BACKGROUND
alzheimer’s disease  is a neurodegenerative disease characterized by cognitive deficits leading to a progressive loss of functional autonomy.
the neuropathological hallmarks of the disease are extracellular amyloid deposits and intracellular neurofibrillar tangles containing phosphorylated tau   <cit> .
according to recently proposed ad diagnostic criteria, diagnosis is based on clinical criteria, medical history, neuropsychological evaluation, morphological neuroimaging, cerebrospinal  biomarkers and/or molecular imaging   <cit> .
ad is biologically characterized by the combination of low aβ1- <dig> peptide levels and high levels of total tau and tau phosphorylated on threonine  <dig>  in the csf  <cit> .
however, new biomarkers are needed to better discriminate ad from non-ad patients and to improve the diagnosis of ad in patients with atypical csf profiles.
amyloid precursor protein  is a key molecule in ad pathology.
proteolytic cleavages of app by the sequential actions of β- and γ-secretases form the neurotoxic amyloid beta  peptide, which typically consists of  <dig> or  <dig> amino acid residues .
an alternative cleavage by α-secretase, which splits the app sequence in the middle of the amyloid peptide sequence, prevents formation of the aβ peptide and releases the resulting app n-terminal ectodomain  <cit> .
the physiological regulation of β and α cleavages is poorly established.
however, recent papers describe an α cleavage following the β cleavage, as the c-terminal fragment  released after β-secretase cleavage can be processed in α-secretase to generate the c <dig> fragment  <cit> .
in addition, truncated aβ peptide of  <dig> to  <dig> residues generated from the sequential actions of β and α-secretases have been identified in the csf  <cit> .
interestingly, an inhibitory domain for γ-secretase activity in the c <dig> fragment has been identified able to down regulate the production of aβ peptide  <cit> .
in addition to its regulation of aβ peptide, α-secretase cleavage appears very important as sappα is involved in several functions  <cit> .
sappα is known for its neurotrophic and neuroprotective properties  <cit> .
in addition, it stimulates the proliferation of neuroblasts from the sub-ventricular zone and increases long-term potentiation  <cit> .
recent studies have investigated sappα as a putative biomarker of ad  <cit>  and tested the hypothesis that dysregulation of the alternative cleavage of app may contribute to the pathophysiology of the disease.
in the past, human sappα has been evaluated by western blotting  <cit> , elisa  <cit> , and multiplex technology  <cit> .
however, some of the findings regarding sappα levels in human csf have been contradictory.
such discordant results signal the need for an ideal test that allows very sensitive and specific detection of sappα in small amounts of human csf.
recently, a new technology   <cit>  requiring no washing and using low sample volume has been applied to sappα  but no results have been published yet.
from a research perspective, sappα quantification in preclinical studies in rodents is also necessary to better understand ad pathophysiology and to develop new therapeutic targets.
different cell lines, which include neuroblastoma cells, are currently being used to study app metabolism in vitro.
although rodent primary neurons produce low amounts of aβ peptide  <cit> , this model remains interesting to study the response of sappα to ad therapeutic drug candidates before their in vivo validation in mice or rats.
to effectively evaluate these models, a sensitive, reproducible and rapid test is needed to accurately assess sappα levels in the conditioned medium of rodent neuron primary cultures and to create high-throughput drug screening assays.
tests are also needed for the in vivo evaluation of candidate drugs identified in in vitro screens, both in wild-type mice and in transgenic ad mouse models.
we describe two new rapid and sensitive sappα tests that are based on homogenous time-resolved fluorescence  and can be used to quantify the levels of: i) endogenous rodent sappα in the conditioned medium of mouse primary neurons, as well as in the csf of wild type mice; ii) human sappα in the csf of ad mouse models; and iii) human sappα in the csf of ad and non-ad patients.
these tests require only 5 μl of conditioned medium from 5 ×  <dig> mouse primary neurons, 1 μl of csf from wild-type and transgenic ad mouse models or  <dig>  μl of human csf.
