならびにこれを用いた分析化学の進展に寄与するところ大である。

よって本論文は博士(工学)の学位請求論文として合格と認められる。