· BLUE CARRIERÒ
USED IN ANTIBODY PRODUCTION

BLUE CARRIERÒ has been successfully used in the development of murine monoclonal antibodies, and in rabbit and goat polyclonal antibodies by Biotech Companies (Santa Cruz Biotechnology, USA[13]; Bios Chile IGSA[12], Chile; INUAL, Chile and BIOSONDA, Chile[9,10,13]). Also, BLUE CARRIERÒ, has been used by research groups at: Mount Sinai, monoclonal antibody facility USA., Catholic University of Chile[8] and the University of Chile[11]. A wide range of antibodies against antigens such as synthetic peptides, human recombinant proteins and haptens, have been achieved with BLUE CARRIERÒ.

· ANTIGEN COUPLING TO BLUE CARRIERÒ

The methods most frequently used for coupling molecules to Hemocyanin are described in Practical Immunology[4] and Current Protocols in Immunology[5].

· IMMUNIZATION OF MICE WITH PEPTIDES BOUND TO BLUE CARRIERÒ

To test the immunogenic capacity of BLUE CARRIERÒ, BIOSONDA performed a series of immunizations of mice with the small hapten Gizzerosine  an adduct of Histamine and Lysine [9,10]. Furthermore, we have used BLUE CARRIERÒ to develop a series of anti-peptide antibodies [8,9,10,11,12] as part of our custom made monoclonal and polyclonal antibody service at BIOSONDA. A representative experimental protocol is summarized below:

1. Mice immunization

Groups of 2 month old BALB/cJ mice were immunized according to the following schedule:

Day 1 100 mg of antigen in Complete Freund's Adjuvant, intraperitoneally
Day 7 20 mg of antigen in Complete Freund's adjuvant, foodpad
Day 27 12,5 mg of antigen in phosphate saline buffer (PBS) pH 7.2, intravenously
Day 47 Boost 100 mg antigen in PBS, intraperitoneally and in foodpad

Before immunization, the animals were tail bled to get pre-immune sera. The humoral immune response was evaluated at days 14, 29, 44 and 60 post- immunization.

2. Evaluation of the immune response

ELISA plates coated with the hapten linked to Bovine Serum Albumin (BSA) were used. As specificity controls we used BSA treated with Glutaraldehyde and BSA-Histamine.

Comment: After the booster, the animals showed a strong and specific immune response against the hapten.

3. Development of monoclonal antibodies to gizzerosine using BLUE CARRIERÒ

Splenic lymphocytes from immunized mice were fused with the NSO/2 cell line using the classical protocol described by Köhler and Milstein [9]. We obtained 18 monoclonal antibodies that reacted with the hapten modified BSA. One of them was selected due to its specificity to Gizzerosine and low reactivity to Histamine in solution.

· 1. Immunization protocol

Two New Zealand rabbits were immunized with the carrier-peptide conjugate according to the following schedule:

Day 1 500 m g of antigen in Complete Freund's Adjuvant, subcutaneous and intradermal.
Day 20 500 m g of antigen in Incomplete Freund's adjuvant, subcutaneous and intradermal.
Day 45 1,000 mg of antigen in Incomplete Freund's adjuvant, subcutaneous and intradermal.
Day 60 1,000 mg of antigen in Incomplete Freund's adjuvant, subcutaneous and intradermal.

Before immunization, the animals were ear bled to get pre-immune sera.

COMPARISON OF  BLUE CARRIERÒ AND KLH: GENERATION OF ANTI-PEPTIDE RESPONSE

To compare BLUE CARRIERÒ immunogenicity side by side with Keyhole Limpet hemocyanin (KLH), we immunized rabbits with a peptide from Connexin 33, a Gap Junction protein[8]. A synthetic fragment of 14 amino acids in lenght (SSDQVVPVGLSSYM) was bound to both carriers using glutaraldehyde[5]. Rabbits were immunized according the protocol described below.   The presence of specific antibodies, was measured by a direct ELISA on a 96-multiwell plate coated with the peptide. The ELISA was developed with anti-rabbit IgG- alkaline phosphatase and revealed by adding p-nitrophenyl phosphate.  The plate was read spectrophotometrically at 405 nm. Figure A show that the immune response against the peptide linked to BLUE CARRIERÒ, was four times more stronger than the response obtained when the same peptide was bound to KLH.

