Nicole Gillain-Martin

Head of department and professor retired; educational activities only now

Questions and Answers

  • Nicole Gillain-Martin added an answer in IgG:
    In ELISA IgG standards curve, why are my OD giving me a non linear increase but when i include concentration I don't get the sigmoid shape?

    I have done dilution series of the total IgG standards 12 times, when I check the od readings from different dilution series (1:2; 1:3; 1:10) they give a sigma shape graph, but when I align with the concentration (500ng) going down, it does not align well hence i loose the shape.

    The two graphs are attached.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Sorry for the late reply. You have acceptable calibration lines in some areas of concentrations (in yellow): Do not work with too high or too low concentrations
    The answer is the same in dilution and in absorbance (see example from your data)
    I find that variations in DO (or absorbance) is low.

  • What importance do you attribute to the type of protein responsible for proteinuria?

    The intensity of proteinuria is an important element to define the degree of chronic renal failure. As a clinician, what importance do you attribute to the type of protein (low/high molecular weight) detected?

  • Nicole Gillain-Martin added an answer in IgG:
    In ELISA IgG standards curve, why are my OD giving me a non linear increase but when i include concentration I don't get the sigmoid shape?

    I have done dilution series of the total IgG standards 12 times, when I check the od readings from different dilution series (1:2; 1:3; 1:10) they give a sigma shape graph, but when I align with the concentration (500ng) going down, it does not align well hence i loose the shape.

    The two graphs are attached.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I do not understand: the ordinate should be the same in the two graphs: the absorbance (as in 2)
    on 2; how concentration (abscissa) can it be zero?

    send me your numbers

  • Which 'one' biomarker should I consider to detect nephrotoxicity induced by Herbicides in general?

    KIM-1, NGAL, NAG, alpha-1 microglobulin, cystatin-c or vitamin-D binding protein.

    Of above all, which 'one' biomarker should I consider to indicate an overall kidney damage in the patients exposed to higher levels of herbicides.

    364      
    Nicole Gillain-Martin · CHR Citadelle Hospital

    Thank you to Carl Wesolowski for this information about  this new approach to the determination of GFR (unknown to me)
    Isotopic techniques are unfortunately not always accessible

  • Which 'one' biomarker should I consider to detect nephrotoxicity induced by Herbicides in general?

    KIM-1, NGAL, NAG, alpha-1 microglobulin, cystatin-c or vitamin-D binding protein.

    Of above all, which 'one' biomarker should I consider to indicate an overall kidney damage in the patients exposed to higher levels of herbicides.

    364      
    Nicole Gillain-Martin · CHR Citadelle Hospital

    NAG has the advantage of being easily measurable by colorimetry and easily automated.

    Consider also the low MW protein assay as alpha1microglobuline. It increases in urine soon after aggression and is also easy to measure. You can also assaying beta2microglobuline, but it is less sensitive. Urine cystatin is not a sensitive marker. 

    About NGAL, this marker is used primarily in the context of AKI, no personal opinion on the utility to detect a toxic effect in the short or long term
     

  • Nicole Gillain-Martin added an answer in Biomarkers:
    Which biomarkers could need a better assay?

    Hi everyone!

    We have found that a mass spectrometry based technology we are working on can do really fast measurements of targeted proteins.

    We would like to demonstrate the technology with established biomarkers that have known troubles with conventional assays (e.g. poor selectivity with exiting ELISA approaches etc).

    But being the dumb chromatography person that I am, I am not too familiar with which markers that are notoriously in need of better assays.

    Therefore: Do you have any suggestions for cool biomarkers to test a proof of concept for a novel MS tech?

    I wish you all a great weekend!

