人類的血紅素結合蛋白 (haptoglobin) 分為三種亞型: Hp 1-1，Hp2-2，以及Hp 2-1。Hp 1-1為單一分子構造，由兩個α1 subunit及兩個β subunit 經雙硫鍵鍵結所構成，Hp2-1及Hp 2-2則是以上述鍵結方式分別聚合α1，α2及β等subunits以多聚體的構造出現於人體中。血紅素結合蛋白和人類的許多具發炎反應之疾病有關，因此在臨床檢測是一有效的指標蛋白質。
本研究中成功地研製出六株haptoglobin的單株抗體，分別命名為8B1、2H3、W1、4A2、G2D及12B1。六株單株抗體中，對於haptoglobin結構中的β subunit具有極高特異性 (specificity) 者有五株，僅W1對α subunit具有特異性。Subtyping測試結果得知這六株抗體均歸屬於IgG1。探討haptoglobin的免疫活性過程中，我們利用單株抗體進行competitive ELISA，結果顯示Hp1-1的免疫活性大於其他haptoglobin亞型；另外，將haptoglobin甲基化改變其雙硫鍵結構做進一步探討，發現G2D單株抗體之免疫活性不受甲基化影響，且可以辨識經過trypsin切過的片段；藉此證據進而推測G2D無法辨識haptoglobin雙硫鍵鍵結之蛋白質相關結構。同時，利用單株抗體進行sandwich ELISA所得之標準定量曲線可以適當地應用於臨床haptoglobin的定量標準。

There are 3 major phenotypes of human haptoglobin (Hp), designated as 1-1, 2-2, and 2-1, respectively. The simplest form Hp 1-1 is a tetrachain structure composed of 2 α1 and 2 β chains linked by disulfide linkages. Whereas, Hp 2-2 and 2-1 are polymerized forms of higher molecular mass. Genetically, there is still no generally accepted or satisfactory mechanism explaining the functional maintenance of genetic polymorphism in human populations.
Six monoclonal antibodies (mAb): 8B1, 2H3, W1, 4A2, G2D, and 12B1, prepared against human Hp 2-1 were produced, purified, and characterized. The specificity of each mAb evaluated by Western blot shows that five of the mAb were specific to the β-chain of Hp. Whereas, only mAb W1 recognized α 1 and α 2 subunits of Hp. Each mAb was then subject for purification on a DEAE-column. Notably, the purity of each mAb was greater than 90%. Subtyping indicated that they were all IgG1 subclass.
To determine the role of cysteine in maintaining the immunochemical structure of Hp, Hp 1-1, 2-1, and 2-2 phenotypes were irreversibly modified by carboxymethylation to block all the disulfide linkages between the α- and β-chains as well as the inter-molecular linkages between each monomer of Hp 2-1 or 2-2. It was of significant interest that one of the β-chain monoclonal antibodies (G2D) possessed the immunoreactivity that was not affected by the blockage of disulfide linkages and trypsin fragmentation of Hp. Since Hp 1-1 represents a monomeric unit of Hp (α1β)2, we speculated that the antigenic epitopes might be more accessible for the antibody binding than that of polymeric Hp 2-1 or 2-2. To test this hypothesis, we determined the immunoreactivity of each phenotype of Hp in a competitive ELISA. We demonstrated that the immunoreactivity of Hp 1-1 was significantly greater (5x) than that of Hp 2-1 and 2-2 using mAb 2H3.
We conclude that: (1) intra-molecular disulfide linkages play an essential role in maintaining the antigenic structure of Hp; (2) Hp 1-1 appears to possess higher immunoreactivity then that of polymeric form Hp 2-1 and Hp 2-2. It further suggests the notion that Hp 1-1 provides more surface area and hence an increase in biological activity.

Contents－－－－－－－－－－－－－－－－－－－－－－－－ i
Abbreviation－－－－－－－－－－－－－－－－－－－－－－ ii
Abstract－－－－－－－－－－－－－－－－－－－－－－－－ iii
Introduction－－－－－－－－－－－－－－－－－－－－－－ 1
Materials and Methods－－－－－－－－－－－－－－－－－－ 5
Results－－－－－－－－－－－－－－－－－－－－－－－－ 26
Discussion－－－－－－－－－－－－－－－－－－－－－－－ 31
References－－－－－－－－－－－－－－－－－－－－－－－ 35
Table－－－－－－－－－－－－－－－－－－－－－－－－－ 39
Figure－－－－－－－－－－－－－－－－－－－－－－－－－ 45