RESULTS
we developed two new sappα assays using an adaptation of the htrf technology: one detects mouse/rodent-sappα  and the other human-sappα  through the application of two different app antibodies .
both tests are single-step homogenous assays that require no washing unlike most elisa procedures.
this absence of multiple washing steps increases the speed and reproducibility of the assay.
the final volume of the test is 20 μl, which allows detection in a 384-well plate for high-throughput investigations.
these tests are extremely easy to use and require few preanalytical preparations.
analytical performance of the htrf sappα assays
the purity of the standard recombinant proteins h-sappα and r-sappα was checked by sds-page .we evaluated the specificity of the h-sappα assay using human-sappβ  and r-sappα recombinant proteins, which both contain the mab  <dig> epitope, and the human peptide aβ1- <dig> , which contains the 6e <dig> epitope; both epitopes are necessary for h-sappα assay.
the signal obtained with 3 − 200 ng/ml of h-sappβ, r-sappα and h-aβ1- <dig> was similar to that obtained for the blank, demonstrating that the h-sappα does not cross-react with any of these negative controls .
we evaluated the specificity of the r-sappα assay using recombinant h-sappα, h-sappβ and the h-aβ1- <dig> peptide.
we detected a cross-reactivity level of 23% with h-sappα, indicating that the rodent c-terminal sappα antibody is less specific than the human sappα 6e <dig> antibody .
no cross-reactivity was observed with h-sappβ or h-aβ1- <dig> .
we obtained a linear response using 3 − 200 ng/ml of recombinant h-sappα or r-sappα, which indicated that the limit of detection was approximately 3 ng/ml .
we then evaluated the linearity of both assays with serial dilutions of human  or mouse  csf.
in both cases, the slopes obtained with h-csf  and r-csf  were parallel to the standard curves generated by recombinant human  and rodent  sappα, respectively.
these results demonstrate that sappα levels in the csf can be accurately evaluated in these assays with only  <dig>  μl  of h-csf or 1 μl  of r-csf .
to assess the intra- and inter-assay imprecision of the h-sappα assay, five h-csf samples were each tested ten times on one plate in the same experiment and were tested three times in three independent experiments.
the mean values for intra- and inter-assay imprecision were <5% with one exception in each experiment.
imprecision was  <dig> % for one csf sample in the intra-assay experiment and  <dig> % for a different csf sample in the inter-assay experiment .
intra-assay imprecision: five csf samples were tested ten times on one plate.
inter-assay imprecision: the same samples were tested three times in three independent experiments.
mean ± sem of h-sappα concentration .
cv: coefficient of variation .
to assess the intra- and inter-assay imprecision of the r-sappα assay, three samples of conditioned media from mouse neuron primary cultures were each tested ten times on one plate in the same experiment and were each tested three times in three independent experiments.
the mean values for the intra- and inter-assay imprecision were below 6% and 7%, respectively, with one exception of  <dig> % for one sample in the inter-assay experiment .
intra-assay imprecision: three conditioned media samples from three different experiments were tested ten times on one plate.
inter-assay imprecision: the same samples were tested three times in three independent assays.
mean ± sem of r-sappα concentration .
cv: coefficient of variation .
we then examined the stability of h-csf sappα.
first, we evaluated the effect of storage in polypropylene or polystyrene tubes.
five samples were divided into polypropylene or polystyrene tubes and stored for  <dig> hours at −80°c.
the h-sappα values of samples stored in polystyrene tubes were not significantly different from those of samples stored in polypropylene tubes , which is a real concern in a clinical practice  <cit> .
five csf samples were divided into polypropylene or polystyrene tubes and stored for  <dig> hours at −80°c.
mean ± sem of h-sappα in ng/ml and  compared to the aliquot stored in polypropylene tubes.
next, we evaluated whether storage temperature affected the stability of h-sappα.
