protein interactions , cellular recognition , 
 and , in particular , cancer development and progression . increasing evidence has indicated that abnormal posttranslational 
 modifications are associated with cancer progression and that potential 
 biomarkers may be identified based on their changes in protein modifications . glycosylation changes in serum proteins have been reported to contribute 
 to the progression of various cancers , including pancreatic , ovarian , hepatocellular , and breast cancer . therefore , analysis of glycosylation changes of serum glycoproteins 
 may provide a promising strategy to identify new diagnostic biomarkers . methods to identify and quantify variations in glycosylation in 
 complex biological samples have been widely established , which are 
 mainly based on the removal of glycans by enzymatic digestion , followed 
 by chromatographic separation and mass spectrometry analysis . these methods can provide detailed information about glycan structures 
 but are not suitable for analyzing a large number of biological samples . 
 because of the lack of glycopeptide standards , srm / mrm assays , which 
 have been widely used to quantify protein level changes , are not optimal 
 for quantifying glycosylation changes of target proteins . because 
 a number of glycosylated proteins in serum have been identified as 
 promising candidate biomarkers , an improved method for quantification 
 of glycosylation changes of proteins from original sera is needed . recently , lectin - based antibody microarrays and lectin - elisa assays 
 have been developed to analyze glycosylation changes of proteins from 
 serum samples . the lectin - based antibody microarrays and the lectin - elisa 
 assay are both based on coating antibodies to the slides or 96-well 
 plates , where the glycans on the antibodies need to be oxidized by 
 sodium periodate ( naio4 ) , followed by derivatization with 
 mpbh and dipeptide solution ( cys - gly ) . unfortunately , periodate oxidation 
 can diminish or inactivate antibody immunoreactivities , which may increase nonspecific binding and lead 
 to inaccurate quantitative results . furthermore , precipitates are 
 formed during the process of oxidation with naio4 , which 
 can increase background if not completely removed . herein , we have developed an alternative method , 
 a reverse lectin - based 
 elisa assay to quantify glycosylated proteins from crude serum samples 
 without the oxidization and derivatization of glycans on the antibodies . because fucosylation / sialylation changes have been reported in a number 
 of cancers and are regarded as promising targets of cancer diagnosis 
 and therapy , in this study , we mainly focused 
 on optimizing the reverse lectin - based elisa assay to quantify fucosylation / sialylation 
 changes of target proteins in ovarian cancer . the reverse lectin - based 
 elisa assay is based on coating lectin on the microtiter plate followed 
 by detection of captured glycoproteins with biotin / hrp - conjugated 
 primary antibodies . the concentration of lectins coated on the 96-well 
 plate was optimized to achieve a low background compared with the 
 current lectin - elisa format . with this method , we confirmed the increased 
 expression of fucosylated haptoglobin in ovarian cancer , especially 
 in early - stage ovarian cancer . our findings suggest that fucosylated 
 haptoglobin could be a promising candidate biomarker , which can supplement 
 the clinically used biomarker ca125 ( cancer antigen 125 ) to detect 
 ovarian cancer with improved sensitivity and specificity . all clinical investigations were conducted 
 according to the principles expressed in the declaration of helsinki . the set of serum samples used in this study comprised 15 healthy controls , 
 12 benign diseases , 21 early - stage ovarian cancers , and 29 late - stage 
 ovarian cancers . all of the healthy controls were provided by the 
 great lakes - new england cvc ( edrnglne ) . serum samples from 12 benign 
 diseases and 17 late stage ovarian cancers were collected preoperatively 
 at the university of michigan as part of an irb - approved tumor banking 
 protocol , while the other samples were obtained from proteogenex ( manhattan 
 beach , ca ) . fibroids 
 and ovarian thecoma for 
 benign diseases ; poorly differentiated adenocarcinoma and fallopian 
 tube carcinoma for ovarian cancer . for some ovarian cancer patients , 
 the grade information was not available . a reverse 
 lectin - based elisa assay was developed to analyze glycosylation changes 
 of proteins from crude serum samples based on the differential binding 
 of glycoproteins to their specific lectins . one hundred microliters 
 of lectin ( 1.25 , 2.5 , 5 , 10 , 50 , 100 g / ml aal or 1 , 5 , 10 , 
 50 g / ml sna ) was added to each well of a 96-well elisa plate 
 ( thermo scientific , il ) and incubated at 37 c for 2 h. after 
 the lectin solution was removed , the plate was washed five times with 
 pbst ( 0.1% tween-20 in pbs ) . the plate was then blocked with 3% bsa 
 in pbst for 1 h. one hundred microliters of each 200-fold diluted 
