Rat IL-4 ELISA Kit (ab100770)

The lab adviced the following:

While serum and plasma samples maybe run undiluted, we do not usually recommend doing so. The reason is not because Assay Diluent A needs to be present in every sample but that testing undiluted serum/plasma samples increases the risk of experiencing matrix effects.


Matrix effects occur when some component of the sample affects immunoreactivity of the target molecule, possibly by sequestering binding sites. Auto-antibodies, binding proteins, albumin, etc. may be possible culprits, but it’s hard to say for sure. The result is that the sample may read falsely high or low in the ELISA test, or may give a non-linear dilution response. Sometimes certain disease states contribute to matrix effects by altering the properties of the blood (highly lipemic samples, for instance); hemolysis may also cause similar problems. Matrix effects can be hard to predict and challenging to overcome.


For this reason, we rarely recommend running plasma or serum samples undiluted, as matrix effects are more likely to occur. The lowest dilution factor we normally recommend starting with is 2-fold, even for the low-abundance cytokines. If the customer is concerned that their sample levels will be below detection, keep in mind the optional overnight standard/sample incubation can be performed to try and maximize the absorbances. If that doesn’t work, the customer may want to consider concentrating their samples or switching sample types (for example plasma) to see if that helps.

Thank you for contacting us.

Yes, Assay Diluent A contains sodium azide which inhibits HRP activity. Therefore in this case 1X Assay Diluent B should always be used to prepare the HRP-Streptavidin and detection antibody regardless of the sample type being tested.

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

I have the following information for you regarding the kits we discussed:


ab46073:
We have no data regarding normal values observed with IL-6 and IL-4 in rat but as free circulating cytokines are rather dangerous for living animal, the normal level is probably below the detection level.


ab119548:
Unfortunately, we have not tested the rat IL-6 ELISA on normal level in rat samples. We have only tested this ELISA using samples spiked with recombinant protein.


ab100770 and ab100772:
1) What is the normal amount of rat IL4 in serum?
We tested the normal amount of IL-4 in healthy rat serum/plasma samples to be undetectable – 2 pg/ml.

2) What is the normal amount of rat IL6 in serum?
We tested the normal amount of IL-6 in healthy rat serum/plasma samples to be below the detectable limits of this ELISA. Thus, this kit may be more suitable for diseased/treated samples; however, there are some attached tips for increasing sensitivity the customer may use - see below.

3) What is the recommended dilution range for rat serum for IL4 and IL6?
We recommend a 2-fold dilution for serum/plasma samples for use with our IL-4 and IL-6 Rat ELISA kits.


Some advice for what to do if samples are reading below detection limit and how the sensitivity of the kit can be increased:


Incubate sample overnight at 4 degrees C
Increase amount of biotinylated ab (by 1.5 fold or so – too much may increase background)
Incubate biotinylated ab overnight at 4 degrees C
Increase amount of HRP-strep (by about 1.5 fold or so – too much may increase background)
Concentrate sample (using a 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.