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High levels in APO-A1 ELISA

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Hi Mabtech,

 

I have a short question about the sample dilution in APO-A1.
We dilute the samples for APO-A1 1:250 000 and the values are still outside the standard curve.
The samples are stored in -20°C. I can’t believe that this will make the measured concentration higher. What do you think about that?
A quick response will help me so much. Thank you for your support!
 
Best regards 
 

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Dear C,

 

Thank you for your question!

 

When plasma/serum samples are stored in -20°C things happens to the lipoproteins in the samples that will affect the apolipoprotein A1 measurements. We have seen this phenomenon several times, and this is why we recommend not to store samples in -20°C (written in the data sheets). I have detected up to 300% (!) higher apoA1 levels in old and badly stored plasma samples. We cannot be totally sure what is really happening in the tube but it is known that apoA1 can form aggregates (look like coin rolls). The avidity of the antibodies to the aggregated apoA1 will be higher, leading to false high levels. We have tried to dissolve the aggregates but we have not managed to do this without affecting the measurements. So I really want to emphasize the importance of not storing samples in -20°C. Even though apoA1 is extreme in this sense, many different types of proteins are affected by handling and storage. This is not due to the methods, it is due to the analytes.

 

So yes, your problem is most likely due to the fact that you have stored the samples in -20°C. To rule out some other possibilities, I have a couple of comments and questions for you. When analyzing apoA1 in plasma you need to do extensive dilutions, I hope you have seen the dilution guidelines at page 10 in the data sheet, the guidelines are also available here: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/apolipoprotein-dilution. It is very important to be precise during the pipetting and to change pipet tips between the steps. To really sort this out it would be great if you can answer the following questions:

 

  1.       How are your standard curves looking?
  2.       How are your blanks looking?
  3.       What kind of samples do you have and for how long do you store them in -20°C?
  4.       How do you wash the plates?

 

Thank you!

 

Lena Beckman

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Dear Lena,

thank you for your quick and detailed answer! Yes we follow the provided guidelines and change pipet tips.

 

1+2)I think our standard curves and blanks looking good, they corresponds very well with how it looks in the data sheets.

 

4)We wash by hand, with a 8-Channel stepper (300µl) 5x

 

3) we have plasma samples, the samples are kept in -20°C around for a year

 

Best regards and thanks a lot

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Dear C,

Thank you for your reply! So you get nice looking standard curves and blanks, and you are following the protocol. This means that the assay is working and you are doing everything right. I think that the high levels you see are due to the storage and handling of the samples. We have done several experiments looking at plasma samples stored in different temperatures for different times. And the conclusion is that apoA1 starts to form these aggregates (leading to false high levels) after a couple of weeks in -20°C and warmer. This will not happen if the samples are fresh, freeze dried or stored in -80°C. The aggregation problem is exaggerated in samples with several freeze-thaw cycles.

 

I would advise you to change the procedure with the samples so that they are stored in -80°C and minimize the number of freeze-thaw cycles.

 

Best regards,

Lena

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Guest Neelika Malavige

Dear C,

 

Thank you for your question!

 

When plasma/serum samples are stored in -20°C things happens to the lipoproteins in the samples that will affect the apolipoprotein A1 measurements. We have seen this phenomenon several times, and this is why we recommend not to store samples in -20°C (written in the data sheets). I have detected up to 300% (!) higher apoA1 levels in old and badly stored plasma samples. We cannot be totally sure what is really happening in the tube but it is known that apoA1 can form aggregates (look like coin rolls). The avidity of the antibodies to the aggregated apoA1 will be higher, leading to false high levels. We have tried to dissolve the aggregates but we have not managed to do this without affecting the measurements. So I really want to emphasize the importance of not storing samples in -20°C. Even though apoA1 is extreme in this sense, many different types of proteins are affected by handling and storage. This is not due to the methods, it is due to the analytes.

 

So yes, your problem is most likely due to the fact that you have stored the samples in -20°C. To rule out some other possibilities, I have a couple of comments and questions for you. When analyzing apoA1 in plasma you need to do extensive dilutions, I hope you have seen the dilution guidelines at page 10 in the data sheet, the guidelines are also available here: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/apolipoprotein-dilution. It is very important to be precise during the pipetting and to change pipet tips between the steps. To really sort this out it would be great if you can answer the following questions:

 

  1.       How are your standard curves looking?
  2.       How are your blanks looking?
  3.       What kind of samples do you have and for how long do you store them in -20°C?
  4.       How do you wash the plates?

 

Thank you!

 

Lena Beckman

Hi,

 

Do you recommend not storing samples at -80 C too? We stored ours at -80 C and got expected results.

 

Neelika

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Guest Neelika Malavige

Dear C,

 

Thank you for your question!

 

When plasma/serum samples are stored in -20°C things happens to the lipoproteins in the samples that will affect the apolipoprotein A1 measurements. We have seen this phenomenon several times, and this is why we recommend not to store samples in -20°C (written in the data sheets). I have detected up to 300% (!) higher apoA1 levels in old and badly stored plasma samples. We cannot be totally sure what is really happening in the tube but it is known that apoA1 can form aggregates (look like coin rolls). The avidity of the antibodies to the aggregated apoA1 will be higher, leading to false high levels. We have tried to dissolve the aggregates but we have not managed to do this without affecting the measurements. So I really want to emphasize the importance of not storing samples in -20°C. Even though apoA1 is extreme in this sense, many different types of proteins are affected by handling and storage. This is not due to the methods, it is due to the analytes.

 

So yes, your problem is most likely due to the fact that you have stored the samples in -20°C. To rule out some other possibilities, I have a couple of comments and questions for you. When analyzing apoA1 in plasma you need to do extensive dilutions, I hope you have seen the dilution guidelines at page 10 in the data sheet, the guidelines are also available here: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/apolipoprotein-dilution. It is very important to be precise during the pipetting and to change pipet tips between the steps. To really sort this out it would be great if you can answer the following questions:

 

  1.       How are your standard curves looking?
  2.       How are your blanks looking?
  3.       What kind of samples do you have and for how long do you store them in -20°C?
  4.       How do you wash the plates?

 

Thank you!

 

Lena Beckman

Hi,

 

Do you recommend not storing samples at -80 C too? We stored ours at -80 C and got expected results.

 

Neelika

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