Hybridoma Technology

Afsaneh,
I appreciate your question, here is my anwers for it.
mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.

Kranthi

Dear Debyani,
Here is my explaination..
With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

Kranthi

Yes there are a wide number of cancer can be cured by using this Mabs. which includes
Prostrate cancer,
colon cancer,
xenografted human carcinoma cancer,
immuniotherapy cancer etc
all this are specific and can be treated with mabs.

Kranthi

Ayush,
i appreciate your question, but most of the biotechnology products are produced in the pharamcy industries only, infact we use the different branches of science in the pharmacy industries. so its very much benfit to pharmacy industires only. Yes we can synthesise the drugs based on this technology
Kranthi

Dear Ks kumar,
there is no chance of myeloma cells reclaiming their antigencity because of the process adopted to make them Mab itself will kill all the myeloma cells.

Kranthi

Santosh kumar,
here is my explaination,
Monoclonal antibodies (Mabs) against white spot syndrome virus (WSSV) were tested for their ability to neutralize WSSV infectivity by immunodot-blot assay and enzyme-linked immunosorbent assay. It was proved that WSSV could bind to haemocyte membranes of Penaeus chinensis, moreover, the result showed that the Mab 2D2 could reduce the binding of WSSV with haemocyte membranes of P. chinensis. The neutralization assay was further conducted in vitro with primary cultured haemocytes of P. chinensis, and the cell culture assay showed that the two monoclonal antibodies, 2D2 and 6B4 could partially neutralize WSSV infection.

Kranthi

Dear Jasmine jose,
I appreciate your question, here is my answer
Factors Affecting MAb Production
The purity of the antigen, the mouse strain which is used, the
age and sex of the mouse, the tolerance of the individual
mouse, and many other factors may affect the outcome of Abproducing
procedures. Because so many variables are present,
one general way to maximize the animal’s immune response is
to use adjuvants. Adjuvants hold the antigen at the site of
immunization and release it over a long period of time.
Freund’s Complete Adjuvant, which is more widely used than
any other adjuvant, includes killed mycobacteria which serve to
increase inflammation upon injection, and thereby enhance the
mouse’s immune response. This inflammation often results in
painful lesions at the injection site

Definately this techniques need a lot of affort, i have given this information in the previous comment pls check them out.
Kranthi

Khushbu hasmukh,
I have already given this information anyway the major steps involved are
1 immunization of a mouse
2 Spleen removal from the mouse
3 Fusion of spleen cells with myeloma cells
4 Spleen cells are mortal but produce antibodies while myeloma cells are immortal but have no ability to produce asntibodies.
5 The resulting hybrid cell will have both of the above properties.
6 Screen the cell population for hybrid cells.
7 clone the isolated hybrid cells
8 Produce these cells in large quantities ( Scale-up )

Kranthi

dear kishore reddy,
I have already mentioned the advantages and disadvantages of monoclonal antibodies, but still i am giving you the side effects associated with it,
Side effects associated with monoclonal antibodies (MOABs) differ depending on the form of monoclonal antibody that is used in treatment. Side effects associated with these drugs include:

Allergic infusion reactions (including fever and chills)
Decreased blood pressure
Facial swelling
Coughing
Hives or rashes and itching
Nausea and vomiting
Headache
Shortness of breath
Swollen tongue
Lower blood counts
Blood clots or bleeding
Chest pain
Loss of coordination
Numbness
Change in mental status
High blood pressure (hypertension)

kindlers.
Kranthi

Dear Gangadhar hari,
NO its not widely used in india , but in other countries,
I have already given explainatin about it, please refer it.
Kranthi

Sirisha,
When we compare the two technologies, we are also need to considere the conditions such as
what type of cancer is it ?
what age is the person to be treated ?
etc
so deponding on these conditions, one will choose a best suitable method.
because these technologies have their own disadvantages and advantages.
but to the most hybridoma technology is used because nano technology is not yet fully came into light..

Kindlers.
Kranthi

SIr,
Yes as you mentioned along with its wide range of advantages, it do carry a major drawback that is its cost factor.
I may not give you the exact figure, how this cost is being reduced. but i can say there is wide range of research is going on aroung the golbe. As number of research is going on one day will see the cost of this monoclonal antibodies will come down such that every person can make use of it, just like pencilin, which is very coslty when it entered into market for the first time, but now its economic and every one is using.
Hope i clarified your question.
Kindlers.
Kranthi

Juhi sharma,
I appreciate your doubt, as i have already mentioned about monoclonal antibodies, This monoclonal antibodies produced through hybridoma technology will not disturbs the host or patient immune system infact it will support our immune system.
Kindlers.
Kranthi

Hi Madhuri,
Thanks for your comment, here is your answe,
This monoclonal antibodie are widely used for cancer curing, but not for all types of cancers. but covers majority of cancers.
and not for disorder.

your second question answer is
Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction. High sensitivity is achieved by using an indicator system (e.g., enzyme label) that results in amplification of the measured product.
and
Fluorescent immunoassay is an immunoassay technique in which antigen or antibody is labeled with fluorescent dye.