· IMMUNOGENICITY OF BLUE CARRIERÒ vs KLH

To compare the immunogenicity of BLUE CARRIERÒ (CCH) with that of Keyhole Limpet hemocyanin (KLH), the sera from the rabbits used to produce anti-peptide antibodies was titrated by a direct ELISA on a 96-multiwell plate coated either with KLH or CCH. The ELISA was developed with anti-rabbit IgG- alkaline phosphatase and revealed by adding p-nitrophenyl phosphate.  The plate was read spectrophotometrically at 405 nm. Figure B show that the immune response against both carrier is almost identical. However, the anti-peptide response when using BLUE CARRIERÒ was four times more stronger than the response obtained when the same peptide was bound to KLH.

                         FIGURE A                                                                 FIGURE B

REFERENCES
 
· General 

1. Immunochemical properties of haemocyanin. Weigle W.O. Immunochemistry 1: 295 - 302 (1964).

2. Immunochemical and immunogenic properties of a purified keyhole limpet haemocyanin. Herscowitz H.B., Harold W.W., Stavitsky A.B. Immunology 22: 51-61 (1972).

3. Quaternary structure of gastropod haemocyanin. Mellena J.E., Klung A. Nature 239: 146-150 (1972).

4. Practical Immunology. Leslie Hudson & Frank C Hay. Third Edition. Chapter 1. Blackwell Scientific Publications (1989).

5. Production of anti-peptide antisera. Colligan J.E., Kruisbeek A.M., Margulies D.H., Shevach E.M., Strober W. In: Current protocols in Immunology, Volume 2, Chapter 9. National Institutes of Health. Wiley Interscience. USA (1993).

6. Continous cultures of fused cells secreting antibody of predefined specificity. Köhler G., Milstein C. Nature 256: 495 - 498 (1975).

7. Hemocyanins. Van Holde K.E., Miller K.I. In: Advances in Protein Chemistry 47: 1 - 81. Academic Press Inc, New York, (1995).

 

· BLUE CARRIERÒ

8. Regulated assembly of connexin 33 and connexin 43 into rat Sertoli cell gap junctions. Tan I.P., Roy C., Sáez J.C., Sáez, CG., Paul, D.L., Risley, M.S. Biol. Reprod. 54: 1300 - 131 (1996).

9. Development of monoclonal antibodies to gizzerosine, a toxic component present in fish meal Becker M.I., Carrasco I., Beltran C., Torres M., Jaureguiberry B., De Ioannes A.E. Hybridoma 17: 373 - 381 (1998).

10. Specific RIA for Gizzerosine and simple procedure labeling method for 125I-Gizzerosine. Torres M., Manosalva H., Carrasco, I., De Ioannes A. E., Becker M.I. J. Agric. and Food Chem. 47: 4231- 4236 (1999).

11. HFE inhibits apical iron uptake by intestinal epithelial (Caco-2) cells. Arredondo, M., Muñoz, P., Mura, C., Núñez, M.T.  FASEB J. 15: 1276-1278 (2001).

12. An in vitro assay to detect paralytic shellfish poison. Córdova, J.L; Jamett, A; Aguayo J; Fauré, M.T; Villarroel O, Cárdenas L. J Shellfish Science 20: 55-61 (2001)

13. Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demostrate common and specific epitopes among subunits Oliva H, Moltedo B, De Ioannes P, Faunes F, De Ioannes A.E., Becker M.I. Hybridoma 2:365-374 (2002).

WORLDWIDE DISTRIBUTOR CATALOGS

1. Santa Cruz Biotechnology Research Antibodies. Catalog # SC-3995 SC-3996 Catalog Pag. 966  (2002). Customer since 1998.

2. Pierce-Endogen Catalog # 77130. BLUE CARRIERÒ Immunogenic protein, Page ? (2003) 

3. Calbiochem Calatog. Hemocyanin from Concholepas concholepas, Catalog # 374802 374803 Page 271 (2000-2001).

4. Sigma-Aldrich Chemical Company.  Catalog # B 8556 AND H 9035. Pag. 999 (2002-2003)