 serum sample or 2 g of purified protein was applied to each 
 well of a 96-well elisa plate . after 1 h of incubation , the plate 
 was washed with pbst five times to remove unbound proteins . one hundred 
 microliters of biotinylated / hrp - conjugated antibodies ( 1:1000 for 
 antihaptoglobin , and 1:50  000 for anti - igg ) purchased from 
 abcam ( cambridge , ma ) was added to bind with their corresponding antigens . the absorbance values were read on a microplate reader ( biotek , synergy 
 ht ) at a wavelength of 450 nm . human full - length 
 proteins of haptoglobin and igg were purchased from abcam ( cambridge , 
 ma ) . the 
 resolved proteins were then transferred onto a pvdf membrane ( bio - rad ) . the membrane was blocked by 3% nonfat milk in pbst ( 0.1% tween-20 
 in pbs ) for 1 h. the membrane was probed with 0.5 g / ml biotinylated 
 aal or biotinylated sna to bind with their preferred oligosaccharides . hrp - conjugated streptavidin ( 1 g / ml ) was then added , and the 
 blot was detected by dab detection kits . the 
 protein abundances of hap , igg , and 
 ca125 in sera of ovarian cancer and control groups were measured by 
 elisa assay . elisa kits for hap , igg , and ca125 were all purchased 
 from genway ( san diego , ca ) . statistical differences were determined 
 using wilcoxon rank - sum test . for all statistical comparisons , p < receiver 
 operating characteristic ( roc ) curves were produced in terms of the 
 sensitivity and specificity of markers at their specific cutoff values . multivariate analysis was also done by logistic regression to find 
 the best - fitting multivariate model for each comparison group . using the reverse lectin - based elisa 
 assay , we found high responses of haptoglobin and igg to aal or sna . 
 to exclude false - positives , we used lectin - blots to verify the presence 
 of fucosylated / sialylated glycans on haptoglobin and igg . aal is a 
 lectin that responds to fucose linked ( -1,6 ) to n - acetylglucosamine or to fucose linked ( -1,3 ) to n - acetyllactosamine related structures , while sna responds to sialic 
 acid attached to terminal galactose in -2,6 or -2,3 
 linkage . as shown in supplemental figure s1 in the supporting information , the fucosylated / sialylated haptoglobin 
 and igg were detected by the aal - blot and sna - blot . the results confirmed 
 the presence of fucosylated / sialylated glycans on the haptoglobin 
 and igg , which had responses to aal / sna detection . the workflow 
 of the reverse lectin - based elisa assay is shown in 
 figure 1 . in this experiment , a number of control 
 determinations with the removal of serum or lectin from the mixture 
 of reverse lectin - based elisa assay were tested to avoid potential 
 interfering factors . to reduce the background caused by nonspecific 
 binding of serum proteins or biotinylated / hrp - conjugated antibodies 
 to the 96-well plate , a buffer containing 1% bsa is used for the dilution 
 of the serum and antibodies . in addition , we also measured the absorbance 
 values when lectin , serum , or antibody was omitted from the reverse 
 lectin - based elisa assay . to measure sialylated haptoglobin by reverse 
 sna - based elisa assay , low absorbance values were obtained when lectin 
 or serum was removed from the assay . the results indicate that there 
 was no nonspecific binding of serum proteins to the plate , and the 
 glycans on the antibodies had no effect on detection of the sialylated 
 haptoglobin . to measure fucosylated haptoglobin by reverse aal - based 
 elisa assay , low absorbance values were obtained when lectin was omitted 
 from the assay , while a slightly higher absorbance value was obtained 
 when serum was left out of the assay , indicating the presence of fucosylated 
 glycans on the antibody . the s / n ratio of the reverse aal - based elisa 
 assay for detecting fucosylated haptoglobin is still higher than 3 , 
 as shown in supplemental table s1 in the supporting 
 information . therefore , the low binding of aal to the glycans 
 on the antibodies did not influence the quantification of fucosylated 
 haptoglobin from the serum samples . diagram of reverse lectin - based elisa 
 assay for the analysis of 
 glycosylation of target glycoproteins . because of the high abundance of igg in the serum , we observed 
 absorbance values when lectin was left out of the reverse sna / aal - based 
 elisa assay . however , compared with the high absorbance values for 
 the sample group , this low background did not influence the accuracy 
 of quantifying fucosylated or sialylated igg from crude serum samples . for detection of fucosylated igg by reverse aal - based elisa assay , high background was observed when serum was omitted from the assay , 
 indicating high binding of aal to the fucosylated glycans on the anti - igg 
 ( fc ) . attempts to reduce this background by oxidizing the glycans 
 on the anti - igg ( fc ) were unsuccessful . finally , we found that using 
 anti - igg ( fab ) in place of anti - igg ( fc ) could dramatically reduce 
