Biotechnologia Acta

ISSN 2410-776X (Online),
ISSN 2410-7751 (Print)

Biotechnologia Acta
V. 8, No 3, 2015

Biotechnologia Acta V. 8, No 3, 2015
DOI: 10.15407/biotech8.03.045
Р. 45-55, Bibliography 25, Ukrainian
Universal Decimal Classification: 557.161.2+612.017.1:616-097

SYNTHESIS OF 25-HYDROXYVITAMIN D₃ CONJUGATE WITH KEYHOLE LIMPET HEMOCYANIN AND OBTAINING OF IMMUNE SERA

А. O. Mazanova, I. O. Shymanskyy, D. M. Petukhov, L. B. Drobot, M. M. Veliky, S. V. Komisarenko

Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kyiv

The study was aimed at obtaining polyclonal antibodies that recognize 25-Hydroxyvitamin D₃, for their further specifications and applications in immunochemical test systems of 25-Hydroxyvitamin D₃, determination in blood serum. We used the methods of chemical synthesis of immunoconjugates (modified carbodiimide method using N?-ethyl carbodiimide), thin layer chromatography, gel filtration and indirect immunoenzyme analysis ELISA. The work describes the stages of the synthesis of 25-Hydroxyvitamin D₃, immunoconjugate with keyhole limpet hemocyanin (KLH), which was used for receiving immune sera. As a result of mouse and rabbit immunization antisera were obtained and antibody titers against 25-Hydroxyvitamin D₃, were tested by immunoenzyme assay. It was demonstrated that the titer of specific antibodies was higher in rabbits compared with mice. The resulting polyclonal antibodies can be used for the development of immunochemical test systems for screening studies of 25-Hydroxyvitamin D₃, content in human blood serum as a marker of vitamin D₃ availability.

Key words: 25-Hydroxyvitamin D₃, haptens, immunoenzyme analysis, polyclonal antibodies.

© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2008

• References

  • 1.  Cooper  H.  M.,  Paterson  Y.  Production  of polyclonal  antisera.  Curr.  Protoc.  Cell  Biol. 2001, 2 (2.4), 1–10.
    doi: 10.1002/0471142735.im0204s13.

    2.  Zegers N. D. Synthetic peptides for antibody production.  Thesis  Erasmus  Universiteit Rotterdam. 1995, 248 p.

    3.  Dobnig H. A review of the health consequences of  the  vitamin  D  deficiency  pandemic.  J. Neurol.  Sci.  2011,  311  (1–2),  15–18. 
    doi: 10.1016/j.jns.2011.08.046.

    4.  Wacker M., Holick M. F. Vitamin D — effects on skeletal and extraskeletal health and the need  for  supplementation.  Nutrients.  2013, 5 (1), 111–148.
    doi: 10.3390/nu5010111.

    5.  Wang Y., Zhu J., DeLuca H. F. Where is the vitamin D receptor? Arch. Biochem. Biophys. 2012,  523  (1),  123–133. 
    doi:  10.1016/j.abb.2012.04.001.

    6.  Haussler M. R., Jurutka P. W., Mizwicki M., Norman  A.  W.  Vitamin  D  receptor  (VDR)-mediated  actions  of  1?,  25(OH)2vitamin D₃:  genomic  and  non-genomic  mechanisms. Best Pract. Res. Clin. Endocrinol. Metab. 2011, 25(4), 543–559.
    doi:  10.1016/j.beem.2011.05.010.

    7.  Takeyama K., Kato S. The vitamin D₃ 1 alphahydroxylase gene and its regulation by active vitamin D₃. Biosci. Biotechnol. Biochem. 2011, 75 (2), 208–213.
    http://dx.doi.org/10.1271/bbb.100684

    8.  White J. H. Regulation of intracrine production of  1,25-dihydroxyvitamin  D  and  its  role  in innate immune defense against infection. Arch. Biochem. Biophys. 2012, 523 (1), 58–63.
    doi: 10.1016/j.abb.2011.11.006.

    9.  Guillot  X.,  Semerano  L.,  Saidenberg-Kermanach N., Falgarone G., Boissier M. C. Vitamin D  and  inflammation.  Joint  Bone  Spine. 2010,  77  (6),  552–557.
    doi:  10.1016/j.jbspin.2010.09.018.

    10.  Ananthakrishnan  A.  N.,  Khalili  H.,  Higuchi L. M., Bao Y., Korzenik J. R. Giovannuc-ci E. L., Richter J. M., Fuchs C. S., Chan A. T. Higher  predicted  vitamin  D  status  is associated  with  reduced  risk  of  Crohn’s disease.  Gastroenterology.  2012,  142  (3), 482–489.
    doi: 10.1053/j.gastro.2011.11.040.

