Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.
Abstract: Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents.
Publication Date: 2006-11-15 PubMed ID: 17109971DOI: 10.1016/j.vetimm.2006.10.010Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper delineates how heterophile antibodies (HAs) in horse plasma can interfere with an immunoassay used for the detection of equine growth hormone, leading to falsely elevated measurements. The study also highlights an occurrence rate of 4.7% in false positive results in the measured 127 horses sample.
Role of Heterophile Antibodies (HAs)
- HAs can interact with animal immunoglobulins, thus they are a critical source of false results in immunoassays. This unpredictability makes diagnosis and detection procedures less reliable.
- In this study, the researchers found that the elevated equine growth hormone (eGH) in one horse (>100 ng/mL) was falsely elevated due to interference from HAs.
- The interfering antibodies were not confined to a single species, indicating broad species-specificity. In particular, they obstructed the eGH immunoassay by forming a bridge between the mouse IgG capture antibody and the rabbit IgG conjugate.
Creation of Artificial Elements
- This study reveals that interference from HAs resulted in the production of artificial “sandwiches”. These falsely elevated the eGH plasma concentration levels.
- While spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma from various species decreased the HAs interference, it did not eradicate the issue entirely.
- The varying capabilities of immunoglobulins and whole plasma to block the interference suggest that HAs could also be identifying proteins other than immunoglobulins in animal sera.
Implications in Equine Clinical Practice
- The researchers tested plasma samples from another 114 horses in an attempt to understand the implications of HAs in regular practice and found that 5 of these samples had plasma HAs.
- Therefore, out of a total of 127 horses examined, 6 had plasma HAs that led to falsely elevated eGH measures, indicating an incidence rate of 4.7%.
Conclusions
- This study is the first to show evidence of HAs in horse plasma interfering with an immunoassay. This has important implications for veterinary diagnosis and detection procedures involving horse plasma.
- It also suggests that those working in veterinary surgery and diagnostic laboratories need to consider this potential for interference in tests on horse plasma using either monoclonal or polyclonal antibody reagents.
Cite This Article
APA
Borromeo V, Berrini A, Gaggioli D, Secchi C.
(2006).
Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.
Vet Immunol Immunopathol, 115(1-2), 1-9.
https://doi.org/10.1016/j.vetimm.2006.10.010 Publication
Researcher Affiliations
- Department of Veterinary Pathology, Hygiene and Health, Biochemistry and Physiology Unit, University of Milan, Via Celoria 10, I-20133 Milan, Italy. vitaliano.borromeo@unimi.it
MeSH Terms
- Animals
- Antibodies, Heterophile / blood
- Dogs
- Enzyme-Linked Immunosorbent Assay / methods
- Female
- Growth Hormone / blood
- Horses
- Male
- Mice
- Rabbits
Citations
This article has been cited 3 times.- Dong B, Bergman D, Holst BS. Prevalence of heterophilic antibodies in serum samples from horses in an equine hospital, and elimination of interference using chicken IgY.. Acta Vet Scand 2021 Mar 12;63(1):10.
- Hsiao YC, Chi LM, Chien KY, Chiang WF, Chen SF, Chuang YN, Lin SY, Wu CC, Chang YT, Chu LJ, Chen YT, Chia SL, Chien CY, Chang KP, Chang YS, Yu JS. Development of a Multiplexed Assay for Oral Cancer Candidate Biomarkers Using Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry.. Mol Cell Proteomics 2017 Oct;16(10):1829-1849.
- Borromeo V, Ferrucci F, Zucca E, Berrini A, Secchi C. Growth hormone secretion in relation to plasma lactate and glucose concentrations during a maximal treadmill exercise test in horses.. Vet Res Commun 2008 Sep;32 Suppl 1:S111-3.
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