Serum creatinine assay troubleshooting - (Jan/20/2013 )
I recently used a serum creatinine assay kit from abcam (link below) and obtained some unexpected results. I was wondering if anyone on this forum could share their experience with measurement of serum creatinine that could possibly shed light on what's going wrong:
Serum samples were used that were stored at -80 degrees Celsius, previously aliquoted, hence at the time of assay at most two or three freeze thaw cycles would've been performed.
This assay is based on conversion of creatinine to sarcosine, which is then oxidised and reacts with a probe to produce red colour measured with a spectrophotometer set at 570nm. The reaction is performed using a reaction mix containing all supplied enzymes and then again without creatininase in the reaction mix to account for background. The concentration is determined from the difference between the sample reading and its background.
I ended up with measurements of serum creatinine around 10pg/microlitre whereas the normal range is 45 to 110pg/microlitre. I tested serum samples from three subjects, and all of them had around 10pg/uL. The background reading was quite high for all of the samples, so it's definitely necessary. The serum samples had varying amounts of haemolysis, but the background reading should account for this. If anything, haemolysis should overestimate the concentration... not underestimate it.
I did not dilute the serum samples.
The standard curve was linear and covered 0 to 200pg/uL, with absorbances from 0.0052 (water + reaction mix in the well) to 1.778, which suggests I didn't dilute the standards incorrectly either (given that our spectrophotometer measures from 0.000 to 4.000).
If anyone could help, it would be much appreciated. Thanks.
Competitive ELISA researching free creatinine: dispense into polystyrene microtiter plate (MTP), 125 µl/well of 10 µg/ml of sheep polyclonal IgG against creatinine(N)-BSA), that recognizes free creatinine in biological fluid or tissue extract, diluted in Tris-HCl buffer 10 mM at pH 8.5, and incubate for 2 hrs at 37 °C. Wash the plate 4 times with wash solution (Tris-HCl buffer 0.1 M at pH 7.4 containing 150 mM NaCl and 0.05% Tween 20) and reverse the wells on blotting paper and tap for dry. Prepare standard solutions from concentrate creatinine standard (20 mg/dl) diluting at redoubling for to obtained series of 10, 5, 2.5, 1.25, and 0.625 mg/dl in dH2O useful for determination in biological fluids, while series from 0 to 10 ng/ml dilute in wash solution for tissue extract. For urine’s this are centrifuged at 1500 rpm for 5min, dilute the resulted supernatant 20 times with dH2O and measure the amount of creatinine in remaining diluted supernatant with stabilized methods(*) for compensation. Procedure : dispense in MTP 50 µl of the whole or diluted sample, 50 µl of standard series and add 75 µl of creatinine conjugate to HRP diluted in conjugate diluents (Tris-HCl buffer 0.1 M at pH7.2 containing 0.01% Proclin 300, 10% di FCS) to each wells. Incubate the plate for 1 hr at 25 °C. Wash the plate 6 times over a 10 minute period with wash solution. Add 125 µl of TMB substrate (dissolve 10 mg <0.5 mM> TMB in 4 ml of dH2O and add to 56 ml of potassium acetate buffer 50 mM at pH 4.5. Just before assay, add 6 µl of the peridrole (*) Creatinine Jaffé stabilized modified method : picric acid which forms the coloured complex reacts unspecifically with interfering serum components so-called pseudo-creatinines. This leads to falsely elevated creatinine values in serum and plasma samples especially in the low measuring range. For to compensate these interferences the calibrator value must be traceable with GC-IDMS and the reagent stabilized (Picric Reagent : dissolve 4.58 g/L <20 mM> picric acid in dH2O slightly warm, stable protect from light at 2-25 °C for 1 year; Alkaline Reagent : dissolve 8.0 g/L <0.2 M> NaOH and 12.08 g/L <33.73 mM> Na2HPO4•12H2O in dH2O, stable at 2-25 °C for 1 year; done by me and place on board analyser Targa 3000
(*) Creatinine Jaffé stabilized modified method : picric acid which forms the coloured complex reacts unspecifically with interfering serum components so-called pseudo-creatinines. This leads to falsely elevated creatinine values in serum and plasma samples especially in the low measuring range. For to compensate these interferences the calibrator value must be traceable with GC-IDMS and the reagent stabilized (Picric Reagent : dissolve 4.58 g/L <20 mM> picric acid in dH2O slightly warm, stable protect from light at 2-25 °C for 1 year; Alkaline Reagent : dissolve 8.0 g/L <0.2 M> NaOH and 12.08 g/L <33.73 mM> Na2HPO4•12H2O in dH2O, stable at 2-25 °C for 1 year; done by me and place on board analyser Targa 3000
do you block unreacted sites after binding the polyclonal anti-creatinine?
The use of the conjugate diluent containing highly saturating component (FCS) made to react in coeval way with the sample it makes not necessary the pre-saturation of the eventually free reactive sites on the wall of the wells after the coating of the antibody. In every case if you wanted to proceed at blocking phase this can be realized with 3% of BSA in TBS and to incubate for 3 hrs at room temperature.
if your polyclonal antibody coating solution is saturated with fcs then how do you ensure that your antibody binds to the well.
you should coat with antibody, without additional proteins, then block any free binding sites in the well.
A certain something in the communication doesn't go!. We see together what is written in the protocol by me posted :
a) in a polystyrene microtiter plate at high binding capacity, polyclonal antibody is distributed pre-diluted in coating buffer that, as you can see, doesn't contain it any protein or blocking component for to block free sites;
b) to appraise the efficiency of the coating is needs to perform some experimental titration tests, through which, employing BioHit plates, the average useful concentration of the polyclonal antibody described, results to be around 10 µg/ml. At this time the wall of the well is coated of the polyclonal antibody with presence of free sites;
c) to stop the free sites can be proceeded in two ways: 1) to follow at the coat phase saturation procedure of the free sites (1-3% of BSA in TBS) as showed in my reply or, 2) using a sample diluent that is able to saturate the free sites (FCS) contemporarily to the formation of the immunocomplex. This last alternative has been from me choice for procedural advantage.
Always available for further reply
ah, i see. it wasn't clear in the original post. thank you.