five aliquots of h-csf were stored for  <dig> hours at −80°c, −20°c, +4°c or room temperature, or were stored at −80°c and subjected to three freeze/thaw cycles during three days.
no significant differences were detected for the different storage conditions or for the samples that underwent the three freeze/thaw cycles.
thus, sappα in csf appears to be very stable .
five csf samples were divided in five aliquots and stored for  <dig> hours at one of several different temperatures including room temperature , or stored at −80°c and exposed to three freeze/thaw cycles  within a period of three days.
the results were normalized  for the results from the aliquot stored at −80°c.
sappα assay development: in vitro applications
we used our sappα assays to measure the sappα levels in the conditioned media of human neuroblastoma cells and mouse neuron primary cultures.
human neuroblastoma cells and mouse primary neurons were plated in 96-well plates.
we first determined whether sappα secretion was proportional to the number of cells per well and to the time of incubation .
sappα secretion from cultured cells can be enhanced in vitro by the pkc-mediated stimulation of the metalloprotease adam <dig>  <cit> .
the neuropeptide pacap  <dig>  <cit>  promotes the α-secretase-mediated cleavage of full-length app through the constitutive adam <dig> metalloprotease.
the green tea polyphenol egcg stimulates either adam <dig> or adam <dig>  depending on the cell type being treated  <cit> .
we treated  <dig> x  <dig> neuroblastoma cells with pdbu  to evaluate its impact on the levels of sappα produced in conditioned media.
we demonstrated that  <dig>  to  <dig>  μm of pdbu treatment caused a dose-dependent increase in the level of h-sappα in the conditioned medium of neuroblastoma cells .
this increase was completely abolished by treatment with the pkc inhibitor gf- <dig> or the α-secretase inhibitor tapi  <dig> .
to further validate the new r-sappα assay, we used mouse primary neurons, at six days in vitro.
we first checked by western blotting the presence of r-sappα in the conditioned medium recognized by both 22c <dig> and rodent specific c-terminal antibodies .
this sappα band at 100 kda is specific of the isoform  <dig> present in neurons  <cit> .
mouse primary neurons were plated in  <dig> well-plates at the density of 5 ×  <dig> cells per well.
we checked that r-sappα secreted was proportional to the number of neurons and to the time of incubation .
treatment of primary neurons with 30 μm egcg or with 1 μm pacap- <dig> produced a strong increase in the level of endogenous r-sappα in the incubation medium .
these results from our rodent test validate previously reported western blotting data from treated neuroblastoma cells  <cit> .
the rodent test detected sappα in only 5 μl of conditioned medium from 5 ×  <dig> primary neurons cultured in 96-well plates, without concentration of the proteins.
five csf samples were divided into polypropylene or polystyrene tubes and stored for  <dig> hours at −80°c.
mean ± sem of h-sappα in ng/ml and  compared to the aliquot stored in polypropylene tubes.
in previously published reports, evaluating sappα in conditioned media required higher numbers of cells combined with trichloracetic acid precipitation of proteins  <cit> , media lyophilization  <cit>  or protein concentration with centrifugal filters  <cit> .
sappα assay development: in vivo applications in mice
to evaluate the feasibility of measuring sappα levels in the csf of mice, we used 14-month-old wild-type mice and 12-month-old ad triple transgenic mice   <cit> .
the maximum volume of csf obtained from one mouse was 5 μl.
sappα assays were performed on each csf sample diluted 1/ <dig> or 1/ <dig>  the mean r-sappα concentrations in the csf were 137 ± 17 ng/ml for wild-type mice and 105 ± 8 ng/ml for 3xtg ad mice.
in the 3×tg ad mice, the h-sappα concentration was 34 ± 6 ng/ml .
in the 3×tg ad mice, the h-sappα derived from the transgene was specifically and distinctly quantified in the csf relative to the endogenous r-sappα, whereas the r-sappα may be contaminated by 23% h-sappα .