    Nicole Gillain-Martin · CHR Citadelle Hospital

    There are many difficulties with the assay of sclerostin with current kits

  • What is the best method for collection of 24 hours urine samples for evaluation of promoters and inhibitors of urinary stone formation?

    medical management of urolithiasis in recurrent stone formation requires estimation of promoters and inhibitors of stone formation in 24 hours urinary and blood evaluation. Can any one give us the best method in collection of 24 hours urinary sample.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    You are asking a question that I do not think anyone really resolved. Indeed, the suggested additive varies depending on the constituent analyzed. The urine stored at room temperature becomes alkaline which may alter the dosage of phosphate and calcium. For the determination of oxalic acid also it is advisable to acidify the urine. But this acidification  can be harmful as it will promote the precipitation of uric acid. In addition, acidification may alter some assays performed with enzymes. In practice, one does not use an additive. The urine should ideally be stored in the refrigerator or in a cool place during the 24H of the collection which is not always possible for the patient. So, if possible, the patient after placing the urine of the night in the container shall be to the laboratory as soon as possible so that the urine may be analyzed the same day. Do not exceed 24 hours. Say to the patient to bring all the bottles he had collected. This is the lab that must collect and homogenize them!

  • What is the best method for collection of 24 hours urine samples for evaluation of promoters and inhibitors of urinary stone formation?

    medical management of urolithiasis in recurrent stone formation requires estimation of promoters and inhibitors of stone formation in 24 hours urinary and blood evaluation. Can any one give us the best method in collection of 24 hours urinary sample.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    It is important to give the patient a suitable container to collect the urine of 24 hours, so a container that can hold at least 2 liters (diuresis may be more important in some patients) and if possible in which he can urinate directly. The patient should start collecting the morning at a definite time. The simplest is when patient rises. He urine as usual in the toilet, but from that moment, he must retain all its urines, absolutely all. If the patient urine once in the toilet, he will have to start all over again! He collects his urine  in the container before going to bed and get up at the same time as the previous day. He collects his urine into the container. He has a complete collection of 24H urine: day and night.
     

  • What is the normal blood concentration of Erythropoietin (Epo)?

    What is the normal blood concentration of Erythropoietin (Epo)? What is the minimum level reported in medical conditions like Chronic Renal Failure etc.? What is the maximum reported concentration in conditions like Polycythemia etc.?

    Kindly provide references.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    The values observed in 170 healthy adults with normal hematocrit are: 3.3 to 23.4 mU/ml
    (from WE and WL Owen Roberts, Clinica Chimica Acta, 2011, 412: 480-482).

  • Nicole Gillain-Martin added an answer in EGFR:
    Is nutrition important in clinical treatment of severe reduction of eGFR?

    when  an  eGFR is 15 - 29 ml/min/1.73 mwhich part of treatment, either clinical action alone or Nutrition part- is really works to get back to 30-59 ml/min/1.73 m2 or the actions for preceding stages? or increase the ca absorption, lipoprotein activity or develop the metabolic consequences?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    That's right, in current practice, nephrologists will not advise a restricted diet intake of proteins; according to them, the patients naturally eat less meat (natural disgust). This avoids denutrition. A restriction is only recommended if the patient has clearly a diet with excess proteins. The guidelines I gave you dated a few years, they are no longer applicable. Sorry for the misinformation.

  • Nicole Gillain-Martin added an answer in Buffer:
    Why is there no current during my electrophoresis experiment?

    During western blotting, there is no current flow (mA and Watt) during electrophoresis. i have checked the connection, ph buffer, make new buffer (1X, 2X and 5X), but still no flow of the current..can anybody help me with this kind of situation...TQ in advance.

    Really need help here.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I guess you place your gel on a plate to which are connected the electrodes. This plate should be wet before placing the gel on the plate (the manufacturer may recommend a product that promotes the flow of current)

  • Nicole Gillain-Martin added an answer in EGFR:
    Is nutrition important in clinical treatment of severe reduction of eGFR?

    when  an  eGFR is 15 - 29 ml/min/1.73 mwhich part of treatment, either clinical action alone or Nutrition part- is really works to get back to 30-59 ml/min/1.73 m2 or the actions for preceding stages? or increase the ca absorption, lipoprotein activity or develop the metabolic consequences?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Yes, nutrition is very important in renal failure, especially protein intake. Following P. Jungers (Necker Hospital, Paris), it must be of the order of 0.7 g / kg body weight / day if creatinine clearance is <20 ml/min/1.73m2,  of 0.8 if cl.creat. between 20 and 40, 1, if cl.creat is  40- 60 and < or = 1.2,  if cl. creat is > 60 ml/min/1.73m2. Not reduce protein intake too much especially in the elderly and ensure that abumine does not drop below 35 g / L. A supplement of vitamins, Ca, the control of the amount of water and ingested Na should be considered depending on the circumstances and the type of kidney disease. 

