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Questions on blood pretreatment and improvement of assay time

Questions on blood pretreatment and improvement of assay time - ELISA Assay Forum

Questions on blood pretreatment and improvement of assay time - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 04-11-2010, 09:44 PM
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Default Questions on blood pretreatment and improvement of assay time



Hi, I am currently studying about ELISA but I have never done one so I would like to ask some questions regarding the testing technique.

1. Are we required any sample pretreatment for blood (ex. removal of red blood cells) before doing ELISA testing? If not, will the red color of the blood hinder the optical analysis of the results (by means of bare eye observation and absorbance measurement for HRP-TMB system)?

2. What are the promising means for improving assay time and sensitivity for the ELISA testing?


Any kind response is appreciated.
Thank you.
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Old 04-22-2010, 11:41 PM
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Default Re: Questions on blood pretreatment and improvement of assay time

You either collect blood as serum/plasma--then use centrifugation (at max ~10min) dilute into buffer or sample diluent. Then proceed with ELISA.

Color from the serum/plasma will end up being washed away long before the detection phase.
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Old 10-28-2010, 03:13 PM
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Default Re: Questions on blood pretreatment and improvement of assay time

Enzymatic methods employed for substrates or enzymes determination in biological fluids suffer of the interference by high levels of hemoglobin, lipids, and bilirubin. In some immunometric direct or indirect methods these interferences can be evident, especially in hemolyzed specimen, so much that asks for a new collection. Nevertheless, in particular situations, this it’s impossible to realize for to occur death of the patient or for great distance from the laboratory. This last event is often verified in Africa countries in which the "clinic mobile", using an modified van, such as “collection unity ”, “vaccination unity” and “medical-surgery unity” for treat directly patients in the villages, while blood collection must be delivered at hospital analysis laboratory. Generally 30 – 40 % of the specimens show hemolysis signs from light-moderate up to marked, derived by not correct collection procedures or traumatism during the transport. Processing of these samples inevitably involves an low accurate of the results. To eliminate such interference, specific precipitation of hemoglobin from solution has been achieved by the use of certain organic solvents, for example alcohols such as ethanol and/or butanol, ketones such as acetone, ethers, e.g. cyclic ethers such as dioxane and tetrahydrofuran, amide solvents such as dimethylformamide or diethylformamide, sulphoxide solvents such as dimethylsulphoxide, hydrocarbon solvents such as toluene, and halogenated hydrocarbon solvents such as chloroform. In past experience, whole blood sample in EDTA-K3, diluted to give approximately 6.5 mg/L hemoglobin and 1.5 mg/L human serum albumin, then to add butanol:ethanol (50:50 v/v) mixture to a final concentration of 9 % (v/v) butanol (0.18 ml mixture + 0.82 ml diluted blood ), 94 % of the hemoglobin was precipitated and only 1 % of the HSA. Similar precipitation of hemoglobin may also be achieved using metallic cations binding to and aggregating proteins. Suitable cations include zinc, copper, and less preferably nickel, cobalt and cadmium. This has the important advantage that any hemoglobin precipitated in this way may easily be re-dissolved by adding a solubilising complexing agent. In our and other experiences using zinc ions, guise of chloridric zinc, with which a substantially specific precipitation of hemoglobin in hemolysates can be obtained. When used a zinc ion concentration of 3.2 mM, don't greater than 3.6 mM, in the same conditions of Hb/HSA a 88 % of hemoglobin and 10 % HSA were precipitate. In both methods, organic solvents/metallic cations, precipitate can be totally or partially separated by means of centrifugation or filtration.
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