Fragile X Screening by Quantification of FMRP in Dried Blood Spots by a Luminex Immunoassay
2013; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2013.02.006
ISSN1943-7811
AutoresGiuseppe LaFauci, Tatyana Adayev, Richard J. Kascsak, Regina Kascsak, Sarah L. Nolin, Pankaj Mehta, W. Ted Brown, Carl Dobkin,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoFragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening. Fragile X is the most common inherited cause of intellectual disability and is frequently associated with autism. The syndrome is due to mutations of the FMR1 gene that result in the absence of fragile X mental retardation protein (FMRP). We have developed a rapid, highly sensitive method for quantifying FMRP from dried blood spots and lymphocytes. This assay uses two new antibodies, a bacterially expressed abbreviated FMRP standard, and a Luminex platform to quantify FMRP. The assay readily distinguished between samples from males with fragile X full mutations and samples from normal males. It also differentiated mosaic from nonmosaic full-mutation male samples. This assay, because of its methodology and minimal cost, could be the basis for newborn or population screening. The fragile X syndrome (FXS) (OMIM 309550) results from the absence of fragile X mental retardation protein (FMRP). This lack of expression is most commonly due to the expansion of a CGG repeat in the 5′-untranslated region of the fragile X mental retardation gene (FMR1) to more than 200 repeats (the full mutation), which leads to hypermethylation of the FMR1 gene promoter and silences transcription.1Pieretti M. Zhang F.P. Fu Y.H. Warren S.T. Oostra B.A. Caskey C.T. Nelson D.L. Absence of expression of the FMR-1 gene in fragile X syndrome.Cell. 1991; 66: 817-822Abstract Full Text PDF PubMed Scopus (1206) Google Scholar, 2De Boulle K. Verkerk A.J. Reyniers E. Vits L. Hendrickx J. Van Roy B. Van den Bos F. de Graaff E. Oostra B.A. Willems P.J. A point mutation in the FMR-1 gene associated with fragile X mental retardation.Nat Genet. 1993; 3: 31-35Crossref PubMed Scopus (501) Google Scholar, 3Coffee B. Ikeda M. Budimirovic D.B. Hjelm L.N. Kaufmann W.E. Warren S.T. 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Individuals with premutation alleles (55 to 200 repeats) have normal or somewhat reduced FMRP levels, but have increased FMR1 mRNA11Tassone F. Hagerman R.J. Chamberlain W.D. Hagerman P.J. Transcription of the FMR1 gene in individuals with fragile X syndrome.Am J Med Genet. 2000; 97: 195-203Crossref PubMed Scopus (166) Google Scholar that is translated with reduced efficiency.12Kenneson A. Zhang F. Hagedorn C.H. Warren S.T. Reduced FMRP and increased FMR1 transcription is proportionally associated with CGG repeat number in intermediate-length and premutation carriers.Hum Mol Genet. 2001; 10: 1449-1454Crossref PubMed Scopus (373) Google Scholar, 13Primerano B. Tassone F. Hagerman R.J. Hagerman P. Amaldi F. Bagni C. Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations.RNA. 2002; 8: 1482-1488PubMed Google Scholar Premutation alleles are highly unstable and may expand to the full mutation in one generation when transmitted by a female. Premutation carrier prevalence in North American populations was estimated at approximately 1 in 151 females and 1 in 468 males in a population-based sample of 6747 Wisconsin adults.14Seltzer M.M. Baker M.W. Hong J. Maenner M. Greenberg J. Mandel D. Prevalence of CGG expansions of the FMR1 gene in a US population-based sample.Am J Med Genet B Neuropsychiatr Genet. 2012; 159: 589-597Crossref Scopus (146) Google Scholar The full-mutation allele, with >200 repeats, has an estimated prevalence of 1 in 3600 to 4000 males and 1 in 4000 to 6000 females (http://www.fragilex.org/fragile-x-associated-disorders/prevalence, last accessed April 25, 2013). Some individuals are mosaic, having both the full mutation and a premutation in blood cells15Rousseau F. Vincent A. Rivella S. Heitz D. Triboli C. Maestrini E. Warren S.T. Suthers G.K. Goodfellow P. Mandel J.L. Toniolo D. Oberle I. Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile-X locus (FRAXA).Am J Hum Genet. 1991; 48: 108-116PubMed Google Scholar (mosaic full mutation). The rate of adaptive skills development is two to four times greater in mosaic cases than in full-mutation cases.16Bailey Jr., D.B. Armstrong F.D. Kemper A.R. Skinner D. Warren S.F. Supporting family adaptation to presymptomatic and "untreatable" conditions in an era of expanded newborn screening.J Pediatr Psychol. 