one μl of mouse csf was sufficient to detect r-sappα as well as h-sappα and detection was linear for serially diluted samples.
this level of sensitivity may allow the levels of the human transgenic sappα to be followed through the progression of the disease, which has never been reported previously.
csf samples  were collected from wild type or 3xtg ad mice and 1/ <dig> or 1/ <dig> dilutions of the csf were tested in the r-sappα and in the h- sappα assays.#: this value includes 23% of h- sappα .
sappα assay development: in vivo applications in human
we then assessed h-sappα levels in the csf of ad and non-ad patients.
high levels of h-sappα in the csf determined using a multiplex technology were reported to be associated with ad  <cit> .
in this study, we evaluated our h-sappα assay using csf samples from patients who had been classified according to validated criteria as either non-ad or ad by a team specialized in the diagnosis of neurodegenerative disorders .
these patients were matched according to sex and age and significantly differ by the mmse test .
we checked by western blotting in some csf samples the presence of two sappα isoforms recognized by both 22c <dig> and 6e <dig> antibodies and the absence of specific detectable cleaved isoforms .
the two sappα isoforms of molecular weights   <dig> and 125 kda detected are similar to that observed in soluble fraction of mouse brains  and correspond to mature glycosylated forms of sappα  <dig>  <cit> .
however we cannot exclude that the higher isoform correspond to the sappα  <dig> containing kpi domain and present in glial cells  <cit> .
the bands of lower mw and lower intensity detected were non specific as recognized by the anti-mouse secondary antibody .
in our population, aβ1- <dig> concentrations were lower  and tau and ptau <dig> concentrations were higher  in the ad group than in the non-ad group.
we found significantly higher sappα concentrations in ad csf compared to non-ad csf , which agrees with recent studies in which the sappα concentrations in csf were determined by a multiplex technology  <cit> .
ad: patients with alzheimer’s disease; non-ad: patients with other dementias; m: male; f: female.
mmse: mini mental state examination.
**: p <  <dig> .
furthermore, our test detected h-sappα in as little as  <dig>  μl of csf, which is a vast improvement over elisa tests and multiplex technology, which require sample volumes of 100 μl and 25 μl, respectively.
the low amounts of csf required by our test will allow large clinical and longitudinal investigations in patients to validate sappα as an ad biomarker.
CONCLUSIONS
this is the first description of two highly sensitive tests that can be used to detect sappα in a variety of samples.
samples include the conditioned media of human neuroblastoma cells and mouse primary neurons, as well as very small samples of biological fluids, such as mouse and human csf.
these new tools will have very wide applications from pre-clinical to clinical studies.
together, their rapidity, sensitivity and small incubation volumes will facilitate high-throughput investigations of drugs capable of increasing the secretion of endogenous sappα from primary neurons.
the tests may be subsequently validated for in vivo analyses by using them to evaluate at different time points sappα levels in the csf of wild-type mice or ad mouse models treated with molecules that exhibit activity in in vitro screening assays.
finally, the human sappα test will allow sappα levels to be measured in the csf of patients participating in large clinical investigations.