    For a recent development, see the clinical pratice guideline for evaluation and menagement of chronic kidney disease (KDIGO 2012)

    www.kidney-international.org

     

  • Nicole Gillain-Martin added an answer in Plating:
    How can I increase reads (OD values) on ELISA tests?

    I am using an ELISA kit to detect BAFF on human serum. The kit has a standard curve that goes from 0.31ng/ml to 20ng/ml. In the literature, I found no article that used the exact same kit, but found that, with the other kits used, the concentration observed in normal "healthy" control individuals is around 1ng/ml (mean of 1.35, median of 0.93 and goes from 0.2 to 4.8 in different articles). No one reported individuals "negative" for the molecule. However, in my tests I am getting very low reads for the samples, most below the lowest concentration point of the curve (0.31ng/ml). Also, the OD values for the lower part of my curve are also low. I have the OD values obtained for the QC done by the company for the kit lot I'm using, and my curves give OD values that are higher for the most concentrated points (should be 1.6, I get 2) and lower for the least concentrated points (should be 0.15, I get 0.09).
    I already tried adding more serum (no dilution, the kit says to dilute 1:3), or washing less times (the kit says to wash 6 times, I tried 4 and 5), but had no satisfactory results.
    One possible critical point is that we do the washes manually. What I do is to use a multichannel pipette to fill the wells (I pipette 190ul twice on each well), then I use the same pipette to take off half the volume and then I turn the plate upside down to get rid of the rest of the volume (using a paper towel to dry the plate before turning it up again). I tried once not to aspirate half the volume, and directly turn the plate upside down, but the result was terrible. Maybe I am using too much or too little strength when turning the plate. Or maybe I am taking too long to pipette twice and then aspirate half the volume before turning the plate (the kit protocol says we should wait about 10-15 seconds before emptying the wells).
    What do you think is the issue here? Can it be solved?

    Just in case someone wants to know, my serum samples are stored at -80°C and the kit is this: http://www.ebioscience.com/human-baff-instant-elisa-kit.htm
    I attached the results for my plates, done with no dilution of the serum and 6 washes.

    Thanks in advance for the help!

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Determine the value of the controls of the company  (or of another company), and the value of samples of known concentration seems essential to ensure that differences in the calibration curve has really an impact on the results.
    You must use controls that have a value close to the standard values,  low and high.

  • What is your experience in the management of a patient with steroid resistant nephrotic syndrome with stage two CKD?
    We have managed 2 cases. Both of them were diagnosed with SSNS at two years, responded to prednisolone, subsequently became steroid resistant, managed with methylprednisolone and cyclosporine. After stopping cyclosporine a recent relapse shows serum creatinine levels of 3 mg/dl. What should we do?
    Nicole Gillain-Martin · CHR Citadelle Hospital

    Have you studied autoimmunity of  your patients?
    the presence of anti-ANCA antibody, anti-PLA2R, anti-DNA ...?

  • Storing serum for a long term?

    What is the ideal temperature for storing serum and lyophilized serum for a long term?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    One can also use freezers at -70 ° C.
    Do not forget to fill as completely as possible the container in which you keep your samples with serum.

  • What fraction of proteins within a cell are of low molecular weight (<15kDa)?

    Any cell type would suffice, just looking for a general reference.  

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Cystatine C has a MW of 13kDa and is present in all nucleated cells

  • Nicole Gillain-Martin added an answer in FACS:
    What is better method to measure the amount/level of antibodies - ELISA or FACS?

    I am working with Indirect ELISA to measure the level of  IgG and IgM in CSF of MS patients. I have 2 dilutions in triplicates.

    Regards,

    Pooja.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Do you have an ELISA kit for the detection of these antibodies or will you have to prepare yourself your test from commercial antigens?
    If you work with  microplates you can decrease the volume of CSF to use and carry a big number of analyzes what seems to be your case

  • Nicole Gillain-Martin added an answer in Lysosome:
    Are there any biochemical kits available for lysosomal enzyme estimation in rat serum samples?

