Human C Reactive Protein ELISA Kit (CRP) (ab99995)

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Overview

  • Product name

    Human C Reactive Protein ELISA Kit (CRP)
    See all C Reactive Protein kits

  • Detection method

    Colorimetric

  • Sample type

    Cell culture supernatant, Serum, Plasma

  • Assay type

    Sandwich (quantitative)

  • Sensitivity

    < 2 pg/ml

  • Range

    34.29 pg/ml - 25000 pg/ml

  • Recovery

    > 100 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 106.2 95% - 116%
    Serum 116.7 106% - 127%
    Plasma 102.2 93% - 112%

  • Assay duration

    Multiple steps standard assay

  • Species reactivity

    Reacts with: Human

  • Product overview

    Abcam’s C Reactive Protein (CRP) Human ELISA (Enzyme- Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human CRP in serum, plasma, cell culture supernatants.


    This assay employs an antibody specific for Human CRP coated on a 96-well plate. Standards and samples are pipetted into the wells and CRP present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human CRP antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of CRP bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    Get results in 90 minutes with Human C Reactive Protein ELISA Kit (ab181416) from our SimpleStep ELISA® range.


     


     

  • Notes

    Optimization may be required with urine samples.

  • Platform

    Microplate

Properties

Associated products

Images

  • Sandwich ELISA- hu CRP (ab99995)
    Sandwich ELISA- hu CRP (ab99995)

    huCRP measured in biological fluids showing quantity (pg) per mL of sample tested.

  • Typical Standard Curve
    Typical Standard Curve

    Representative Standard Curve using ab99995.

References

Publishing research using ab99995? Please let us know so that we can cite the reference in this datasheet.

ab99995 has been referenced in 19 publications.

  • Qu S  et al. MicroRNA-194 reduces inflammatory response and human dermal microvascular endothelial cells permeability through suppression of TGF-ß/SMAD pathway by inhibiting THBS1 in chronic idiopathic urticaria. J Cell Biochem 121:111-124 (2020). PubMed: 31190349
  • Mateos J  et al. Serum proteomics of active tuberculosis patients and contacts reveals unique processes activated during Mycobacterium tuberculosis infection. Sci Rep 10:3844 (2020). PubMed: 32123229
  • Zhong H & Zhong M LINC00311 is overexpressed in ankylosing spondylitis and predict treatment outcomes and recurrence. BMC Musculoskelet Disord 20:278 (2019). PubMed: 31170957
  • Tan Y  et al. Serum YKL-40 positively correlates with MMP-9 and CRP in patients with acute ST segment elevation myocardial infarction following emergency treatment. Medicine (Baltimore) 98:e17950 (2019). PubMed: 31764795
  • Cao S  et al. The upregulation of miR-101 promotes vascular endothelial cell apoptosis and suppresses cell migration in acute coronary syndrome by targeting CDH5. Int J Clin Exp Pathol 12:3320-3328 (2019). PubMed: 31934175
View all Publications for this product

Customer reviews and Q&As

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1-10 of 15 Abreviews or Q&A

Question


What is the shelf life of your assay kits?

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Answer


The shelf life of each of our kits is indicated in the individual protocol booklet.

ab99995 may be stored for up to 6 months at 2 to 8°C from the date of shipment. Standard (recombinant protein) should be stored at -20°C or -80°C (recommended at –80°C) after reconstitution. Opened Microplate Wells or reagents may be store for up to 1 month at 2 to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge. Note: the kit can be used within one year if the whole kit is stored at -20°C. The same is true for ab100610 and ab113326.

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Question

For all those tests that I explained in previous email, I incubate them over night at 4C.
and dilution for HRP was once 300 fold (as described in the manual) and also when I ran the test
in parallel with HRP form albumin kit, I diluted 200 fold, but still no changes.

The data from one of the experiment is attached. this is the one I used the concentrated antibody that I prepared the day before and stored in 4C for further use.

As you see there is no changes between each Standards and also my samples (A,B, and C) didn't show any response, which
is not right because my samples have CRP. (all samples including standards are duplicate as 1,2 )

When I ran this test and I saw no color change on standards (no blue at all) I realized it's probably because of antibody since
I used the concentrated one stored the day before.

In previous tests I didn't used the concentrated stored in the fridge and at least I saw slightly color change for 600pg/ml.

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Answer

The lab sent the following comments and questions. Please let me know if you have the answers to those. I think the lab is trying to understand where exactly the problem might lie.

But I still think the standard curve should look better than what you had obtained the first time. Could you try the standard curve with the new kits you had sent and maybe a few sample dilutions - just to get some preliminary data?