1. Beaty J.D, Beatty B.G. and Vlahos W.G.. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J. Immunol Methods 100: 173-179 (1987)
2. Bowman B H, Kurosky A. Haptoglobin: the ecolutionary product of gene duplication, unequal crossing over and point mutation. Adv Hum Genet, 12:189-261. (1982)
3. Dobryszycka. W. Biological functions of haptoglobin-New pieces to an old puzzle. Eur J Clin Chem Clin Biochem;. 35(9): 647-654 (1997)
4. Delanghe J J, Langlois M, Ouyang J. Effect of haptoglobin phenotypes on the growth of Streptoccocus pyogens. Clin Chem Lab Med, 36:691-696. (1998)
5. Dirk D B, Guy D B, Michel L, Joris D, Hugo K, Marcel K. Haptoglobin polymorphism as a risk factor for coronary heart disease mortality. Atherosclerosis, 157: 161-166. (2001)
6. El Ghmati S M, Van Hoeyvald E M, Van Strijp J A G, Ceuppens J L, Stevens EAM. Identification of haptoglobin as an alternative ligand for CD11b/CD18. J Immunol, 156:2542-2552. (1996)
7. Friguet B., Djavadi-Ohaniance L., J. Pages, A. Bussard and Goldberg M.l. A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Journal of Immunological Methods, 60: 351-358 (1983)
8. Hanly W. C., Artwohl J.E., and Bennett B.T.. Review of polyclonal antibody production in mammals and poultry. ILAR Journal, 37(3): 93-118. (1995)
9. Harlow and Lane D. Antibodies, a laboratory manual. Cold Spring Harbor Laboratory, New York. (1988)
10. Hanasaki K, Powell L. D, Varki A. Binding of human sialoglycoproteins by the B cell specific lectin CD22. J. Biol Chem, 270:7543-7550. (1995)
11. http://www.pharma.ethz.ch/bmm/protocols
12. http://www.biotech.ufl.edu/~hcl/protocols.htm
13. Jackson L.R. and Fox J.G. Institutional policies and guidelines on . adjuvants and antibody production. ILAR Journal, 37(3): 141-152 (1995.)
14. Jennings V.M.. Review of selected adjuvants used in antibody production. ILAR Journal, 37(3):119-125. (1995)
15. Kohler G. and Milstein C.. Continuous cultures of fused cells screting antibodies of predefined specificity. Nature 256:495-497 (1975)
16. Katnik I., Steuden I., Pupek M., Wiedlocha A., Dobryszycka.W. Monoclonal antibodies against human haptoglobin. Hybridoma, 8. (5) 551-560 (1989).
17. Kakui T., Ishibashi Y., Miyake A., Terano Y., and Nakatani K.. Development of monoclonal antibody sandwich-ELISA for determination of beer foam-active proteins. J.Am. Soc. Brew. Chem. 56(2): 43-46 (1998)
18. Kino K, Tsuno H, Higa Y, Takamai M, Hamaguchi H and Nakajima H. Hemoglobin-haptoglobin receptor in rat plasma membrane. J. Biol. Chem, 255: 9616-9620. (1980)
19. Langlois M.R., Delanghe J.R.. Biological and clinical significance of haptoglobin polymorphism in humans. Clinical Chemistry 42:10 (1996)
20. Lew A.M.. The effect of epitope density and antibody affinity on the ELISA as analysed by monoclonal antibodies. J. Immunol. Methods 72, 171-176. (1984)
21. Liau C.Y., Chang T.M., Pang R.P., Chen W.L., Mao S.J.T.. Purification of human haptoglobin using hemoglobin affinity column. J Chromatogr B Analyt Technol Biomed Life Sci, 25; 790 (1-2):209-216. (2003)
22. Lange V, Der Haptoglobin polymorphisms. Anthropol Anz, 50:281-302. (1992)
23. Langlois M R, Delanghe J R, de Buyzere M L, Bernard D R and Ouyang. Effect of haptoglobin on the metabolism of vitamin C. Am J Clin Nutr, 66:606-610. (1997)
24. Louagie H K, Brouver J T, Delanghe J R. Haptoglobin polymorphism and chronic hepatitis C. J Hepatol, 25: 4-10. (1996)
25. McGuill M W and Rowan A. N. Refinement of monoclonal antibody production and animal well-being. ILAR Journal, 31(1):7-10. (1989)
26. Nimmo G.R., Lew A.M., , Stanley C.M. and Steward.M.W. Influence of antibody affinity on the performance of different antibody assay. J. Immunol. Methods 72, 177-187. (1984)
27. Pound J. D. . Immunochemical protocols. Second edition. (1998)
28. Rantappa D S, Beckman G, Beckman L. Serum protein markers in systemic lupus erythematosus. Hum Hered, 35: 207-211. (1988)
29. Steward M.W., and Lew A.M.. J. The importance of antibody affinity in the performance of immunoassays for antibody. Immunol. Methods 78, 173-179 (1985 )
30. Underwood P.A.. Practical considerations of the ability of monoclonal antibodies to detect antigenic differences between closely related variants. J. Immunol. Methods 85, 309-323.(1985)
31. Wassell J.. Haptoglobin: Function and Polymorphism. Clin. Lab. 46:547-552 (2000)
32. Zola H.. Monoclonal antibodies. Springer. (2000)