Naturally Occurring Anti-albumin Antibodies Are Responsible for False Positivity in Diagnosis of Autoimmune Premature Ovarian Failure

Abstract

Autoimmunity is a well-established causative factor of premature ovarian failure (POF), and evidence for the same has been well reported in the literature. Detection of specific autoantibodies remains the most practical clinical research marker of any autoimmune disease. Variation in efficiency and specificity in the detection of ovarian autoantibodies has been reported. However, the frequency of false positivity and a solution to overcome this has not yet been reported. Herein, we report autoantibody to albumin as the likely responsible agent for false positivity. Our data indicate that presence of naturally existing autoalbumin antibodies in the circulation of normal women is responsible for the false signal seen in SDS-PAGE Western blot analysis and in immunohistochemistry (IHC). Having shown the presence of anti-albumin antibody in normal women as well as in the sera of POF patients, we have developed a novel blocking agent to overcome this problem. A high titer polyclonal antibody against human serum albumin was generated. This antibody showed immunoreactivity to albumin obtained from various sources. Preincubation of Western blots and IHC sections with this antibody drastically reduced background signals. The advantage of using this blocking was evident by identification of specific anti-ovarian antibodies in a group of POF patients. This blocking procedure made it possible to obtain a clear indication of the ovarian antibody status in women presenting with autoimmune POF.

Can anti-ovarian antibody testing be useful in an IVF-ET clinic?

“…This indicates that not all women respond uniformly to the treatment. The 66 kDa reactivity is the reaction of naturally occurring abundant auto-antibodies to albumin was commonly seen in all patients and controls (lane C) and as described earlier [13]. A 'no primary antibody control' showed no immunoreactivity (lane NC).…”

“…Although various investigators have demonstrated that there is multiplicity of molecular and cellular targets in human ovarian autoimmunity, there is still a need to identify all specific ovarian targets [29,30]. However, from our earlier findings we have investigated the cause of the non-specificity and were able to demonstrate true molecular and histochemical targets [5,13]. Once the patient tests positive for AOA by Western blot analysis, the serum is screened by us to identity the cell types which are targeted, using IHC which employs a novel blocking recipe [13].…”

“…However, from our earlier findings we have investigated the cause of the non-specificity and were able to demonstrate true molecular and histochemical targets [5,13]. Once the patient tests positive for AOA by Western blot analysis, the serum is screened by us to identity the cell types which are targeted, using IHC which employs a novel blocking recipe [13]. Our AOA combo test (Western blotting followed by IHC) is very useful as it not only demonstrates whether the patient has AOA but also highlights the cellular targets of these antibodies.…”

“…Ovarian tissues were fixed and processed for immunohistochemistry (IHC) as per the protocol described earlier [13] which involved a novel blocking recipe. Briefly, Bouin's fixed paraffin embedded ovarian sections was melted at 56°C, deparaffinized in xylene, quenched endogenous peroxidase activity and rehydrated.…”

“…Specificity of AOA testing has been questioned and the available tests reported in literature have been challenged [12] due to high rates of false positives seen using sera from normal fertile controls. This problem of non-specificity has been overcome in a specific, simple and non-invasive diagnostic test developed by us [13]. Using this test we could show new and true molecular and histochemical targets in the ovary in case of ovarian autoimmunity [5,14].…”