    Kindly get me a supplier who can deal with biochemical kits - lysosomal enzymes.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    previously, i assayed lysozyme in human serum using a kit based on a radial immunodiffusion and sold by Siemens without problem.
    The question is whether this test is still marketed and is the immune serum able to recognize  the rat lysozyme

  • Nicole Gillain-Martin added an answer in FACS:
    What is better method to measure the amount/level of antibodies - ELISA or FACS?

    I am working with Indirect ELISA to measure the level of  IgG and IgM in CSF of MS patients. I have 2 dilutions in triplicates.

    Regards,

    Pooja.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Want you to determine total IgG and IgM? then a nephelometric assay is sufficient (BNII  of Siemens for exemple) or do you want determine IgG and IgM specific for a given virus or bacteria? than ELISA is interesting; see Enzygnost  of Siemens

  • Can a hemagglutination inhibition (HIA) assay be correlated to IgG concentrations for long term immunity?

    I'm looking for some general information on correlates between these two diagnostic tests, as well as, that which pertains specifically to canine parvovirus. I have HIA titres but how does that relate to long term immunity? Thanks in advance

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Perhaps these articles will help you to solve your very specific problem.
    Remember that a hemagglutination inhibition test informs you about the existence of exposure to the virus but does not give you any indication of how recently or active is this infection  
     

  • Can a hemagglutination inhibition (HIA) assay be correlated to IgG concentrations for long term immunity?

    I'm looking for some general information on correlates between these two diagnostic tests, as well as, that which pertains specifically to canine parvovirus. I have HIA titres but how does that relate to long term immunity? Thanks in advance

    Nicole Gillain-Martin · CHR Citadelle Hospital

    hemagglutination inhibition has long be used for the determination of acquired immunity following infection by measles virus or rubella virus

    For parvovirus, I do not know because now, ELISA kits detecting specifically either IgG or IgM antibodies are used. In principle, there must be a relative correspondence between tests
     
     
     
     

  • How can I quantify immunoglobulins by electrophoresis?

    Need to quantify Ig in a serum sample.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    As T Kiessig, I would say that you can estimate the total gamma globulin by electrophoresis and this is a common analysis, performed daily.
    If you want determine concentration of a particular immunoglobulin that is impossible with conventional electrophoresis method unless it is a monoclonal immunogobuline. You can estimate it  by measuring the peak area. To do this it must be in sufficient quantity or isolated from other immunoglobulins, otherwise it is impossible

  • Nicole Gillain-Martin added an answer in Pyruvates:
    Is anyone familiar with photometric pyruvate kinase assay?

    I am trying to determine pyruvate kinase activity from crucian carp (Carassius carassius) muscle and liver. I am working on a photometric assay, where the formation of pyruvate and ATP from PEP and ADP by pyruvate kinase is coupled with the formation of lactate and NAD+ from pyruvate and NADH by LDH. The concentration of NADH is measured photometrically at 340 nm. As NADH is oxidized the absorption should decrease. However, in most of my tests, it has increased. I have checked all my reagents and have no idea what is causing this and how to fix it. Has anyone used this assay (and had similar problems)?

    The incubation has been done at 25˚C, and the tissue samples have been homogenized in 20X volume of triethanolamine buffer (0,1M, pH=7,6). The concentrations in the test solution are the following:

    • Triethanolamin 85,6mM
    • PEP 0,54mM
    • MgSO4 2,5mM
    • KCl 10mM
    • ADP (neutralized with KOH) 4,7mM
    • NADH 0,2mM
    Nicole Gillain-Martin · CHR Citadelle Hospital

    hello Joonas,

    I have not used your technique, but I had a similar problem with a similar reaction using both NADH / NAD: absorbance increased instead of decreasing and only on some samples! I realized that slight turbidity appeared during the reaction. I was forced to abandon the technique.
    The problem may be a molecule of the tissue extract you analyze
    Why not use a commercial kit? MAK072 Sigma Aldrich. These products are generally very reliable

  • Does somebody have an opinion about the value of specific IgG 4 during desensitization?

    Some pulmonologists want to follow IgG4 concentration of the allergen during desensitization because IgG4 must increase for good result. This test is often contested.

107 Following View all

43 Followers View all