2009; 34: 648-661Crossref PubMed Scopus (25) Google Scholar At present, there is no newborn screening for fragile X in the United States, although pilot projects are underway.16Bailey Jr., D.B. Armstrong F.D. Kemper A.R. Skinner D. Warren S.F. Supporting family adaptation to presymptomatic and "untreatable" conditions in an era of expanded newborn screening.J Pediatr Psychol. 2009; 34: 648-661Crossref PubMed Scopus (25) Google Scholar, 17Skinner D. Choudhury S. Sideris J. Guarda S. Buansi A. Roche M. Powell C. Bailey Jr., D.B. Parents' decisions to screen newborns for FMR1 gene expansions in a pilot research project.Pediatrics. 2011; 127: e1455-e1463Crossref PubMed Scopus (35) Google Scholar Despite the availability of commercial molecular fragile X diagnostic testing, the average age at which the fragile X syndrome is diagnosed in males has remained unchanged, at approximately 36 months.17Skinner D. Choudhury S. Sideris J. Guarda S. Buansi A. Roche M. Powell C. Bailey Jr., D.B. Parents' decisions to screen newborns for FMR1 gene expansions in a pilot research project.Pediatrics. 2011; 127: e1455-e1463Crossref PubMed Scopus (35) Google Scholar, 18Bailey Jr., D.B. The blurred distinction between treatable and untreatable conditions in newborn screening.Health Matrix Clevel. 2009; 19: 141-153PubMed Google Scholar Delay in diagnosis prevents early therapeutic intervention for affected individuals and genetic counseling for their families. Early detection may become even more critical if pharmacological therapies specific for fragile X that are currently in phase 2 or 3 clinical trials19Michalon A. Sidorov M. Ballard T.M. Ozmen L. Spooren W. Wettstein J.G. Jaeschke G. Bear M.F. Lindemann L. Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice.Neuron. 2012; 74: 49-56Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar prove effective. Here, we describe an inexpensive immunoassay based on a Luminex (Austin, TX) platform that detects FMRP in dried blood spots (DBS), as well as in fresh human lymphocytes and other tissue samples. The assay accurately measures FMRP in a DBS sample and can identify males with the full mutation with sensitivity and specificity approaching 100%. This highly accurate test could be the basis for a fragile X test for newborn screening, as well as for population studies of fragile X. Blood samples were obtained from individuals being evaluated for fragile X at the New York State Institute for Basic Research in Developmental Disabilities (IBR) or were samples received at the Specialty Clinical Laboratory at IBR for fragile X DNA analysis. Control blood samples were obtained from IBR staff volunteers who were confirmed to be of normal genotype by DNA analysis. This study was approved by the Institutional Review Board of IBR. Blood was spotted onto ID bloodstain cards BFC180 (WB100014; GE Healthcare, Piscataway, NJ), using a syringe with an 18-gauge needle. Cards were dried overnight and stored in low-gas-permeable plastic bags with desiccant packs, according to DBS guidelines and published protocols.20Mei J.V. Alexander J.R. Adam B.W. Hannon W.H. Use of filter paper for the collection and analysis of human whole blood specimens.J Nutr. 2001; 131: 1631S-1636SPubMed Google Scholar, 21Behets F. Kashamuka M. Pappaioanou M. Green T.A. Ryder R.W. Batter V. George J.R. Hannon W.H. Quinn T.C. Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa, Zaire.J Clin Microbiol. 1992; 30: 1179-1182PubMed Google Scholar Moisture, heat, and direct sunlight are detrimental to the stability of DBS.20Mei J.V. Alexander J.R. Adam B.W. Hannon W.H. Use of filter paper for the collection and analysis of human whole blood specimens.J Nutr. 2001; 131: 1631S-1636SPubMed Google Scholar, 22Adam B.W. Hall E.M. Sternberg M. Lim T.H. Flores S.R. O'Brien S. Simms D. Li L.X. De Jesus V.R. Hannon W.H. The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States.Clin Biochem. 