 the background caused by the binding of aal to fucosylated glycans 
 on the antibodies . the high background obtained by anti - igg ( fc ) is 
 due to the presence of fucosylated oligosaccharide in its fc portion 
 ( supplemental table s1 in the supporting information ) . antibodies with the f(ab ) portion have been used in several studies 
 to eliminate the binding of lectin to glycans on the antibodies . to 
 reduce the background and increase the s / n ratio of the assay , 
 we further optimized the amount of lectin for well coating . the data 
 on optimization of aal concentration used for well coating are shown 
 in figure 2 . when the amount of aal for well 
 coating increased , higher intensities for both test and control ( no 
 serum ) group were obtained ( figure 2a ) . the 
 highest s / n ratio ( > 5 ) was obtained when 5 g / ml aal was 
 used 
 for coating the plate ( figure 2b ) . therefore , 
 5 g / ml aal was chosen for the well coating for the reverse 
 aal - based elisa assay . optimizing the amount of lectin for well coating . ( a ) 
 to obtain 
 the highest s / n ratios for the reverse aal - based elisa assay , we measured 
 the od values for the sample ( with serum samples / purified haptoglobin 
 incubation ) and control ( no serum / haptoglobin incubation ) groups after 
 different amounts of aal were coated on the microtiter plates . ( b ) 
 s / n ratios for reverse aal - based elisa assay with different amounts 
 of aal coated on the microtiter plates . ( c ) od values for sample and 
 control groups were determined after different amounts of sna were 
 coated on the microtiter plates . ( d ) s / n ratios for the reverse sna - based 
 elisa assay . compared with reverse 
 aal - based elisa assay , lower background was 
 obtained for the reverse sna - based elisa assay ( figure 2c ) , which may be due to the lower sialylation level of the 
 tested antibodies . the s / n ratio reached the maximum when 10 g / ml 
 sna was applied for well coating , and the higher concentration of 
 sna ( 50 g / ml ) did not increase the amount of bound protein . therefore , 10 g / ml sna was used for well coating for reverse 
 sna - based elisa assay , as shown in figure 2d . to verify that aal specifically bound to fucosylated 
 glycans on the glycoproteins rather than nonspecifically interacted 
 with other glycan structures , we measured aal binding performance 
 in the presence of competing sugars , l - fucose . as shown in 
 figure 3a , preincubation of aal with competing 
 sugar , l - fucose , resulted in a dramatic reduction in aal 
 binding to fucosylated haptoglobin after serum / protein incubation . the reduced binding of aal to fucosylated proteins using 
 its competing sugar indicates the specific binding of aal to fucosylated 
 glycans on the captured proteins . determining the specificity of the reverse 
 lectin - based elisa assay . ( a ) for reverse aal - based elisa assay , od values were measured after 
 the lectin coated on the microtiter plates had or had not been preincubated 
 with 100-fold molar excess of a competing sugar . ( b ) for reverse sna - based 
 elisa assay , od values were measured with removal of the sialic acid 
 on the glycoproteins . to study the specificity of the reverse sna - based elisa assay , 
 we tested the lectin binding to the captured proteins by treating 
 samples with neuraminidase to remove the sialic acid of the glycoproteins . after samples were treated with neuraminidase , no proteins were found 
 to bind to sna when detected by antihaptoglobin ( hrp ) , while serum / protein 
 without sialic acid removal showed the high binding to sna , as shown 
 in figure 3b . the results confirmed that lectins 
 specifically bind to glycans rather than nonspecifically interact 
 with proteins . the specificity of reverse aal / sna - based elisa assays 
 were also confirmed by testing protein igg ( supplemental figure s2 
 in the supporting information ) . our results 
 indicated that the reverse lectin - based elisa assay showed the specific 
 glycosylation changes of proteins . to further address the specificities 
 of the reverse lectin - based 
 elisa assay , we tested the specificity of the antibodies of haptoglobin 
 and igg used in this study . serum samples with haptoglobin- and igg - depleted 
 were used for the reverse lectin - based elisa assay to check whether 
 these two proteins can still be detected with their antibodies . as 
 shown in supplemental figure s3 in the supporting 
 information , a significant reduction in antibody bindings to 
 their antigens the results further confirmed the specificity of the reverse 
 lectin - based elisa assay to quantify the fucosylation / sialylation 
 changes of haptoglobin and igg from crude serum samples . to compare samples analyzed on different microtiter 
 plates or in different laboratories , standard samples need to be used 
 to normalize the results . because most of standard samples of differentially 
 fucosylated / sialylated proteins are not commercially available , purified 
 proteins were used to establish the standard curves in this study . 