 alzheimer’s disease is a neurodegenerative disease characterized by cognitive deficits leading to a progressive loss of functional autonomy. the neuropathological hallmarks of the disease are extracellular amyloid deposits and intracellular neurofibrillar tangles containing phosphorylated tau. according to recently proposed ad diagnostic criteria, diagnosis is based on clinical criteria, medical history, neuropsychological evaluation, morphological neuroimaging, cerebrospinal biomarkers and/or molecular imaging. ad is biologically characterized by the combination of low aβ1- peptide levels and high levels of total tau and tau phosphorylated on threonine in the csf. however, new biomarkers are needed to better discriminate ad from non-ad patients and to improve the diagnosis of ad in patients with atypical csf profiles. amyloid precursor protein is a key molecule in ad pathology. proteolytic cleavages of app by the sequential actions of β- and γ-secretases form the neurotoxic amyloid beta peptide, which typically consists of or amino acid residues. an alternative cleavage by α-secretase, which splits the app sequence in the middle of the amyloid peptide sequence, prevents formation of the aβ peptide and releases the resulting app n-terminal ectodomain. the physiological regulation of β and α cleavages is poorly established. however, recent papers describe an α cleavage following the β cleavage, as the c-terminal fragment released after β-secretase cleavage can be processed in α-secretase to generate the c fragment. in addition, truncated aβ peptide of to residues generated from the sequential actions of β and α-secretases have been identified in the csf. interestingly, an inhibitory domain for γ-secretase activity in the c fragment has been identified able to down regulate the production of aβ peptide. in addition to its regulation of aβ peptide, α-secretase cleavage appears very important as sappα is involved in several functions. sappα is known for its neurotrophic and neuroprotective properties. in addition, it stimulates the proliferation of neuroblasts from the sub-ventricular zone and increases long-term potentiation. recent studies have investigated sappα as a putative biomarker of ad and tested the hypothesis that dysregulation of the alternative cleavage of app may contribute to the pathophysiology of the disease. in the past, human sappα has been evaluated by western blotting, elisa, and multiplex technology. however, some of the findings regarding sappα levels in human csf have been contradictory. such discordant results signal the need for an ideal test that allows very sensitive and specific detection of sappα in small amounts of human csf. recently, a new technology requiring no washing and using low sample volume has been applied to sappα but no results have been published yet. from a research perspective, sappα quantification in preclinical studies in rodents is also necessary to better understand ad pathophysiology and to develop new therapeutic targets. different cell lines, which include neuroblastoma cells, are currently being used to study app metabolism in vitro. although rodent primary neurons produce low amounts of aβ peptide, this model remains interesting to study the response of sappα to ad therapeutic drug candidates before their in vivo validation in mice or rats. to effectively evaluate these models, a sensitive, reproducible and rapid test is needed to accurately assess sappα levels in the conditioned medium of rodent neuron primary cultures and to create high-throughput drug screening assays. tests are also needed for the in vivo evaluation of candidate drugs identified in in vitro screens, both in wild-type mice and in transgenic ad mouse models. we describe two new rapid and sensitive sappα tests that are based on homogenous time-resolved fluorescence and can be used to quantify the levels of: i) endogenous rodent sappα in the conditioned medium of mouse primary neurons, as well as in the csf of wild type mice; ii) human sappα in the csf of ad mouse models; and iii) human sappα in the csf of ad and non-ad patients. these tests require only 5 μl of conditioned medium from 5 ×  mouse primary neurons, 1 μl of csf from wild-type and transgenic ad mouse models or  μl of human csf.  we developed two new sappα assays using an adaptation of the htrf technology: one detects mouse/rodent-sappα and the other human-sappα through the application of two different app antibodies. both tests are single-step homogenous assays that require no washing unlike most elisa procedures. this absence of multiple washing steps increases the speed and reproducibility of the assay. the final volume of the test is 20 μl, which allows detection in a 384-well plate for high-throughput investigations. these tests are extremely easy to use and require few preanalytical preparations. analytical performance of the htrf sappα assays the purity of the standard recombinant proteins h-sappα and r-sappα was checked by sds-page.we evaluated the specificity of the h-sappα assay using human-sappβ and r-sappα recombinant proteins, which both contain the mab epitope, and the human peptide aβ1-, which contains the 6e epitope; both epitopes are necessary for h-sappα assay. the signal