Recombinant DNA technology (Immunological screening)

Immunological Screening

1. Immunological screening Submitted TO : present by : Name : Mala. P name : Mahalakshmi . B UNIVERCITY NAME : Pmist , vallam , thanjavur Register no :123011356012 Designation : Assistant professor,Department of biotechnology year : 1 year MSC District : Thanjavur branch :Biotechnology

2. Contents INTRODUCTION IMMUNOSCREENING TYPES METHODS SCREENING OF RECOMBINANTS IMPORTANCE OF IMMUNOLOGICALSCREENING MCQ REFERENCE

3. Introduction Immunoscreening is a method of biotechnology used to detect a polypeptide produced from a cloned gene. The term encompasses several different techniques designed for protein identification, such as Western blotting, using recombinant DNA, and analyzing antibody-peptide interactions. cDNA is cloned Into the expression Vector under the Control of the Bacterial lac promotor.

4. Immunoscreening Recombinant phages are plated out onto a suitable bacterial host on agar plates. Incubated untill small plaques appear Place a nitrocellulose sheet soaked in IPTG on top of the plaques. It bind the proteins produced by the E. Coli cells after lyse by the phage infection. Peel off the nitrocellulose membrane. Incubate membrane with a specific Antibody Incubated membarane with a labelled secondary antibody to detect the presence of the bound primary antibody.

5. Types Immunological techniques, such as ELISA [1], RIA [2] and fluorescence immunoassays [3], and chemoluminescence immunoassays [4] have been conventionally applied in a wide range of areas, including disease diagnosis, clinical therapy, environmental monitoring, and food analysis. METHODS OF IMMUNOSCREENING : The term encompasses several different techniques designed for protein identification, such as Western blotting, using recombinant DNA, and analyzing antibody-peptide interactions.

6. ELISA The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries. BLODS : A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a carrier (for example, a nitrocellulose, polyvinylidene fluoride or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane

7. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining (for example, silver staining of proteins), autoradiographic visualization of radiolabelled molecules (performed before the blot), or specific labelling of some proteins or nucleic acids RIA : The full form of RIA is Radioimmunoassay. Radioimmuno assay is a highly sensitive method to determine an antigen’s concentration in a sample. This technique was introduced by Rosalyn Yalow and Solomon Berson for the detection of insulin in human blood.

8. Importance Of immunological screening These methods have been used extensively for the detection and quantification of foreign proteins in materials derived from a single major commodity, and to a lesser extent to assist with establishing the pattern of sensitivity of a given individual to the different proteins in a given food. Immunological tests can also be used to detect viruses. Examples include hepatitis C, HIV or HPV viruses.

9. Mcq 1) immunological screening is detect a___________ produced from a cloned gene A) Polypeptide B) Antigen C) Nucleotide 2) IPTG used for? A) gene cloning B) metabolic prosses C) protein expression 3) ELISA is commonly used for? A) detection of mutation B) Detection of pathogens

10. 4) Which of the following techniques is used in recombinant identification?
A) Ligation b) Isolation c) Replica plating

11. Answers A) polypeptide C) protein expression B) Detection of pathogen C) Replica plating

12. Reference Razavi , Morteza ; Pope, Matthew E.; Soste , Martin V.; Eyford , Brett A.; Jackson, Angela M.; Anderson, N. Leigh; Pearson, Terry W. (2011-02-01). “MALDI immunoscreening ( MiSCREEN ): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics ”. Journal of Immunological Methods. 364 (1–2): 50–64. doi:10.1016/j.jim.2010.11.001. ISSN 1872-7905. PMC 3018550. PMID 21078325 Mukerjee S, McKnight ME, Glassy MC. Immunoscreening protocols for the identification of clinically useful antibodies and antigens. Expert Opin Investig Drugs. 1998 Mar;7(3):373-89. doi : 10.1517/13543784.7.3.373. PMID 15991979.

13. Karam , James (November 26, 1990). Methods in Nucleic Acids Research. CRC Press. P. 309. ISBN 0849353114.

14. Thank you