2011; 44: 1445-1450Crossref PubMed Scopus (60) Google Scholar Three 6.9-mm-diameter disks (37.4 mm2) were cut from each card with a paper punch (MCG503, 1/4 inch; McGill, Marengo, IL) and then were transferred into a microtube. Proteins were eluted in 200 μL of extraction reagent (Pierce M-PER mammalian protein extraction reagent; Thermo Fisher Scientific, Rockford, IL) containing 150 mmol/L NaCl, 10 μg/mL chymostatin, 10 μg/mL antipain, and 1× protease inhibitor cocktail (Complete mini tablets, EDTA-free; Roche Applied Science, Indianapolis, IN) by shaking for 3 hours at room temperature. After a brief centrifugation at 10,000 × g, the eluates were decanted by pipette, and 50 μL was used in the assay. This 50 μL corresponds to an eluate from 28.0 mm2 (ie, 25% of three 6.9-mm disks). The assay was reliably performed with eluates from samples as small as a 3-mm disk (7.1 mm2). A 3-mm-diameter disk was cut from each ID card with a Harris Micro-Punch (LabSource, Romeoville, IL). Each disk was placed into a well of low-protein-binding Durapore MultiScreen 96-well filter plates (EMD Millipore, Billerica, MA), and protein was eluted with agitation at 4°C overnight in 50 μL of extraction reagent (as above). Eluates were collected by centrifugation into a 96-well catch plate and were used in the Luminex assay. We compared the Luminex results for eluates from 30 randomly chosen 3-mm disks with the 28.0-mm2 eluate. The FMRP levels detected in the 3-mm disk eluate were consistently 25% of the FMRP detected in the 28-mm2 eluate (r = 0.91). Blood samples (6 to 8 mL) were collected in Vacutainer CPT tubes with citrate anticoagulant (BD, Franklin Lakes, NJ), and lymphocytes were isolated within 2 hours of blood collection, according to the manufacturer's instructions. Cell pellets were stored at −70°C. For protein preparation, pellets were lysed in extraction reagent (as above). After a brief sonication, cell debris was removed by centrifugation at 16,000 × g for 15 minutes; the protein concentration in the supernatant was determined using a Pierce BCA protein assay (Thermo Fisher Scientific). Long-term lymphoblastoid cells were collected at 400 × g, washed in PBS, and frozen at −70°C. Extracts were prepared as described for lymphocytes. The mouse monoclonal antibody (mAb) 6B8 was generated by immunizing mice with a human FMRP expressed in Sf9 insect cells infected by a recombinant baculovirus.23Chen L. Yun S.W. Seto J. Liu W. Toth M. The fragile X mental retardation protein binds and regulates a novel class of mRNAs containing U rich target sequences.Neuroscience. 2003; 120: 1005-1017Crossref PubMed Scopus (133) Google Scholar The baculovirus (generously provided by M. Toth) included the entire human FMRP open reading frame, which was in-frame with six copies of a His tag; this baculovirus was used to prepare high-titer viral stocks. Sf9 cells (grown in suspension at 27°C) were infected with the baculovirus and cells were harvested at 72 hours after infection. Recombinant FMRP was purified from lysed cells by nickel-nitrilotriacetic acid column chromatography (Ni-NTA purification system; Life Technologies-Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Three male FVB Fmr1tm1Cgr mice were immunized four times at 3-week intervals by subcutaneous injection of 100 μg of recombinant FMRP in TiterMax adjuvant (CytRx, Norcross, GA). Mice received additional injections of 50 μg antigen in PBS for three consecutive days before animal sacrifice and spleen removal. After splenocyte fusion to cells of the NSO myeloma cell line (ATCC, Manassas, VA), cells were processed as described previously to clone hybridomas producing anti-FMRP mAbs.24Kascsak R.J. Fersko R. Pulgiano D. Rubenstein R. Carp R.I. Immunodiagnosis of prion disease.Immunol Invest. 1997; 26: 259-268Crossref PubMed Scopus (45) Google Scholar, 25Spinner D.S. Kascsak R.B. Lafauci G. Meeker H.C. Ye X. Flory M.J. Kim J.I. Schuller-Levis G.B. Levis W.R. Wisniewski T. Carp R.I. Kascsak R.J. CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils.J Leukoc Biol. 2007; 81: 1374-1385Crossref PubMed Scopus (34) Google Scholar Several anti-FMRP mAbs were isolated, characterized (LaFauci et al, unpublished data), and purified from ascites fluid by protein G spin column chromatography (Pierce kit 89979; Thermo Fisher Scientific) according to the manufacturer's instructions. Clone 6B8, used in the present study, recognized full-length human FMRP with high affinity and specificity. Rabbit anti-FMRP polyclonal antibody R477 was obtained by immunizing rabbits with the oligopeptide DDHSRTDNRPRNPREAK, which corresponds to a region of the carboxyl