Lab response:
Thank you for the update. In this recent test, none of the absorbances are high enough above background to be considered as real signals. As stated by the customer, this may have been due to using the already reconstituted antibody from the day before. With this being said, the first two tests did show very strong and consistent standard curves. The highest OD reading of ˜1.67 is definitely strong enough for creating a wide enough standard curve to incorporate a range of sample absorbances. Therefore, we do not believe this is a kit or lot related issue. The unfortunate conclusion may be that the amount of CRP in the cell lysate samples is simply too low to be detected by the ELISA kit. I understand the frustration of the customer for not obtaining useful data from the samples and if there truly is high amounts of this protein in the samples, there may be certain sample preparation issues that are contributing to them not being detected.

How were the HepG2 cells prepared?
Did the lysis buffer used contain any denaturing or reducing agents?
How long were the samples stored for before testing?
Were there any freeze-thaw cycles?



Thank you so much for your help and understanding.

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Question

For all those tests that I explained in previous email, I incubate them over night at 4C.
and dilution for HRP was once 300 fold (as described in the manual) and also when I ran the test
in parallel with HRP form albumin kit, I diluted 200 fold, but still no changes.

The data from one of the experiment is attached. this is the one I used the concentrated antibody that I prepared the day before and stored in 4C for further use.

As you see there is no changes between each Standards and also my samples (A,B, and C) didn't show any response, which
is not right because my samples have CRP. (all samples including standards are duplicate as 1,2 )

When I ran this test and I saw no color change on standards (no blue at all) I realized it's probably because of antibody since
I used the concentrated one stored the day before.

In previous tests I didn't used the concentrated stored in the fridge and at least I saw slightly color change for 600pg/ml.

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Answer

Thank you so much for the additional information. I'll pass it on to the lab.

Should they have more suggestions, I will let you know.

I hope the 2 new kits will work as expected.

Please let me know if you have further questions.

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Question

I ran few more tests again based on what your lab recommended.

1- I used another kit with standards and my samples without dilution :
Result: same situation as previous kit for STD and still no detection for my samples.
my samples are high in CRP but still no detection were observed and for STD I got the same range values
as 1.67 for 600 pg/ml and 0.07 for 66.67 pg/ml and lower concentration plus my samples.
but no blue color was observed for concentration less than 200pg/ml.

2- as far as I know if HRP is exposed to the light at room temperature, it turns blue in few minutes.
this is what usually happen with albumin ELISA kit. But I noticed that when I the HRP form this kit doesn't turn blue
even after 10min.
So then I used once HRP from albumin kit (that I had as left over) in parallel with CRP kit.
Result: still no changes in absorption. Everything was the same as before for STDs and samples

Then I did the same way for TMB. I used TMB from albumin kit in parallel with CRP kit, but the result was still
the same, no detection.

3- The most important thing is I ran the test again with higher concentration of antibody (item F).
I used the concentrated antibody that I kept in the fridge the day before and diluted 60-fold instead of 80-fold
But I used the new STDs solution (not the left over kept in the freezer). base on the storage information, the concentrated
antibody can be stored at 4C for 5 days, but I used it in 48hrs.

Result: I didn't get any detection at all. For all STDs and samples, there was no change in color , it didn't turn blue at all, even the high concentration STD that I observed before.
but I still waited for 30min and added the stop solution, and of course nothing.

The absorbance values for all of them including the samples were 0.03.

based on these results I believe the problem is either because of the antibody or the treated plates.
My samples should be high in CRP. The samples are collected media from HepG2 cells treated with cytokines (IL-1b and IL-6)
in high concentration. But I shouldn't get the same value or even the same range for my samples either, since they were
also different in some amino acids.

I wasted 2 kits plus 4 days for nothing. Honestly, I'm not happy at all.
I'm going to graduate in 3 weeks and this is the last test that I need to do and it was such a wasting time and money.

Please either consider a refund for 2 kits (one of them is still unused) or replace them with new and
freshly produced kits. I'm running out of samples too, I cannot waste any more sample.

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Answer

Thank you for your emails.

I apologize that I could not get back to you earlier, but our office was closed over the holidays.

I am very sorry to hear that your effort and attempts to try to get the kits to work were unsuccessful. I will pass your comments on to the lab for their consideration.

In the meantime, I have arranged for a free of charge replacement for the 2 kits with another, newer lot number. The order number is xxx, and you should receive the 2 kits tomorrow.

I really hope the new kits will work as expected and they will be helpful for finishing your research before graduation

Please also do let me know if you have more questions and how the new kits are working for you.

Best of luck to you!

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Question

I confirm our PO 2527 of 11/09/2012.
So please send us new vial of RP-streptavidin concentrate item.