obtained with 3 − 200 ng/ml of h-sappβ, r-sappα and h-aβ1- was similar to that obtained for the blank, demonstrating that the h-sappα does not cross-react with any of these negative controls. we evaluated the specificity of the r-sappα assay using recombinant h-sappα, h-sappβ and the h-aβ1- peptide. we detected a cross-reactivity level of 23% with h-sappα, indicating that the rodent c-terminal sappα antibody is less specific than the human sappα 6e antibody. no cross-reactivity was observed with h-sappβ or h-aβ1-. we obtained a linear response using 3 − 200 ng/ml of recombinant h-sappα or r-sappα, which indicated that the limit of detection was approximately 3 ng/ml. we then evaluated the linearity of both assays with serial dilutions of human or mouse csf. in both cases, the slopes obtained with h-csf and r-csf were parallel to the standard curves generated by recombinant human and rodent sappα, respectively. these results demonstrate that sappα levels in the csf can be accurately evaluated in these assays with only  μl of h-csf or 1 μl of r-csf. to assess the intra- and inter-assay imprecision of the h-sappα assay, five h-csf samples were each tested ten times on one plate in the same experiment and were tested three times in three independent experiments. the mean values for intra- and inter-assay imprecision were <5% with one exception in each experiment. imprecision was % for one csf sample in the intra-assay experiment and % for a different csf sample in the inter-assay experiment. intra-assay imprecision: five csf samples were tested ten times on one plate. inter-assay imprecision: the same samples were tested three times in three independent experiments. mean ± sem of h-sappα concentration. cv: coefficient of variation. to assess the intra- and inter-assay imprecision of the r-sappα assay, three samples of conditioned media from mouse neuron primary cultures were each tested ten times on one plate in the same experiment and were each tested three times in three independent experiments. the mean values for the intra- and inter-assay imprecision were below 6% and 7%, respectively, with one exception of % for one sample in the inter-assay experiment. intra-assay imprecision: three conditioned media samples from three different experiments were tested ten times on one plate. inter-assay imprecision: the same samples were tested three times in three independent assays. mean ± sem of r-sappα concentration. cv: coefficient of variation. we then examined the stability of h-csf sappα. first, we evaluated the effect of storage in polypropylene or polystyrene tubes. five samples were divided into polypropylene or polystyrene tubes and stored for hours at −80°c. the h-sappα values of samples stored in polystyrene tubes were not significantly different from those of samples stored in polypropylene tubes, which is a real concern in a clinical practice. five csf samples were divided into polypropylene or polystyrene tubes and stored for hours at −80°c. mean ± sem of h-sappα in ng/ml and compared to the aliquot stored in polypropylene tubes. next, we evaluated whether storage temperature affected the stability of h-sappα. five aliquots of h-csf were stored for hours at −80°c, −20°c, +4°c or room temperature, or were stored at −80°c and subjected to three freeze/thaw cycles during three days. no significant differences were detected for the different storage conditions or for the samples that underwent the three freeze/thaw cycles. thus, sappα in csf appears to be very stable. five csf samples were divided in five aliquots and stored for hours at one of several different temperatures including room temperature, or stored at −80°c and exposed to three freeze/thaw cycles within a period of three days. the results were normalized for the results from the aliquot stored at −80°c. sappα assay development: in vitro applications we used our sappα assays to measure the sappα levels in the conditioned media of human neuroblastoma cells and mouse neuron primary cultures. human neuroblastoma cells and mouse primary neurons were plated in 96-well plates. we first determined whether sappα secretion was proportional to the number of cells per well and to the time of incubation. sappα secretion from cultured cells can be enhanced in vitro by the pkc-mediated stimulation of the metalloprotease adam. the neuropeptide pacap promotes the α-secretase-mediated cleavage of full-length app through the constitutive adam metalloprotease. the green tea polyphenol egcg stimulates either adam or adam depending on the cell type being treated. we treated x neuroblastoma cells with pdbu to evaluate its impact on the levels of sappα produced in conditioned media. we demonstrated that to  μm of pdbu treatment caused a dose-dependent increase in the level of h-sappα in the conditioned medium of neuroblastoma cells. this increase was completely abolished by treatment with the pkc inhibitor gf- or the α-secretase inhibitor tapi. to further validate the new r-sappα assay, we used mouse primary neurons, at six days in vitro. we first checked by western blotting the presence of r-sappα in the conditioned medium recognized by both 22c and rodent specific c-terminal antibodies. this sappα band at 100 kda is specific of the