Thank you for your reply.
I have been in touch with our colleagues at Mitosciences who have kindly provided the following information:
To answer your question directly, The antibody coating the plate supplied with Kit MTOX1 ab109903 does not cross-react with rat samples (it is human/bovine only). If you would like to test complex I activity in Rat mitochondria, it would be better to purchase kit ab109121.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109121).
However, the drawback of kit ab109121 is that it is not rotenone sensitive as it is only the dehydrogenase activity of the enzyme and does not follow the complete reaction (dehydrogenase-ubiquinol reductase). This is explained in detail in the protocol on the datasheet.
To answer the complete question, we have had a substantial experience testing mitochondrial toxicity due to antiretrovirals. It looks to me that you are treating animals with an antiretroviral compound for HIV? The effect on mitochondria due to antiretroviral therapy varies tremendously depending on the regime used and to my knowledge the most prominent causes of mito toxicity are :
(1) inhibition of chain elongation and/or exonuclease activity of the mtDNA polymerase γ,
(2) inhibition of thymidine kinases,
(3) oxidative stress,
(4) impairment of the adenine nucleotide translocase and
(5) reduction in mitochondrial membrane potential (Δψm).
The easiest and most economical way of looking at such a wide spread of potential effects in mitochondria is by testing cultured cells exposed to the compound with the following sets of kits: ab113849 ATP luminescent detection kit = check the scientific support tab as well as the protocol for further insight into mitochondrial toxicity testing. This can test the mitochondrial bioenergetic state of the cell by comparing treatment in glucose vs. galactose based media.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=113849).
ab113850 JC1 or ab113852 TMRE. Both of these test membrane potential. The protocol also has insights as to the effect of low membrane potential in the overall mitochondrial metabolism.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=113850).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=113852).
ab113851 DCFDA = which measures ROS.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=113851).
ab110217 and ab110216 Mitobiogenesis In-cell ELISA kits (colorimetric or IR) = These will measure the effect of a compound on the protein levels of a mitochondrial DNA encoded protein (typically affected by nucleoside reverse transcriptase inhibitors) in comparison to the protein levels of a nuclear DNA encoded protein (not affected by NRTIs).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=110217).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=110216).
The kits above mention can be used with any mammalian cell. The mitobiogenesis kits can be used with human, rat, mouse or bovine cells. The protocols explain in detail how to set the experiments. If you haven’t tested the compound with the first basic acute tests on cells, I would recommend to do this. It will help to narrow down the potential cause of toxicity or at least help have a viable hypothesis of what could be happening in the animal. If the first four are normal (after acute treatment) I would suggest for to move ahead with the mitobiogenesis kit after treating the cells for at least 5 – 7 passages. If they have a very good reason to suspect (due to the chemical structure of the compound) that the effect will be only on the mtDNA polymerase, then my suggestion is to go directly to the ab110217 to confirm the hypothesis.
Once you have a reasonable hypothesis of the effect of the compound in-vitro, then choosing the test for in-vivo testing will be much easier. i.e if the retroviral has an important effect on ROS production then they may want to follow up with antibodies that target ROS induced post-translational effects such as anti-nitrotyrosine,
Click here (or use the following: https://www.abcam.com/index.html?datasheet=110282).
If galactose sensitizes the cells to compound toxicity as measured by the ATP luminescent assay, then very likely the compound is affecting the enzyme activity/assembly/levels of one of the complexes of the electron transport chain. In this instance, then they will need to determine if it is a direct or indirect effect on the enzymes.
The MTOXC kits (5 kits) will test whether the compound affects directly any of the enzymes using bovine mitochondria (Note that if a compound directly inhibits the transfer of electrons, by in-vitro testing, in bovine mitochondria, it will very likely affect the transfer in of electrons in other mammalian species such as rat). MTOXC will test activity in the presence of compound (while the assay runs) The following MS kits: Complex I Enzyme Activity Microplate Assay Kit ab109721, Complex II Enzyme Activity Microplate Assay Kit ab109908, Complex IV Rodent Enzyme Activity Microplate Assay Kit ab109911 and ATP synthase Enzyme Activity Microplate Assay Kit ab109714, all of which are rat reactive) will test indirect effect of the compound in the activity of enzymes (post-translational modifications, assembly defects) in tissues of treated animals.
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109721).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109908).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109911).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=109714).
These MS kits test activity of tissues or cell extracts after they have been exposed to an experimental condition. For example if you test NRTI compounds with MTOXC, all results will be normal. However if you test with the MS kits tissues from animals treated with NRTIs for a prolonged period of time, you will see decrease in activity. You could use the following antibody cocktails:
ab110413 MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail
Click here (or use the following: https://www.abcam.com/index.html?datasheet=110413).
ab110414 MitoProfile® Membrane Integrity WB Antibody Cocktail
Click here (or use the following: https://www.abcam.com/index.html?datasheet=110414).
If you believe that the structural membrane integrity of the mitochondria (of treated animals) can have an important impact in complexes assembly and protein levels in specific compartments, then these cocktails may be a good choice.
Note that the ATP synthase Enzyme Activity kit ab109714 measures the reverse reactions (ATP hydrolysis) and not ATP synthesis, however the ATP hydrolysis is still oligomycin sensitive. A decrease in oxygen consumption by the oxygraph could be due to effects in any of the complexes and not just ATPase. I.e. rotenone affects oxygraph results, but it is a complex I specific inhibitor. So choosing this early one MS541 kit may lead them into a wild goose chase. You would need to look at mitochondria in a more holistic way and not just as a measure of ATP synthesis.
I hope this information will be helpful to you. if you have any further questions, please do not hesitate to contact us.