Thanks in advance and kind regards

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. It has not been possible to obtain the single component of the kit from our source in this case, I have issued a free of charge replacement of the kit with the order number 1208938.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

When the new kit is used, it would be beneficial to briefly centrifuge the HRP-streptavidin vial before opening to ensure maximum recovery (Sometimes the liquid can adhere on the sides and cap of the vial).

Since excess volume of the HRP-streptavidin is provided, please inform the customer that 40ul of the HRP-streptavidin is still more than enough to complete the entire microplate. 40ul X 300 = 12ml of 1X working HRP-streptavidin solution. (Only 9.6ml is needed.) Please encourage the customer to use their current HRP-streptavidin vial and also remind them to centrifuge the vial before opening to ensure maximum recovery (if not doing so already).

Thank you for your help and cooperation in this case. Please let me know if you have any further questions.

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Question

I was just wondering if there is any progress in your investigation or not yet.
I appreciate if you please let me know as soon as you can, since I need to test my samples
as soon as it's possible.

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Answer

Thank you for your patience.
I have heard back from the lab today with the following response:

"I am not quite sure what exactly the issue is with the standard curve. I plotted the values in a linear regression model and curve looks fine, see attachment. The highest standard signal strength is strong at ˜1.6 OD, the linearity is high (R2 >0.98), the background is low (0.08 OD), and the duplicate CV is low. Overall I’d say the standard curve is acceptable.

The customer is missing the 22.22 pg/ml and 7.407 pg/ml standard points though so I would certainly recommend the customer loads these. I’m also a little confused about the ODs for the 6.66 pg/ml and 2.469 pg/ml – were the OD values measured exactly the same value? This seems unlikely.
The issue to me seems not to be the standard curve but that the sample ODs are very low (0.8 OD) which is below detection, near background. There are a few different options the customer could try in order to get their sample signals within the detectable range of the assay.
Performing the sample/standard incubation overnight at 4ºC with gently rocking.
Increase amount of biotinylated detection antibody (by 1.5 fold or so – too much may increase background)
Increase amount of HRP-streptavidin (by about 1.5 fold or so – too much may increase background)
Concentrate the sample in a non-denaturing method (for example, using a chilled spin column)

Please note: it’s best to try just one of these modifications at a time. Implementing too many of these changes at once may cause high background."



I replied to the lab regarding the standard curve, but please let me know if these tips will be of help and improve your results.

You might not need to dilute your samples by a factor of 10. Maybe try undiluted samples and a dilution by factor of 2 and 4.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

antibody vials seem to be empty
lot GR91493-2 and lot GR95066-1

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Answer

Thank you for your phone message.

I have emailed the lab yesterday but have not yet heard back. I will follow up with them today.

As for the antibody vial:

Item F detection antibody comes as a very concentrated, small volume liquid. Usually the volume is very small, ˜2ul, so it may be difficult to see. This is why we recommend customers spin down the vial before use to ensure no liquid has adhered to the inside cap or sides of the vial. The small volume is also the reason the biotinylated antibody solution is prepared inside the vial and why the customer should not try to pipette the 1-2ul out.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Dear Colleagues,
a customer of ours bought your AB99995 lot GR91493-2 (our PO 2527), but she found less HRP-streptavidin concentrate (Item G) than expected.
In fact she found only 40ul instead of 200ul (300X).
Can you please send us a new vial of HRP-streptavidin concentrate (Item G)?

Thanks in advance and kind regards

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Answer

Thank you for your message. I am sorry to hear there was a shortage of the HRP-streptavidin concentrate item in the ab99995 kit.

If this has been purchased in the last 6 months, I will be pleased to provide a replacement of this component. In order for me to arrange this, please confirm the order number and date of purchase.

Thank you for your cooperation. I look forward to hearing from you.

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Question

a customer of ours has purchased your kit ab99995 (lot GR79069-3) but after dilution of buffer B, this buffer appears to be "opalescent"!.
Is this normal? Does the customer can use it? Will the kit work?

Please note that the customer works in a pharmaceutical industry so we would exclude a problet with the distilled water used to dilute the buffer.

Kind regards

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Answer

Thank you for contacting us.

This is very normal, the buffer contains proteins which gets precipitated at low temperature. Please keep the buffer at room temperature for few minutes and then vortex it. The white opaque residue will go away.

The kit is absolutely fine so please use it without any hesitation.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

The two most recent CRP ELISA kits we ordered gave no signal at all with urine samples or the standards in the kit. An earlier kit from a different lot worked as expected.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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1-10 of 15 Abreviews or Q&A

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