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Non specific reactions

Non specific reactions - ELISA Assay Forum

Non specific reactions - 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 03-12-2010, 11:32 AM
Pipette Filler
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Default Non specific reactions



Hi all,
Can someone please brief me about reasons for non specific reaction colour in negative samples, with regard to composition of diluents for the sample and probes? I am detecting human IgG antibodies using mouse anti-human primary probe and a goat anti-mouse HRP conjugate as secondary probe. Can stabilising agents/ serum be added to the diluents to reduce the non specific binding? Or will stabilising proteinous agents lead to saturation of protein in the system and non specific reactions in turn?
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Old 10-19-2010, 09:52 AM
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Default Re: Non specific reactions

hi i think you have to reduce the concentration of the 2ndry antibody maybe it will solve the proplem


i am still new in this techniqe so don't mad if it not worked.
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Old 10-19-2010, 09:13 PM
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Default Re: Non specific reactions

Adjust concentration and time of incubation of secondary antibody. You can also try adjusting the time of incubation with substrate and read within 15 min after you add the stop solution.

Some protocols call for a correction in the OD readings, but I will not talk about it since I don't understand that procedure very well. It suposely makes you have lower absorbance levels correcting for optical imperfections of the plate.
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Old 11-22-2010, 10:07 AM
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Default Re: Non specific reactions

A typical two-site immunoassay involves two antibodies recognizing different epitopes on the antigen to be quantized. One antibody is immobilized on a solid support and the other is conjugated to a detectable label. The analyte present in a specimen links the two antibodies, so that the amount of label bound to the solid support is proportional to the analyte concentration in the specimen. One limitation of this assay configuration is the false increase in results caused by heterophilic antibodies (HAB) in a test specimen. The HAB are human anti-animal antibodies (HAAA), especially those directed against mouse monoclonal antibodies (human anti-mouse antibodies - HAMA), is of particular importance. Thus, a “heterophilic sample“ is a serum or plasma sample which contains antibodies that are able to bind to the animal antibodies used in immunochemistry assays. The HAB binds to both the detection and the capture antibody, simulating the presence of analyte in its absence and resulting in a false positive result or a false elevated result if analyte is also present. The very rare type of heterophile interaction, which leads to a false negative result. The HAB binds to the capture (or the detection) antibody and prevents antigen-antibody interaction. This type of interference occurs in less than 10% of cases of HAB interference. The incidence of such erroneous results was reported to be from 9.12 % to 40 % in normal populations and could reach 74% in patients receiving murine monoclonal antibodies for therapeutic and diagnostic purposes. Anti-animal antibodies may also arise as a result of incidental or occupational exposure to foreign proteins (e.g. veterinarians, farm workers, food preparers) or due to the presence of domestic animals in the home environment. Eliminating the interference from heterophilic antibodies in two-site immunoassays has been achieved by adding normal mouse serum, unrelated murine monoclonal antibodies, polyclonal antibodies from other animal species, and polymerized monoclonal or polyclonal murine antibody to the immunoassay reagents. The use of immobilized and conjugated antibodies from two different animal species and immunoreactive antibody fragment as one of the assay reagents have also been recommended. Similar solutions for rabbit- and goat-antibody-based two-site immunoassays were reported. Many immunoassays use animal proteins such as bovine serum albumin and casein to block reactive sites of the microtiter plate or polystyrene microspheres. These blockers provide another potential source of assay interference, as elevated values, false-positive results, and high background readings may occur as a result of HAAA binding directly to the blocking protein. The HAAA matter, as you can see, it is therefore very sensitive and it is still a long way off for being completely resolved. I have noticed a significant reduction some interference, when antibodies are research with the capture method in which mouse monoclonal antibodies are used, adding in serum and conjugated diluents these ingredients : 10 % (v/v) fetal bovine serum (FBS – Sigma cod. F2442), 5% (v/v) pooled normal mouse serum (Sigma cod. M5905),and 2.5 % (v/v) pooled normal rat serum (Sigma cod. I4131).
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