 as shown in figure 3 , haptoglobin showed a 
 linear response in sna / aal binding between 5 and 80 ng / ml ( r = 0.991 ) . although this type of standard curve 
 is not ideal to calculate the absolute concentrations of the glycosylated 
 proteins , it could be successfully used to enable comparisons of samples 
 analyzed on different microtiter plates or in different laboratories . the intra- and interassay precision of the reverse lectin - based 
 elisa assay was determined by repeated analysis of the serum samples 
 on the same ( in triplicate ) and different microtiter plates ( in duplicate ) . the coefficient of variation ( cv ) for intra- and interassay precision 
 was 3.6 and 5.7% , respectively . one of the most important applications of the developed 
 reverse lectin - based elisa assays is to measure glycosylation changes 
 on serum proteins from a large cohort of patients . the fucosylation / sialylation 
 changes of haptoglobin and igg in sera of patients with ovarian cancer 
 were measured in duplicate by reverse lectin - based elisa assay . we 
 found the increased fucosylation levels of haptoglobin in ovarian 
 cancer , especially in early - stage cancer compared with healthy controls 
 or benign diseases ( figure 4 ) , while the sialylated 
 haptoglobin and igg as well as fucosylated igg showed no significant 
 changes ( supplemental figure s4 in the supporting 
 information ) . levels of ca125 , haptoglobin , and fucosylated haptoglobin 
 in sera 
 of patients with ovarian cancer were determined by elisa and reverse 
 lectin - based elisa assays . ca125 and fucosylated haptoglobin showed 
 significant changes between noncases ( normal and benign ) and cases 
 ( early - stage and late - stage ovarian cancer ) ( p < 
 0.01 ) . roc curves were constructed for 
 the changes in fucosylated glycoproteins 
 to distinguish cases ( late stage and early stage cancers ) from noncases 
 ( healthy controls and benign diseases ) . the clinically used marker 
 ca125 obtained the highest auc ( 0.86 ) to differentiate cancer from 
 noncases . the combination of ca125 and fucosylated haptoglobin 
 had an auc of 0.88 with specificity of 96.3% at a sensitivity of 78% , 
 which improved both sensitivity and specificity when compared with 
 ca125 alone ( figure 5 ) . it should be noted 
 that the fucosylated haptoglobin had an auc of 0.741 to distinguish 
 early stage from noncases , which was comparable to ca125 ( 0.795 ) . 
 the combination of ca125 and fucosylated haptoglobin resulted in an 
 auc of 0.855 , which outperforms ca125 to distinguish early - stage cancer 
 from noncases ( figure 5 ) . roc analyses for ca125 
 and fucosylated haptoglobin to differentiate 
 ovarian cancer from noncases . we performed a power analysis to determine the power of our 
 experiments . 
 at the given sample size , the variance of expression values , and the 
 difference we want to detect ( two - tailed , 0.05 ) the powers at the calculated differences 
 of the means ( delta mean ) of comparison groups of differentially expressed 
 fucosylated haptoglobin and ca125 are higher than 99% , which provides 
 the statistical support for the number of samples included in our 
 study . haptoglobin , a glycosylated protein , is mainly produced 
 in the 
 liver and composed of two  and two  subunits . increased fucosylated haptoglobin has also been observed in various 
 types of cancers , such as pancreatic cancer , hepatoma , prostate cancer , lung cancer , and 
 ovarian cancer . there are several key 
 advantages of the reverse lectin - based elisa method compared with 
 other methods . in these previous studies , to quantify fucosylated 
 haptoglobin , several high - abundance proteins such as igg were depleted , 
 or haptoglobin needed to be purified from serum samples before mass 
 spectrometry or lectin blotting analysis . also , a large quantity of 
 purified glycoprotein ( micrograms to milligrams ) is required for glycan 
 analysis using mass spectrometry , which needs at least 10 l 
 of sera , while for the reverse lectin - based elisa 
 assay , nanogram levels of protein or < 0.5 l serum is sufficient 
 to analyze the glycosylation changes of protein . because glycans need 
 to be released from glycoprotein purified from depleted serum samples 
 before ms analysis , contaminants from other glycoproteins may interfere 
 with glycan quantification of target proteins . in contrast , our results 
 showed high specificity of reverse lectin - based elisa assay for analyzing 
 the glycosylation changes of target proteins ( figure 3 and supplemental figure s3 in the supporting 
 information ) . recently , using glycopeptides cid ms / ms 
 and glycan database search , 
 chandler et al . have studied site - specific 
 n - glycosylation microheterogeneity of haptoglobin , which provided 
