Department of Immunobiology and Hybridoma Technology
Projects- Hybridoma Technology Section
Completed Projects:
1. Development of monoclonal antibodies for ABO reagents.
Year of Commencement: 1989
Duration/year of Completion: 3years / 1992
Funding agency: ICMR
Outcome:
A Balb/c mice colony was established at the Institute and an immunization programme was undertaken for producing monoclonal antibodies for ABO reagents.
2. Establishment of hybridoma technology.
Year of Commencement: 1991
Duration/year of Completion: 3years / 1994
Funding agency: ICMR
Outcome:
During earlier years infrastructure was created for this purpose and methodologies were established to detect monoclonal antibodies in culture supernatants. The K- 562 cell line was obtained from the CRI and was maintained in culture for about three months. Cultured cells were used for freezing and thawing experiments and about 98% viability was achieved.
3. Detection of weaker variants of A/B.
Year of Commencement: 1988
Year of Completion: 1990
Duration: 2years.
Funding agency: ICMR
Outcome:
The well established ELISA using Penicillinase linked Anti-A/ Anti-B, was further used to investigate five more cases of weaker variants, of which one was A w and the remaining four B w’s . The results confirmed the earlier observations of the utility of this assay in detecting weaker variants.
4. Studies on the structure and sequential expression of ABH antigens in health and malignancy
Year of Commencement: 1994
Year of Completion: 1997
Duration: 3years.
Funding agency: ICMR
Outcome:
Several fusions were performed successfully, out of which 7 clones secreting potent antibodies were expanded. The analysis of 437 adult and 59 cord bloods of different ABO groups, carried out with these monoclonal antibodies suggested that they were reactive with certain epitopes of A and B antigens only. 45 cases of different types of leukaemia were tested for antigenic expression and 10 patients having reduced expression were selected to study sequential expression during the culture. Two-phase liquid culture procedure was employed for culture and antigenic expression was studied by flowcytometry.
5. Study of colony forming units (CFUs) by methyl cellulose assay in certain haematological disorders.
Year of Commencement: 1996
Year of Completion: 1999
Duration: 3years.
Funding agency: ICMR
Outcome:
Colony forming units were studied invitro by culturing aspirated bone marrow cells in a semisolid methyl cellulose assay system in certain haematological disorders like aplastic anemia, polycythemia vera, and other myeloproliferative disorders.
6. Production of monoclonal antibodies to B group erythrocytes.
Year of Commencement: 1995
Year of Completion: 1999
Duration: 4years.
Funding agency: ICMR
Outcome:
Six clones having specific reactivity with B group erythrocytes were isolated. After recloning, two clones having a good titre and avidity were identified and further tested for specificity with panel of red cells at 37 0C and 4 0C.To establish its efficacy to be used as a potent blood grouping reagent, the clones were submitted to International blood group reference laboratory (IBGRL) U.K. They have confirmed them as potent clone to be used as a blood-grouping reagent.
7. Production of monoclonal antibodies to foetal haemoglobin.
Year of Commencement: 1999
Year of Completion: 2001
Duration: 3years.
Funding agency: ICMR
Outcome:
Fusing spleen cells from Balb/c mice immunized with cord cells and SP2/0 Ag 14 mouse myeloma cell line produced monoclonal antibody to foetal haemoglobin. After twice recloning two clones expressing anti-HbF antibody were successfully isolated and tested by flow cytometry using fluorochrome conjugated goat anti-mouse antibody. High fluorescence was obtained when cord RBC were used as opposed to adult RBC. Besides flow cytometry, immunoblotting using PAGE electrophoresis confirmed the presence of anti-HbF. This clone (4F9C5) was then patented with assistance from NCL, Pune.
8. Monoclonal antibodies to Factor VIII
Year of Commencement: 1999
Year of Completion: 2001
Duration: 3years.
Funding agency: ICMR
Outcome:
3 clones expressing antibody activity against factor VIII antigen were produced, out of which one showed very potent anti-factor VIII activity. The Haemostasis department of our Institute has carried out standardization experiments using this antibody to determine the level of Factor VIII antigen present in a sample by ELISA.
9. Monoclonal antibodies to activated platelets
Year of Commencement: 1999
Year of Completion: 2001
Duration: 3years.
Funding agency: ICMR
Outcome:
Balb/c mice were immunized with 1x10 9 ADP activated platelets over a 6 months period. Spleenic lymphocytes from Balb/c mice were fused with SP2/0 myeloma cells. The fusion yielded 3 clones expressing anti-platelet activity and 6 clones expressing anti-HLA antibodies.