It is not widely use in india, but in most of the developed countries its usage is increased.

Hope my answers satisfied you.
Kindlers.Kranthi

dear haritha,
Its a good question, This hybridoma technology is very effective and helpful in present medical field infact this technology is widely used in some countries such as in europian and american countries. but still there is lots of research is going on around the glob.
The monoclonal antibody (mAb) market has grown rapidly in recent years, reaching sales of $14bn in 2005, an increase of 36.5% from 2004 sales of $10.3bn. Köhler and Milstein developed the hybridoma method of murine antibody production in 1975, which allowed the production of the first mAb to market; Johnson & Johnson’s Orthoclone OKT3 (muromonab) in 1986. The mAb market is highly innovative and a key trend has been the move from murine to humanized and fully human antibodies. As technology has progressed these humanized mAbs have prevented immune responses (HAMA), thus having a larger market potential. The traditional therapy areas in the mAb market are oncology and autoimmune and inflammatory disorders (AIID), however this is forecast to change with the emergence of other therapy areas including infectious disease and ophthalmology. The clear leader in the mAb market is Genentech with 5 marketed drugs, with sales totaling $4,116.4m in 2005. A key theme of this report is the high level of innovation, as demonstrated by advancements in antibody engineering with the introduction of chimeric, humanized and fully human mAbs. Other innovation in antibody technology include advancements in non-invasive drug delivery technology, which is predicted to lead to a huge boost in sales in the long-term once drugs that utilize this technology come to market

YOur second question answer Yes its widely used in cancer thereapy but with all this advantages there is a disadvantage that is its high cost. but in near futur. with increase in research hopefull it cost will reduce, so that every common man can access..

Hope your doubts are clarified.

Kindlers.
Kranthi

Dear Vedika Gulati,
I appreciate your question here is my explaination for this question,
The ultimate objective of immunosuppressive therapy is to block transplant recipient reactivity to allograft incompatibilities while sparing other responses. Increased clarification of rejection mechanisms has made possible the precise suppression of specific elements of the immune response using murine anti-human monoclonal antibodies.
In addition, recombinant DNA technology has made available novel agents including humanized, bispecific, or toxin-conjugated molecules, which avoid some of the limitations of murine reagents.
Using such agents, donor-specific tolerance has been induced in experimental models after a limited course of therapy directed against selected effector cell surface-associated molecules such as CD4, CD25, and CD54.
It remains to be determined how such observations can be successfully transferred to the human situation. It seems likely, however, that as new molecular agents are developed, increasingly effective suppression of specific cellular targets will become an essential element of clinical protocols. Such agents may provide long-term immunosuppression with limited periods of immunosuppressive agent administration.

This technology is first instigated in european countries.

I hope your doubts are clarified.

Kranthi

Dear sir,
Thank you for the comment you gave, Yes as you said this technology is very costly, at the same time time consuming and one small mistake will may lead to complete spoilage of the product.
But still there is lot of research is going on in this technology and this monclonal antibodies are produced in many countries to meet the need because this Mabs are more specific and target orientated.
As the out come product is very useful,many industries has started doing research over it.
As the number of industries working on it increasing, hopefully may be not now but in near future this monoclonal antibodies will also be avilable to common man, even in India. just like the pencilin injection right now. as you know when it first came to market it is not available to common man. but now every one can use it.

Yes as i have already mentioned the main test performed to test this Mab s is ELISA test.

Hope i have clarified your comments.

Thank you sir, for your encouragement.

Kranthi

HI Himanshu saurabh,
Yes there are alternative methods for the production for Mabs, In fact the production of monocloan antibodis have started very long time back.
There are three principal steps in production of monoclonal antibodies: 1) immunization, 2) hybridoma formation and 3) MAb production. Each has its own potential for adoption of alternative methods. Although currently done primarily as an in vivo procedure using a few animals, it is possible, especially with human MAbs, to conduct the immunization process entirely in cell cultures. Some technical difficulties remain to be solved before this becomes a routine non-animal-based procedure.

