3
$\begingroup$

If the goal is to generate a rapid assay for an enzyme of plant source what are the typical options?

i.e. Could one do something like: Generate an antibody to the enzyme and then use it to create an ELISA? Would animal-injection be the way to generate the specific antibody needed?

If not, what is typically done in such cases. How does one go about creating an assay for a new enzyme of plant source. Are there alternative approaches that avoid the antibody creation?

$\endgroup$
  • $\begingroup$ I think a very important distinction needs to be made here. An ELISA will allow you to detect the presence of a specific enzyme and to quantify the AMOUNT of that enzyme present. Your ELISA assay will be specific for a given protein, which happens to be an enzyme This is not the same as developing an assay for your enzyme based on the reaction it catalyzes. You might develop a spectrophotometeric assay based on the decrease or increase in absorbance of a product or substrate. Consider the difference where the enzyme is 'dead' because you have extracted at too low a pH. $\endgroup$ – user1136 Apr 8 '16 at 14:41
3
$\begingroup$

I want to add something on top of Chris answer.

The production of an antibody it is usually a quite slow (and expensive) process, an alternative that worth to consider is phage display (https://en.wikipedia.org/wiki/Phage_display). Once you find the phage that effectively bind your protein of interest, you can use it instead of an antibody in what is called a Phage-ELISA assay. However, mind that the ELISA (or Phage-ELISA) will give you information about the presence and the concentration of the enzyme, it will not give you any hint about its activity.

$\endgroup$
  • $\begingroup$ Thank you. I did not know about this. Sounds like this could be what I need. $\endgroup$ – curious_cat Apr 8 '16 at 17:43
  • $\begingroup$ Feel free to contact me for more info. I got quite a lot of experience with phage display. $\endgroup$ – alec_djinn Apr 14 '16 at 12:04
  • $\begingroup$ you can send me an email at alec_djinn-at-yahoo.com $\endgroup$ – alec_djinn Apr 14 '16 at 15:31
2
$\begingroup$

I think the most promising routes use antibodies. You could either develop an ELISA or do western blot analysis of plant material - both need a good and specific antibody. To generate these, the protein of interest (or at least parts of it) are injected into animals (typically mice or rabbit) and then antibodies are pourified from the blood of these animals. These antibodies are polyclonal, but this approach is rather fast and can be done in a few weeks.

If you want to use a more sustainable source of antibodies, the antibody producing cells from these animals are isolated, immortalized and characterized as single clones to get monoclonal antibodies.

Since you are using an enzyme, you could also think about activity assays. So either a chromogenic or fluorogenic substrate is metabolized, or you could use coupled reactions where your enzyme uses a substrate which is refilled by another reaction - classical examples here are coupled reactions which use NADH or ATP.

It is also possible to measure the metabolic rate if the substrate or the product of your enzyme shows fluorescence. Then you can either measure the decrease of your substrate or the increase of your product.

$\endgroup$
  • $\begingroup$ Thanks Chris! Can you elaborate more about the chromogenic / flurogenic substrate part? The enzyme I'm interested in converts FPP (Farnesyl Diphosphate) to a specific terpenoid. $\endgroup$ – curious_cat Apr 7 '16 at 6:31
  • 1
    $\begingroup$ In paragraph 2 you talk about the production of monoclonal antibodies, but isn't the process more like that mAb antibodies are antibodies which are derived from a B cell clone, then an animal is injected with the antigen which thus produces plasma cells, those are hybridised with tumour cells (because of endless divisions), and the resulting hybridoma is then synthesising large quantities of specific antibodies, or am I misunderstanding something? $\endgroup$ – Ebbinghaus Apr 7 '16 at 8:09
  • 1
    $\begingroup$ @JordiZambrino I am not sure if I get your question completely. Monoclonal antibodies are made from animals where the animal is vaccinated with the antigen of choice, then the spleen is removed and from it lymphocytes are isolated. These are then grown into single clones and the secreted antibodies are characterized. These are then fused to make hybridoma cells which produce large amounts of antibodies. $\endgroup$ – Chris Apr 7 '16 at 8:17
  • 1
    $\begingroup$ Thank you for the explanation. The characterisation of antibodies could be seen as a structural specialisation, right? Where the composition and sequence of AA are determined? $\endgroup$ – Ebbinghaus Apr 7 '16 at 8:22
  • 2
    $\begingroup$ @JordiZambrino if you have specific questions about antibodies, please ask, as the topic is quite broad. Sequencing is not necessarily a part of monoclonal antibody characterization, unless you are planning on producing it recombinantly. Plenty of mouse monoclonal antibodies out there for research use are grown in ascites where all you need is a healthy hybridoma culture. I used to work for an antibody company producing research reagents, so I have some experience :) $\endgroup$ – MattDMo Apr 7 '16 at 14:06

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.