isoform present in neurons. mouse primary neurons were plated in well-plates at the density of 5 ×  cells per well. we checked that r-sappα secreted was proportional to the number of neurons and to the time of incubation. treatment of primary neurons with 30 μm egcg or with 1 μm pacap- produced a strong increase in the level of endogenous r-sappα in the incubation medium. these results from our rodent test validate previously reported western blotting data from treated neuroblastoma cells. the rodent test detected sappα in only 5 μl of conditioned medium from 5 ×  primary neurons cultured in 96-well plates, without concentration of the proteins. five csf samples were divided into polypropylene or polystyrene tubes and stored for hours at −80°c. mean ± sem of h-sappα in ng/ml and compared to the aliquot stored in polypropylene tubes. in previously published reports, evaluating sappα in conditioned media required higher numbers of cells combined with trichloracetic acid precipitation of proteins, media lyophilization or protein concentration with centrifugal filters. sappα assay development: in vivo applications in mice to evaluate the feasibility of measuring sappα levels in the csf of mice, we used 14-month-old wild-type mice and 12-month-old ad triple transgenic mice. the maximum volume of csf obtained from one mouse was 5 μl. sappα assays were performed on each csf sample diluted 1/ or 1/ the mean r-sappα concentrations in the csf were 137 ± 17 ng/ml for wild-type mice and 105 ± 8 ng/ml for 3xtg ad mice. in the 3×tg ad mice, the h-sappα concentration was 34 ± 6 ng/ml. in the 3×tg ad mice, the h-sappα derived from the transgene was specifically and distinctly quantified in the csf relative to the endogenous r-sappα, whereas the r-sappα may be contaminated by 23% h-sappα. one μl of mouse csf was sufficient to detect r-sappα as well as h-sappα and detection was linear for serially diluted samples. this level of sensitivity may allow the levels of the human transgenic sappα to be followed through the progression of the disease, which has never been reported previously. csf samples were collected from wild type or 3xtg ad mice and 1/ or 1/ dilutions of the csf were tested in the r-sappα and in the h- sappα assays.#: this value includes 23% of h- sappα. sappα assay development: in vivo applications in human we then assessed h-sappα levels in the csf of ad and non-ad patients. high levels of h-sappα in the csf determined using a multiplex technology were reported to be associated with ad. in this study, we evaluated our h-sappα assay using csf samples from patients who had been classified according to validated criteria as either non-ad or ad by a team specialized in the diagnosis of neurodegenerative disorders. these patients were matched according to sex and age and significantly differ by the mmse test. we checked by western blotting in some csf samples the presence of two sappα isoforms recognized by both 22c and 6e antibodies and the absence of specific detectable cleaved isoforms. the two sappα isoforms of molecular weights and 125 kda detected are similar to that observed in soluble fraction of mouse brains and correspond to mature glycosylated forms of sappα. however we cannot exclude that the higher isoform correspond to the sappα containing kpi domain and present in glial cells. the bands of lower mw and lower intensity detected were non specific as recognized by the anti-mouse secondary antibody. in our population, aβ1- concentrations were lower and tau and ptau concentrations were higher in the ad group than in the non-ad group. we found significantly higher sappα concentrations in ad csf compared to non-ad csf, which agrees with recent studies in which the sappα concentrations in csf were determined by a multiplex technology. ad: patients with alzheimer’s disease; non-ad: patients with other dementias; m: male; f: female. mmse: mini mental state examination. **: p < . furthermore, our test detected h-sappα in as little as  μl of csf, which is a vast improvement over elisa tests and multiplex technology, which require sample volumes of 100 μl and 25 μl, respectively. the low amounts of csf required by our test will allow large clinical and longitudinal investigations in patients to validate sappα as an ad biomarker.  this is the first description of two highly sensitive tests that can be used to detect sappα in a variety of samples. samples include the conditioned media of human neuroblastoma cells and mouse primary neurons, as well as very small samples of biological fluids, such as mouse and human csf. these new tools will have very wide applications from pre-clinical to clinical studies. together, their rapidity, sensitivity and small incubation volumes will facilitate high-throughput investigations of drugs capable of increasing the secretion of endogenous sappα from primary neurons. the tests may be subsequently validated for in vivo analyses by using them to evaluate at different time points sappα levels in the csf of wild-type mice or ad mouse models treated with molecules that exhibit activity in in vitro screening assays. finally, the human sappα test will allow sappα levels to be measured in the csf of patients participating in large clinical investigations. 