    11.  Huber E., Becker J., Kraus W., Kyriatsoulis A., Vogel  R.,  Horn  N.,  Antibodies  against 25-hydroxyvitamin D. European Patent 1 931 711 B1, April 8, 2009.

    12.  Stokes A. N., Williams B. L., French S. S. An improved competitive inhibition enzymatic immunoassay  method  for  tetrodotoxin quantification.  Biol.  Proced.  Online.  2012, 14 (3), 1–5. doi: 10.1186/1480-9222-14-3.

    13.  Prakasha K. C., Raghavendra G. M., Horisa R., Chang Gowda D. Design, synthesis and antimicrobial screening of amino acid conjugated 2-amino  4-aryltiazole  derivatives.  Int.  J. Pharm. Pharm. Sci. 2011, 3 (3), 120–125.

    14.  Chun L., Xiaoping Y., Guangrong H., Hong W. Preparation and characterization of antigenic properties of gramicidin A- keyhole limpet hemocyanin  and  gramicidin  A-  ovalbumin conjugates. Afr. J. Biotechnol. 2009, 8 (24), 7051–7058.

    15.  Huisman  H.,  Wynveen  P.,  Setter  P.  W. Studies  of  the  immune  response  and preparation  of  antibodies  against  a  large panel of conjugates neurotransmitters and biogenic amines: specific polyclonal antibody response and tolerance. J. Neurochem. 2010, 112  (3),  829–841. 
    doi:  10.1111/j.1471-4159.2009.06492.x.

    16.  Maleimidyl  activated  Protein  Carriers Available  at:  http://www.interchim.fr/ft/8/86695A.pdf (accessed 15 February 2013)
    http://dx.doi.org/10.5582/bst.2012.v6.2.52

    17.  Hu K., Huang X., Jiang Y., Qiu J., Fang W., Yang  X.  Influence  of  hapten  density  on immunogenicity  for  anti-ciprofloxacin antibody production in mice. BioSci. Trends. 2012, 6 (2), 52–56.

    18.  Su?rez  A.  M.,  Rodri?guez  J.  M.,  Hern?ndez  P.  E.,  Azcona-Olivera  J.  I.  Generation of  polyclonal  antibodies  against  nisin: Immunization strategies and immunoassay development. Appl. Environ. Microbiol. 1996, 62 (6), 2117–2121.

    19.  Zhao  C.,  Liu  W.,  Ling  H.,  Lu  S.,  Zhang  Y., Liu J., Xi R. Preparation of Antiatifloxacin Antibody  and  Development  of  an  Indirect Competitive Enzyme-Linked Immunosorbent Assay  for  the  Detection  of  Gatifloxacin Residue in Milk. J. Agric. Food Chem. 2007, 55 (17), 6879–6884.
    http://dx.doi.org/10.1021/jf070978g

    20.  Hong  J.  Y.,  Kim  J.-H.,  Choi  M.  J.  Hapten synthesis and Influence of coating ligands on Enzyme-linked Immunoreaction of DDT. Bull. Korean Chem. Soc. 2002, 23 (10), 1413–1419.
    http://dx.doi.org/10.5012/bkcs.2002.23.10.1413

    21.  Zhou Y., Li Y. S., Pan F. G., Liu Z. S., Wangc Z. Identification  of  tetrodotoxin  antigens and  a  monoclonal  antibody.  Food  Chem. 2009,  V.  112,  P.  582–586.  doi:10.1016/j.foodchem.2008.06.022.

    22.  Pravetoni M., Naour M. L., Harmon T. M., Tucker A. M., Portoghese P. S., Pentel P. R. An oxycodone conjugate vaccine elicits drug-specific  antibodies  that  reduce  oxycodone distribution to brain and hot-Plate analgesia. J. Pharmacol. Exp.Tther. 2012, 341 (1), 225–232.
    doi: 10.1124/jpet.111.189506.

    23.  Jiang Y., Huang X., Hu K., Yu W., Yang X., Lv  L.  Production  and  characterization of  monoclonal  antibodies  against  small hapten-ciprofloxacin.  Afr.  J.  Biotechnol. 2011, 10 (65), 14342–14347.
    doi: 10.5897/AJB11.1546.

    24.  Harlow E., Lane D. Antibodies. A laboratory manual.  Cold  Spring  Harbor  Laboratory. 1988, 726 p.

    25.  Li  Z.,  Song  S.,  Xu  L.,  Kuang  H.,  Guo  S., Xu  C.  Development  of  an  Ultrasensitive Immunoassay  for  Detecting  Tartrazine. Sensors.  2013,  13  (7),  8155–8169.
    doi: 10.3390/s130708155.