terminus of human FMRP spanning residues 554 to 570. This oligopeptide was conjugated to KLH and injected subcutaneously to two 8-week-old, 5-pound (∼2.27 kg) female New Zealand rabbits (Charles River Laboratories International, Wilmington, MA) in Freund's complete adjuvant, followed by four immunizations at 2- to 3-week intervals. Rabbits were tested for the presence of anti-FMRP antibodies by enzyme-linked immunosorbent assay using 96-well plates coated with 5 μg/mL of oligopeptide conjugated to OVA. One rabbit's serum had the highest titer (R477). This antibody was purified from serum using a Pierce Melon Gel IgG purification system (Thermo Fisher Scientific) according to the manufacturer's instructions. Both mAb 6B8 and R477, are available on request. Chemicon anti-FMRP mAb 1C3 (MAB2160) and mouse anti-GAPDH (MAB374) were purchased from EMD Millipore. Goat anti-rabbit phycoerythrin-conjugated IgG (P2771MP) was purchased from Life Technologies-Invitrogen. A glutathione S-transferase (GST) fusion protein carrying the epitopes of mAb 6B8 and R477 was constructed in two steps. First, a double-stranded oligomer encoding a nine-amino-acid sequence of FMRP (amino acids 344 to 352) that includes the mAb 6B8 epitope was cloned into vector pGEX-4T (GE Healthcare) using the BamHI and EcoRI sites. Second, the resulting plasmid was modified to include the R477 epitope by ligating (at EcoRI and XhoI sites) an FMR1 sequence corresponding to amino acids 546 to 605. The latter sequence was obtained by PCR using a cloned FMR1 cDNA and primers 5′-CGGAATTCCGTGGAGGAGGCTTCAA-3′ and 5′-CCCTCGAGCAGCCGACTACCTTCCACTG-3′ (forward and reverse, respectively). Plasmids in bacterial clones that expressed proteins recognized by both antibodies (pGEX-hFMR1-SR7) were isolated and expressed in E. coli strain BL21 by isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. The fusion protein, GST-SR7, was purified by glutathione-Superflow resin (Clontech Laboratories, Mountain View, CA) according to the manufacturer's instructions. Eluted GST-SR7 was dialyzed against 25 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl buffer, concentrated in an Amicon Ultra-15 10K centrifugal filter device (EMD Millipore), aliquoted, lyophilized, and stored at −70°C. The mAb 6B8 was coupled to 5 × 106 xMAP MicroPlex microspheres (Luminex) according to the manufacturer's instructions. Assays were prepared in low-protein-binding Durapore MultiScreen 96-well filter plates (EMD Millipore). Each well contained a total volume of 100 μL, including 50 μL DBS eluate or cell lysate (3 μg total protein in 50 μL) and 3000 mAb 6B8-coupled microspheres resuspended in 50 μL assay buffer [PBS pH 7.4 containing 1% bovine serum albumin (Biosource; Life Technologies-Invitrogen) 0.05% Tween 20, and 0.05% sodium azide]. Plates were incubated in the dark with shaking in a Multi-MicroPlate Genie mixer (Scientific Industries, Bohemia, NY) for 5 hours at room temperature. The supernatant was removed using a vacuum manifold (MultiScreen HTS vacuum manifold kit; EMD Millipore), and the microspheres were washed and incubated with the detecting antibody R477 (1.76 μg/mL in 100 μL assay buffer) at 4°C overnight. Supernatant was aspirated, and the microspheres washed and incubated with 100 μL of 2 μg/mL goat anti-rabbit IgG conjugated to phycoerythrin for 2 hours at room temperature with agitation. Finally, microspheres were resuspended in 100 μL assay buffer and were analyzed (in duplicate) using a Luminex 200 system. Dilutions of GST-SR7 were used to generate a standard curve for each plate using MiraiBio MasterPlex QT quantitative analysis software for protein assay (version 2.5; Hitachi Solutions America, South San Francisco, CA). Median fluorescence intensity was plotted against GST-SR7 concentrations. The amount of FMRP in the DBS was reported as concentration (pmol/L) in the DBS extract. A concentration of 1 pmol/L FMRP in the assay well is equivalent to 5.7 pmol/L in the original blood sample. Protein samples (15 μg) were analyzed on precast 4% to 15% polyacrylamide Criterion Tris-HCl gels (BioRad Laboratories, Hercules, CA) that were run at 200 mV for 1 hour. Separated proteins were transferred onto 0.22-μm polyvinylidene difluoride membranes (BioRad Laboratories) in transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, pH 8.3) using a semidry electroblotter (OWL HEP-1; Thermo Scientific, Waltham, MA) for 1 hour at 10 V. Membranes were incubated in 5% nonfat dry milk in 0.01 mol/L Tris pH 7.5, 0.137 mol/L NaCl, 0.05% Tween 20 and then with either anti-FMRP antibodies or a mouse anti-GAPDH mAb. After washing, membranes were incubated for 1 hour with the conspecific alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, MO). Proteins were detected with CDP-Star reagent (New England Biolabs, Ipswich, MA) according to the manufacturer's instructions. Fragile X analysis of DNA isolated from blood samples was performed by PCR and Southern blot as described previously26Brown W.T. The FRAXE Syndrome: is it time for routine screening?.Am J Hum Genet. 1996; 58: 903-905PubMed Google Scholar, 27Nolin S.L. Brown W.T. Glicksman A. Houck Jr., G.E. Gargano A.D. Sullivan A. Biancalana V. Bröndum-Nielsen K. Hjalgrim H. Holinski-Feder E. Kooy F. Longshore J. Macpherson J. Mandel J.L. Matthijs G. Rousseau F. Steinbach P. Väisänen M.L. von Koskull H. Sherman S.L. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles.Am J Hum Genet. 2003; 72: 454-464Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 28Nolin S.L. Ding X.H. Houck G.E. Brown W.T. Dobkin C. Fragile X full mutation alleles composed of few alleles: implications for CGG repeat expansion.Am J Med Genet A. 2008; 146A: 60-65Crossref PubMed Scopus (17) Google Scholar or with AmplideX FMR1 PCR (RUO) reagents (Asuragen, Austin, TX) and capillary electrophoresis29Chen L. Hadd A. Sah S. Filipovic-Sadic S. Krosting J. Sekinger E. Pan R. Hagerman P.J. Stenzel T.T. Tassone F. Latham G.J. An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis.J Mol Diagn. 2010; 12: 589-600Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar according to the manufacturer's instructions. DBS DNA was isolated with a DNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions and was concentrated by precipitation with ethanol. DBSs from blood received more than 3 days after collection were excluded from the analysis because of protein degradation. A cord-blood sample DBS was excluded because of a very high FMRP level and because of the unique nature of this sample. Premutation male data are given in Table 1, but were not included in the analysis because there was only one of these samples. Data were analyzed with either SPSS 11 Statistics (SPSS, Chicago, IL) or SigmaPlot version 11 (Systat Software, San Jose, CA) software.Table 1Capture Immunoassay Quantification of FMRP in DBS Extracts Derived from 215 Individuals with Normal, Premutation, and Full-Mutation Fragile X GenotypeGenotype∗Genotype determined by PCR and Southern analyses.No.Protein concentration (pmol/L)MeanMedianSDMinMax95% CIMale (n = 103) Normal8525.824.810.38.651.523.6-28.0 Premutation1NDNDND29.029.0ND Full mutation171.70.71.70.26.60.8-2.6 Nonmosaic100.60.60.30.21.20.4−0.7 Mosaic73.32.91.61.86.61.8-4.8Female (n = 112) Normal4926.025.38.611.646.323.5-28.4 Premutation5923.022.57.09.938.721.2-24.8 Normal + premutation10824.323.67.99.920.122.8-25.8 Full mutation417.216.72.015.420.114.0-20.5M normal + F normal13425.925.09.68.651.524.2-27.5F, female; M, male; CI, confidence interval; Max, maximum; Min, minimum; ND, not determined.∗ Genotype determined by PCR and Southern analyses. Open table in a new tab F, female; M, male; CI, confidence interval; Max, maximum; Min, minimum; ND, not determined. To develop a DBS immunoassay, we needed one antibody to capture FMRP and another to detect it within a complex background of other proteins. Preliminary experiments suggested mAb 6B8 and R477 as a candidate pair. The specificity of these antibodies was characterized by Western blot analysis of extracts from normal (male and female), premutation (female), and full-mutation (male) lymphocytes (Figure 1A). Three FMRP bands (68 to 80 kDa) were recognized by the mAb 6B8 with short exposure in normal and premutation samples, whereas no bands were detected in the full-mutation FXS male extract (Figure 1A). This indicated that 6B8 has little if any cross-reactivity with the closely related proteins FXR1P and FXR2P,30Tamanini F. Kirkpatrick L.L. Schonkeren J. van Unen L. Bontekoe C. Bakker C. Nelson D.L. Galjaard H. Oostra B.A. Hoogeveen A.T. The fragile X-related proteins FXR1P and FXR2P contain a functional nucleolar-targeting signal equivalent to the HIV-1 regulatory proteins.Hum Mol Genet. 