10. Monoclonal antibodies to N blood group antigen
Human antibodies to MN blood group antigens are naturally occurring cold agglutinins. During one of the fusion experiments using SP2/0 cell lines and spleenic lymphocytes we identified an antibody in the culture supernatant by panel of red cells expressing anti-N specificity. This antibody was of interest since it was pH dependent reacting only in presence of medium containing sodium bicarbonate. Further investigation on this antibody showed that using neuraminidase treated cells enhances its activity, which is contrary to the characteristic of MN antibodies. The antibody specificity is directed toward ‘Cryptic N` or (ISBT-MNS 30) since it is also trypsin resistant.Glycophorin A carries M and N specificities and is sensitive to trypsin. On the other hand Glycophorin B harbours ‘Cryptic N` or (ISBT-MNS 30) which is located on the N terminal end and is resistant to trypsin. Hence the monoclonal anti-‘N` probably is situated on Glycophorin B having anti-‘N` specificity.
11. Production of monoclonal anti-human globulin reagent
Year of Commencement: 2001
Year of Completion: 2002
Duration: 1year.
Funding agency: ICMR
Outcome:
Monoclonal anti-human globulin reagent (AHG) plays an important part in blood banking for the detection of atypical and incomplete antibodies. In the past AHG also known as Coomb’s reagent was produced by immunizing species like rabbits, sheep, goat etc with human serum or purified gamma globulin. With hybridoma technology, AHG was raised which ensured a continuous supply of antibody with same specificity. Two out of 6 clones showed a potent activity with a titre of 1:32-64. These reagents also showed a good reactivity with cells coated with anti-Duffy and anti-Kidd antibodies.
12. Monoclonal antibodies against H antigen
Year of Commencement: 2002
Year of Completion: 2004
Duration: 2years.
Funding agency: ICMR
Outcome:
During the year, while raising a monoclonal antibody against A group red cells by fusing spleen cells from Balb/c mice immunized with A group red cells with SP2/0 Ag 14 mouse myeloma cell line. We encountered an antibody showing anti-H specificity. However, the agglutination titre of this antibody was 1:16 suggesting dosage effect. To verify this, it was suggested by SAC to raise a similar antibody using O group erythrocytes as immunogen. Following a conventional protocol, we isolated two stable clones (3E8H11, 3E8A10), which showed anti-H specificity and has agglutinating titre of 1:128 with O group erythrocytes. The reactivity is also confirmed with A 1, A 2, A 1B, A 2B and B group erythrocytes.
13. Monoclonal antibody against ‘A` group antigen
Year of Commencement: 2005
Year of Completion: 2006
Duration: 1year.
Funding agency: ICMR
Outcome:
A murine monoclonal antibody against ‘A` red cells was generated using a conventional protocol. The antibody thus produced was used as a blend with anti-A murine monoclonal previously generated. The titre of this antibody is 1:64 with A 1 cells and 1:8 with A 2 cells.
On Going Projects:
1. Production of monoclonal antibodies against Rh antigen
Year of Commencement: 2005
Year of Completion: 2008
Duration: 3years.
Funding agency: ICMR
Expected Outcome:
Generation of murine monoclonal antibodies pertaining to the needs of the Institute is an ongoing programme. In the past we reported raising of murine monoclonal against A, B and blood group antigens.
The main objective of the present programme is to produce in-house Rh monoclonal antibodies by hetero-hybridoma. Indigenously generated antibodies besides being cost effective also circumvents the epitope difference that exists in monoclonals procurred commercially. This work is still in progress
2. Production of Murine monoclonal antibody against HbA 2 for detection of Hemoglobinopathies
Year of Commencement: 2007
Year of Completion: 2010
Duration: 3 years.
Funding agency: ICMR
Expected Outcome:
Presently the ideal laboratory methods for carrier detection include measurement of red blood cells (RBC) indices using automated cell counters followed by High performance liquid chromatography (HPLC) analysis of hemoglobin.HPLC is primarily an automated tool and is not suitable for mass screening purposes. With limited resources, naked eye single tube red cell osmotic fragility test (NESTROFT), followed by RBC indices and cellulose acetate electrophoresis are also used. However, this is time consuming and the results are not always reproducible. Hence, there is a need to develop a sensitive and rapid assay like ELISA using antibody against HbA 2. However, this antibody is not commercially available. Hence, raising HbA2 antibody indigenously will be helpful for the identification of β thalassaemia cases. With limited financial resources and available facilities such an antibody will be a useful tool in identifying the β thalassaemia cases and also cost effective.
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