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Technical questions for ApoB ELISA Pro kit

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Dear Sir/Madam:

 

I am interested in using your ApoB ELISA Pro kit to quantify ApoB in cell culture supernatant.  I have a few technical questions to ask.  My colleague ordered the development kit, but I also noticed you sell one that is fully assembled (pro kit).  Both protocols say it can be used for supernatents, but it seems the directions are geared towards plasma/serum samples so I was wondering if technical support can offer some input.

 

  1. For supernatants, which detergent is best?  I saw that the protocol suggests Tween-20, Triton X-100, or NP40
  2. Do you have any suggestions on how long to incubate after adding TMB substrate solution?
  3. The pro kit suggests using “Apo ELISA Buffer” to dilute all standards, samples, and primary antibodies, and the SA-HRP comes with its own diluent. However, the development kit says to use incubation buffer (0.05% tween-20 + 0.1% BSA in pH7.4) instead.  Is one buffer better than the other for supernatents?

 

I am trying to decide if I should use the pro kit or development kit for our supernatents.

 

Best regards,

S

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Dear S,

 

Thank you for your questions!

 

Yes you have absolutely right, we have two different type of ELISA kits: the development kit and ELISAPRO kit. The development kit includes the antibody-pair, standard and the SA-conjugate (either ALP or HRP) and is best suited for researchers that are used to coat ELISA plates, and wish to adjust and customize their own ELISA. The ELISAPRO kit, on the other hand, is a complete kit with pre-coated plates, Apo ELISA buffer, standard, detection antibody, SA-HRP, HRP-diluent, wash buffer, substrate and stop solution. The ELISAPRO kit is a more convenient and straightforward kit which saves time and reduces both inter- and intra-assay variation. Read more about the two different formats on the following link: https://www.mabtech.com/knowledge-center/product-guide/elisa-kits

 

The detailed dilution guidelines for human serum/plasma samples are based on serum samples from fasting healthy subjects. The levels of apoB in cell line supernatants are influenced by several parameters such as type of cell line, cell confluency etc. why it is harder to give detailed dilution guidelines for such samples. The reason why we give detailed descriptions on how to handle human serum/plasma samples is also due to two other important factors:

1) To prevent interference by different LDL-particle sizes, all serum/plasma derived samples need to be diluted 2x in Triton X-100, followed by vortex for 5 seconds (this procedure is specific for when analyzing apoB). This is not necessary for cell line supernatants.  

2) Serum /plasma samples may contain heterophilic antibodies that can, by cross-linking the assay antibodies used, result in false positive signals. The Apo ELISA buffer prevents the heterophilic antibodies for cross-linking. Heterophilic antibodies are not present in cell line supernatants.

 

To answer your specific questions:

 

1)      It is important to use a detergent in the buffers, I cannot say that one detergent is the best, in the development kit we leave this decision to the researcher. But as written in the protocol it is 0.05% Tween-20 in the “incubation buffer”. The “incubation buffer” can be used to dilute cell supernatants, standard, detection antibody and SA-HRP.

2)      I incubate for approximately 15 min.

3)      Due to the factors written above we always recommend to dilute serum/plasma samples in Apo ELISA buffer. When diluting cell supernatants it is also possible to use “incubation buffer”. However,  it is important to use Tween-20 and BSA of high quality, and all our buffers are tested, validated and quality controlled.

 

It is absolutely possible to use the development kit for assaying your cell supernatants, and to dilute the samples using an “incubation buffer”. But if you want to spare time (the time with preparing the buffers and coating the plates) and reduce inter- and intra-assay variability, I would recommend you to use the ELISAPRO kit.

 

This is a link to our apoB ELISAPRO kits: http://www.mabtech.com/products?field_category_tid=66&field_analyte_tid[]=71&field_kit_plate_format_tid[]=1345&items_per_page=20&page=0

I really hope this answer can help you!

 

Please let me know if I can be of any further assistance!

 

Best regards,

Lena Beckman

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