2000; 9: 1487-1493Crossref PubMed Scopus (44) Google Scholar as seen with long exposure (overexposure) (Figure 1A). Bands of the same size (68 to 80 kDa) were detected by R477 in all extracts except the male full-mutation FXS (Figure 1B). This antibody also detected a few faint bands, especially one at approximately 65 kDa, that were visible on overexposure (Figure 1B), indicating that R477 has weak cross-reactivity to a few other proteins. Comparison with the pattern detected by the commercially available anti-FMRP mAb 1C3 (Figure 1C) suggests that the mAb 6B8 and R477 are both highly specific for FMRP. Thus, mAb 6B8 and R477 both recognize FMRP without having any other targets in common. We evaluated the capacity of this antibody pair to capture and detect FMRP in Luminex immunoassays of extracts from normal human lymphoblastoid cell lines. Microspheres were coupled to the mAb 6B8 to capture FMRP, and R477 was used to detect its presence. To evaluate the assay, we used 1.2 to 80 μg of protein extracted from normal and full-mutation male lymphoblastoid cell lines. The level of FMRP in normal cells was proportional to the amount of sample (Figure 1D), and there was a linear (r2 = 0.98) response up to approximately 40 μg of extract. Only background fluorescence values were detected in wells containing up to 80 μg of male full-mutation extracts. To gauge the effectiveness of the Luminex assay, we analyzed the level of FMRP among blood samples from different normal individuals. Repeated assays of 19 lymphocyte extracts were highly correlated (r = 0.96), indicating that the assay is reliable (data not shown). In addition, the relative levels of FMRP detected by densitometric quantification of the short-exposure mAb 6B8-stained bands (Figure 1A) very closely matched (within ≤7%) the relative levels in those samples measured by the Luminex assay. The assay demonstrated a broad FMRP distribution in 11 lymphocyte samples from normal individuals; mean fluorescence intensity ranged from 1194 to 2375 (SD, 405.1). A lymphocyte extract from a full-mutation male showed only background fluorescence value (data not shown) and was easily distinguished from normal samples. We found that lymphocyte samples are very sensitive to the length of time between blood draw and lymphocyte isolation, as well as to blood storage conditions, as has also been observed by others.31Iwahashi C. Tassone F. Hagerman R.J. Yasui D. Parrott G. Nguyen D. Mayeur G. Hagerman P.J. A quantitative ELISA assay for the fragile X mental retardation 1 protein.J Mol Diagn. 2009; 11: 281-289Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Only lymphocyte samples that were isolated within a few hours of blood draw gave dependable results. The requirements for rapid lymphocyte isolation from freshly drawn blood made lymphocyte samples impractical for FMRP screening. Because of these limitations and in view of reports that proteins are stable for at least 6 months in DBS that are stored with desiccant,20Mei J.V. Alexander J.R. Adam B.W. Hannon W.H. Use of filter paper for the collection and analysis of human whole blood specimens.J Nutr. 2001; 131: 1631S-1636SPubMed Google Scholar we tried using DBS eluates; we were surprised to find that FMRP was easily detectable in these samples. In an initial experiment, DBS from four normal individuals and three premutation females averaged approximately 100-fold higher than three male full-mutation samples and approximately 10-fold higher than a male mosaic full-mutation sample (data not shown). The level of FMRP detected in DBS also depended on storage conditions. Proteins are stable for at least 6 months when DBS are stored as recommended (with desiccant at 22°C or 4°C).20Mei J.V. Alexander J.R. Adam B.W. Hannon W.H. Use of filter paper for the collection and analysis of human whole blood specimens.J Nutr. 2001; 131: 1631S-1636SPubMed Google Scholar, 22Adam B.W. Hall E.M. Sternberg M. Lim T.H. Flores S.R. O'Brien S. Simms D. Li L.X. De Jesus V.R. Hannon W.H. The stability of markers in dried-blood spots for recommended newborn screening disorders in the United States.Clin Biochem. 2011; 44: 1445-1450Crossref PubMed Scopus (60) Google Scholar Even in the absence of desiccant, DBSs (n = 8) retained on average 66% of the original FMRP after 1 year of stora
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