 detailed glycosylation patterns of haptoglobin . by site - specific glycan 
 analysis with lc however , these studies analyzed haptoglobin 
 glycans in a qualitative instead of a quantitative manner , which are 
 not applicable to quantify glycosylation changes of haptoglobin from 
 individual samples . quantification methods such as ms and lectin blotting 
 lack sensitivity , accuracy and high sample throughput , which may impede their application in clinical 
 examination . a system that is suitable for analyzing a large number 
 of specimens is required . in this study , the developed reverse lectin - based 
 elisa assay provides a reproducible method to quantify glycosylation 
 changes of proteins from crude serum samples . because biotinylated / hrp - conjugated 
 antibodies are required for the reverse lectin - based elisa assay , 
 the glycoproteins that lack commercial biotinylated / hrp - conjugated 
 antibodies because the abundance changes of the underlying protein 
 could account 
 for the detected glycosylation changes , an elisa assay was used to 
 measure the underlying protein concentrations of haptoglobin from 
 the original serum samples . as shown in figure 4 , fucosylated haptoglobin showed significant changes between cancer 
 and noncases , while its protein levels showed no significant changes 
 between cancer and control groups . the results support previous findings 
 that potential biomarkers may be identified based on their changes 
 in protein modifications rather than changes in protein abundance . our 
 study shows an effective method for the analysis of glycosylation 
 changes on glycoproteins from serum samples . this method can be used 
 not only to analyze the high - abundance serum proteins , but also to 
 quantify moderate - abundance proteins that are potential markers , as 
 described in our previous study . nevertheless , using this novel reverse lectin - based elisa assay , we 
 found that fucosylated haptoglobin could be a potential candidate 
 biomarker , which can be used to supplement ca125 for detecting ovarian 
 cancer with improved sensitivity and specificity . the method has distinct 
 advantages over current lectin - elisas that require oxidation of glycans 
 on the iggs to minimize interaction with the lectins . however , even 
 with this procedure , there is often significant background in these 
 lectin - elisa experiments compared with our current platform . in future work , there are various ways in which the reverse lectin - based 
 elisa method could be expanded and improved . it could be expanded 
 to a 384 well format to increase the throughput for a larger number 
 of samples . also , there has been recent work to improve the response 
 of lectin arrays based on oriented lectins using an immobilization 
 method . these oriented lectin arrays have been shown to markedly 
 increase the response of some 
 lectins to glycoproteins . nevertheless , this strategy has worked for many lectins and 
 could help in expanding this method to lower abundance glycoproteins .
altered 
 glycosylation in glycoproteins is associated with carcinogenesis , 
 and certain glycan structures and glycoproteins are well - known markers 
 for tumor progression . to identify potential diagnostic candidate 
 markers , 
 we have developed a novel method for analysis of glycosylation 
 changes of glycoproteins from crude serum samples using lectin - based 
 glycoprotein capture followed by detection with biotin / hrp - conjugated 
 antibodies . 
 the amount of lectin coated on the microplate well was 
 optimized to achieve low background and improved s / n compared with 
 current lectin elisa methods . in the presence of competing sugars 
 of lectin aal or with sialic acid removed from the glycoproteins 
 , 
 we confirmed that this method specifically detects glycosylation changes 
 of proteins rather than protein abundance variation . using our reverse 
 lectin - based elisa assay , increased fucosylated haptoglobin was observed 
 in sera of patients with ovarian cancer , while the protein level of 
 haptoglobin remained the same between cancers and noncases . the combination 
 of fucosylated haptoglobin and ca125 ( auc = 0.88 ) showed improved 
 performance for distinguishing stage - iii ovarian cancer from noncases 
 compared with ca125 alone ( auc = 0.86 ) . in differentiating early - stage 
 ovarian cancer from noncases , 
 fucosylated haptoglobin showed comparable 
 performance to ca125 . 
 the combination of ca125 and fucosylated haptoglobin 
 resulted in an auc of 0.855 , which outperforms ca125 to distinguish 
 early - stage cancer from noncases . 
 our study provides an alternative 
 method to quantify glycosylation changes of proteins from serum samples , 
 which will be essential for biomarker discovery and validation studies .