I ran the cytotoxicity test of the fresh serum sample containing polyclonal antibodies by incubating with the specific antigen and i found that the antibodies lysed the antigen within 5 minutes. I stored the remaining serum in -80 refrigerator for two days. After two days i thawed the serum to check the cytotoxicity activity against the antigen but there were no results even after 2 hours of incubation.
I guess, you study complement-dependent cytotoxicity of polyclonal antibodies with any mammalian cells as the antigen. Obviously, this reaction requres three components, which should be mixed before incubation at 37 degrees: 1) cell suspension; 2) appropriately diluted sample of antibody (or heat-inactivated antiserum); 3) external source of complement (fresh or only once thawed serum of rabbit or guinea pig). In your description I see only 1 and 2. What kind of the complement do you use? If you do not use any complement, the most probable explanation for your result is the presence of active complement in your fresh serum, which was used as a source of antibodies. The activity of complement greatly declines after repeated cycles of freezing-thawing. Therefore, you could not see cytotoxicity after freezing-thawing. If you use external complement in your experiments, the most probably, it does not work and you see residual activity of complement from antiserum (as above). To determine cytotoxic antibodies, correct set of reagents should include: 1) heat-inactivated antiserum as a source of antibodies 2) viable target cells, expressing antigen of interest 3) fresh external complement, thoroughly tested on activity and the absence of unspecific toxicity - fresh serum would be best (or, you can freeze and thaw aliquotes only once). Obviously, freezing slightly affects activity of antibodies, but is critical for complement activity.
Polyclonal antibodies obtained from large animals are stable for about one year at - 80 oC and such storage slightly affects antigen-antibody binding activity.
Yes Sheela,
You can increase the stability of the polyclonal antibodies addicting a commercially avaliable protein stabilizing cocktail.
Best,
Edward
I agree with a previous post that fequent freeze and thaw is an issue, moreover if you take too long to do it and at high temperature, since you may inactivate complement. I would also suggest to aliquot the serum in order to keep your stock and not to thaw it above 37ºC.
The antibodies in the serum should be active. However, you should avoid repeating circles of thawing / freezing - aliquot the samples is a good suggestion.
Fresh antiserum contain complements. Therefore it is possible that antigen-antibody complexes activated the complement cascade. After storage in -80 freezer, it is possible that the complement component or components was/ were denatured. Was your antigen on cells when you wrote that the antibodies lysed the antigen within 5 minutes? Or was the antigen a free antigen? I will have to know more of what you have done.
Usually -80 is a good temperature for keeping the sera which contain antibodies. However, repeated freezing and thawing the serum, decreases the titer of antibody to some extend. For instance, you will get a lower OD value in an ELISA system each time when you freeze and thaw the serum.
I fully agree with Dmitry Kazansky as far as C-mediated cytotoxicity assay is concerned. In our experience, there is no significant effect on antibody titres measured by ELISA and other serological tests even after 5 years of serum storage at -80C. Of course, we aliquot the sera samples in one ml or so.
-80c is the recommended temperature for serum storage but the best practice is to aliquot the sera, usually in about one ml. Avoid repeated freeing and thawing . This can help to preserve the polyclonal antibody for long period. As for complement activity I completely agree with Dmitry.
The activity of antibodies depends on the isotype. IgM antibodies stored at -80
lose activity after thawing. Ekaterina
I agree with previous answers, aliquote serum sample and store at -80C, avoid repeated freezing and thawing. From our experience, there is no significant effect on antibody titres and other serological tests measured by ELISA even after few years of serum storage at -80C.
Thank you for your kind answers all. The details of my project as explained below:
-I have raised the antibodies by injecting 3 mices with the solubilised antigen A. After several injections, i sacrificed all 3 mices and obtained 1ml of pooled serum. The fresh serum which contains the polyclonal antibodies were diluted into several different dilutions and were tested immediately by incubating the antigen A cells:serum at 1:1 ratio and incubated at 37oC. Since the exact binding time is unknown so i started with every 5 mins intervals to check on the cell viability and i found that in the first 5mins, the antibodies lysed the antigen A even at the 1:1600 serum concentration . I stored the concentrated serum at -80oC and thawed the next day to repeat the experiment and found that it was not killing the antigen cells anymore even after 2 hours of incubation. I did not do the freeze and thaw several times but once which was one day after the storage. The antibody presence were confirmed ealier through ELISA test.
Yes, stable. But the samples must aviod repeated freezing and thawing.
Dear Sheela Devi, 1) had you negative control to check viability of cells, incubated without antiserum and control with normal (non-immune) serum? 2) The setting described does not allow detecting cytotoxic antibodies - you need to add active complement to see antibodies. 3) Sequence of operations is very important: first, you should dilute antiserum; second, you should add cell suspension beginning from the control wells and the wells containing the highest dilution of antiserum (do not use the same cell suspension repeatedly); third, you should add complement in fixed, preliminary determined dilution, also starting from the control wells and wells with the highest dilution of antiserum. Use new tip for each operation. Hope, this will be helpful.
Have you stored the sera in aliquots and tested the target activity with how many alliquots. Freezing at -80 does not harm the poly clonal antibodies and the potency is retained. Please follow the suggestions of Dmitry Kazansky. good luck. Lalitha kabilan
Antibodies can be stored for years at -80 as long as you keep it in aliquot a and don't freeze thaw or let samples dry out. However the complement in the serum could be losing activity. Are you using a separate source of complement or depending upon what is in the samples? You might test this by adding a n external source of fresh complement.
Antisera can be stored at -80 as long as you keep it in aliquots and don't freeze thaw or let samples dry out. Antibodies of IgM isotype after thawing usually lose their activity.
The antibodies will be stable. -80 °C is for sure below the eutectic point of plasma (about -25 °C). In most cases -30 °C is sufficient. Only storage at the eutectic point will destroy many proteins, especially when during the storage the temperature at the eutectic point is passed many times. At temperatures above this point, you can add some protease inhibitors.
Sure, this is one of the best way for antibody storage, but multiple freeze and thawing should be avoided.
If you are using the antibody in serum to lyse bacteria or cells you need to add fresh complement! Complement does not store well . Antibody does. For fresh complement you can add fresh normal serum. Most labs use rabbit serum. I suggest that you read up on anybody- complement mediated lysis.