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Short Communication Volume 9 Issue 1
Recombinant protein (anti-HRP) issued from the sea star igkappa cloning by the use of cho: its antibody specificity revealed by Elisa
Michel Leclerc 
Immunology of Invertebrates, Orléans University, France
Correspondence: Michel Leclerc, Immunology of Invertebrates, Orléans University, France
Received: December 29, 2023 | Published: January 12, 2024
Citation: Leclerc M. Recombinant protein (anti-HRP) issued from the sea star igkappa cloning by the use of cho: its antibody specificity revealed by Elisa. J Stem Cell Res Ther. 2024;9(1):1-2. DOI: 10.15406/jsrt.2024.09.00164
Abstract
It allows, by the use of CHO (Chinese Hamster Ovarian) protocol cloning to produce the «Young Protein» or anti-HRP (Horse-Radish Peroxydase) from the sea star IGKappa gene which corresponds to the IPA: (Invertebrate Primitive Antibody). Two elisas confirm the anti-HRP activity.The first Elisa was done with Protein L-HRP antigen. The second one with Streptavidine-HRP at classical concentrations which are used for Mammals.
Keywords: chinese hamster ovarian, protocol cloning, horse-radish peroxydase, mammals
Introduction
10 years ago, we tried to clone, for the first time, the Asterias rubens sea star IGKappa gene by the use and the help of E.coli as amplificator.1 It allowed, in a second time, to verify that the Young Protein, or anti-HRP Protein recognizes the HRP antigen.1
But, this verification of the affinity between the IPA (Invertebrate Primitive Antibody) and the antigen, seemed unclear at that time, for many of us.
Thus, we decided to operate a new cloning2 of the IGKappa gene with new parameters and new affinity tests. This second one did not allow to obtain the protein of interest or «Young Protein».
A third assay was attempted:
It used a CHO method, as described in various experiments3 and Elisa to verify the specificity of our « antibody » against the HRP antigen.
Material and methods
We use the « Young protein » or anti-HRP protein as primitive antibody.
Secondly, two Elisas are performed in classical microplates coated with BSA to saturate the bottom of each well.
Classical dilutions (half by half) are done.
In the first microplate we add Protein L HRP as antigen, in the second one, Streptavidine-HRP as antigen.
At last, in each well, we place anti-His protein, then substrat TMB to reveal the reaction.
Results
As shown in Figure 1, by the use of Protein L-HRP antigen, a positive reaction occurs for the first dilutions of the IPA in «sensibility» to the antigen. A similar positive reaction was obtained with the antigen: Streptavidine-HRP.
Figure 1 Elisa with STAR or sea star antibody anti-HRP against Protein L-HRP as antigen.
Note the affinity specially with the yellow curve for an IPA 0,034 dilution.
It indicates that the «Young Protein» has a ANTIBODY BEHAVIOUR towards the antigen HRP or HRP complex.
Of course it was possible to perform an Elisa test directly against HRP antigen, it is why in a next assay we ‘ll realize first : an immobilization of His protein in microplates.
The immobilization of Histidine will allow to determine better the specificity of the reaction in our conditions of manipulation.
Discussion and conclusion
We think now that our primitive antibody (IPA) anti-HRP recognizes the antigen HRP-either directly.4
We think now, to immunize other sea stars with anti-tumoral antigens to product specific nanobodies5 from sea stars, against specific cancer proteins (in a general way) Sequencing and cloning, it’’ll allow us to obtain a specific recombinant specific protein we ‘ll test against cancerous cells.
On the other hand, we‘ll try to determine other parameters of the IPA such as CDR 1 and CDR2 with these new invertebrate primitive antibodies.
Acknowledgments
None.
Conflicts of interest
The author declares that there are no conflicts of interests.
Funding
None.
References
- Leclerc M, Otten P. Immune properties corroborated by A. Rubens sea star Igkappa gene. SAJ Biotechnol. 2014;1:104.
- Leclerc M. Other sea star Igkappa gene cloning assay in E. coli with new parameters. J Stem Cell Res Ther. 2023;8(1):25–26.
- Ryu J, Eun‑Jung K, Joo‑Kyung K, et al. Development of a CHO cell line for stable production of recombinant antibodies against human MMP9. BMC Biotechnol. 2022;22:8.
- Leclerc M. Thesis (Doctorat es Sciences) Orléans University. 1974.
- Leclerc M. The nanobodies and its relations to IPA (Invertebrate Primitive Antibodies). Existence of at least 2 classes of IPA in sea Star Immune system. Mathews J Immunol Allergy. 2022;6(1):14.
©2024 Leclerc. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and build upon your work non-commercially.