these μl-volume tests quantify the levels of: i) endogenous mouse sappα in the conditioned medium of mouse neuron primary cultures, as well as in the csf of wild-type mice, ii) human sappα in the csf of ad mouse models, and iii) human sappα in the csf of ad and non-ad patients. finally, the human sappα test could strengthen the biological diagnosis of ad in large clinical investigations. taken together, these new tests have a wide field of applications in preclinical and clinical studies. amyloid precursor protein, a key molecule in alzheimer’s disease, is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in ad pathology or the soluble form of app. alzheimer’s diseasesoluble amyloid precursor protein alphahomogeneous time-resolved fluorescencerodenthumancerebrospinal fluidprimary neuronssensitivity the high sensitivity of the mouse sappα test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sappα in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sappα in the csf of treated wild-type mice. we describe two new rapid and sensitive tests for quantifying mouse and human sappα. active molecules could then be tested in the ad mouse models by quantifying human sappα in the csf through the progression of the disease. to this end, very sensitive tests for preclinical and clinical research are required. these tests require only 5 μl of conditioned medium from 5 ×  mouse primary neurons, 1 μl of csf from wild-type and transgenic mice, and  μl of human csf.
 amyloid precursor protein, a key molecule in alzheimer’s disease, is metabolized in two alternative cleavages, generating either the amyloidogenic peptides involved in ad pathology or the soluble form of app. the level of amyloidogenic peptides in human cerebrospinal fluid is considered to be a biomarker of ad, whereas the level of sappα in csf as a biomarker has not been clearly established. sappα has neurotrophic and neuroprotective properties. stimulating its formation and secretion is a promising therapeutic target in ad research. to this end, very sensitive tests for preclinical and clinical research are required. methods the tests are based on homogenous time-resolved fluorescence and require no washing steps.  we describe two new rapid and sensitive tests for quantifying mouse and human sappα. these μl-volume tests quantify the levels of: i) endogenous mouse sappα in the conditioned medium of mouse neuron primary cultures, as well as in the csf of wild-type mice, ii) human sappα in the csf of ad mouse models, and iii) human sappα in the csf of ad and non-ad patients. these tests require only 5 μl of conditioned medium from 5 ×  mouse primary neurons, 1 μl of csf from wild-type and transgenic mice, and  μl of human csf.  the high sensitivity of the mouse sappα test will allow high-throughput investigations of molecules capable of increasing the secretion of endogenous sappα in primary neurons, as well as the in vivo validation of molecules of interest through the quantification of sappα in the csf of treated wild-type mice. active molecules could then be tested in the ad mouse models by quantifying human sappα in the csf through the progression of the disease. finally, the human sappα test could strengthen the biological diagnosis of ad in large clinical investigations. taken together, these new tests have a wide field of applications in preclinical and clinical studies. alzheimer’s diseasesoluble amyloid precursor protein alphahomogeneous time-resolved fluorescencerodenthumancerebrospinal fluidprimary neuronssensitivity 