Formation of the hybridoma cells has always been an in vitro technique. However, final production of the monoclonal antibodies involves use of either the ascites or in vitro alternative approaches. The present review briefly focuses on this last step, with an emphasis on the availability of multiple alternative MAb production techniques, suitable for the small-to-medium-scale research and commercial laboratories.

Kranthi

HI Himanshu saurabh,
Yes there are alternative methods for the production for Mabs, In fact the production of monocloan antibodis have started very long time back.
There are three principal steps in production of monoclonal antibodies: 1) immunization, 2) hybridoma formation and 3) MAb production. Each has its own potential for adoption of alternative methods. Although currently done primarily as an in vivo procedure using a few animals, it is possible, especially with human MAbs, to conduct the immunization process entirely in cell cultures. Some technical difficulties remain to be solved before this becomes a routine non-animal-based procedure.

Formation of the hybridoma cells has always been an in vitro technique. However, final production of the monoclonal antibodies involves use of either the ascites or in vitro alternative approaches. The present review briefly focuses on this last step, with an emphasis on the availability of multiple alternative MAb production techniques, suitable for the small-to-medium-scale research and commercial laboratories.

Kranthi

Dear zarrinfaria,
I I have already mentioned the advantages and disadvantages of monoclonal antibodies, but still i am giving you the side effects associated with it,
Side effects associated with monoclonal antibodies (MOABs) differ depending on the form of monoclonal antibody that is used in treatment. Side effects associated with these drugs include:

Allergic infusion reactions (including fever and chills)
Decreased blood pressure
Facial swelling
Coughing
Hives or rashes and itching
Nausea and vomiting
Headache
Shortness of breath
Swollen tongue
Lower blood counts
Blood clots or bleeding
Chest pain
Loss of coordination
Numbness
Change in mental status
High blood pressure (hypertension)

And your second question answer is, This monoclonal antibodies are as potential as our immune system antibodies. Infact some time even more effect than our body immunesystem.
this Mabs are even more specific to an antige.

Hope your doubts are clarified.
Kranthi

Dear zarrinfaria,
I I have already mentioned the advantages and disadvantages of monoclonal antibodies, but still i am giving you the side effects associated with it,
Side effects associated with monoclonal antibodies (MOABs) differ depending on the form of monoclonal antibody that is used in treatment. Side effects associated with these drugs include:

Allergic infusion reactions (including fever and chills)
Decreased blood pressure
Facial swelling
Coughing
Hives or rashes and itching
Nausea and vomiting
Headache
Shortness of breath
Swollen tongue
Lower blood counts
Blood clots or bleeding
Chest pain
Loss of coordination
Numbness
Change in mental status
High blood pressure (hypertension)

And your second question answer is, This monoclonal antibodies are as potential as our immune system antibodies. Infact some time even more effect than our body immunesystem.
this Mabs are even more specific to an antige.

Hope your doubts are clarified.
Kranthi

Dear Harpreet singh,
I have already mentioned the advantages and disadvantages of monoclonal antibodies, but still i am giving you the side effects associated with it,
Side effects associated with monoclonal antibodies (MOABs) differ depending on the form of monoclonal antibody that is used in treatment. Side effects associated with these drugs include:

Allergic infusion reactions (including fever and chills)
Decreased blood pressure
Facial swelling
Coughing
Hives or rashes and itching
Nausea and vomiting
Headache
Shortness of breath
Swollen tongue
Lower blood counts
Blood clots or bleeding
Chest pain
Loss of coordination
Numbness
Change in mental status
High blood pressure (hypertension)

Kranthi

Dear Sudha manohar,
I appreciate your question here is my explaination for this question,
The ultimate objective of immunosuppressive therapy is to block transplant recipient reactivity to allograft incompatibilities while sparing other responses. Increased clarification of rejection mechanisms has made possible the precise suppression of specific elements of the immune response using murine anti-human monoclonal antibodies.
In addition, recombinant DNA technology has made available novel agents including humanized, bispecific, or toxin-conjugated molecules, which avoid some of the limitations of murine reagents.
Using such agents, donor-specific tolerance has been induced in experimental models after a limited course of therapy directed against selected effector cell surface-associated molecules such as CD4, CD25, and CD54.
It remains to be determined how such observations can be successfully transferred to the human situation. It seems likely, however, that as new molecular agents are developed, increasingly effective suppression of specific cellular targets will become an essential element of clinical protocols. Such agents may provide long-term immunosuppression with limited periods of immunosuppressive agent administration.

Kranthi