cervical cancer is the second most frequent cancer in women in the world , comprising about 10% of all human cancers . a current summary report from who indicates that there are about 500 thousand women diagnosed with cervical cancer , of whom about 270 thousand women die from the disease every year . in china ,
a total of 500 million women are at risk of cervical cancer . after more than 30 years of research into the infectious nature of cervical cancer
, it is well established that human papillomaviruses ( hpvs ) are the primary cause for cervical cancer .
up till now , over 130 different types of hpv have been found , where hpv-16 and hpv-18 are of high risk , contribute to over 70% of all cervical cancer cases .
therefore , prevention of hpv-16 and hpv-18 's infection among women becomes an important issue for scientists and epidemiologists to lower the incidence of cervical cancer .
hpvs are double - stranded dna viruses , which target the basal cells of squamous epithelia for infection .
the structure of its circular dna genome are composed of 2 major oncogenes e6 and e7 , 2 major structural protein genes l1 and l2 . based on these elements ,
the l1 protein of hpv expressed recombinantly in vitro can self - assemble into virus - like particles ( vlps ) .
vlps have hpv type - specific conformational neutralizing epitopes and thus induce type - specific neutralizing antibodies to protect against hpv infections . in line with this fact ,
large pharmaceutical companies like merck and gsk have developed vlp - based vaccine products and successfully put them into the market , using yeast and insect cell expression systems , respectively . both of these two products include vlps for hpv-16 and hpv-18 . in our company ,
an l1 vlp - based vaccine candidate aiming at hpv-16 and hpv-18 was also produced using yeast protein expression system [ 9 , 10 ] .
it includes vlp-16 and vlp-18 and thus should prevent against over 70% of cervical cancers . for l1 vlp - based vaccines ,
the efficacy is largely dependent on the number of neutralizing antibodies induced by the conformational neutralizing epitopes existing on vlp surfaces .
the more the neutralizing antibodies are induced , the more the vaccine should be effective to protect against hpv infections . to date , different methods have been developed to quantitate the type - specific neutralizing antibodies for hpvs , such as pseudoneutralization assays   and crias .
for this reason , several studies have used a luminex - based competitive immunoassay [ 13 , 14 ] which could be used to quantify different type - specific neutralizing antibodies in a single reaction simultaneously . in the assay ,
different types of vlps were bound with different kinds of microspheres , then the known , hpv type - specific , phycoerythrin - labeled neutralizing monoclonal antibodies ( mabs ) were mixed with vlp - coupled microspheres and the vaccine challenged serum samples for reaction , where the pe - labeled neutralizing mabs compete with the serum antibodies to bind to the conformational epitopes on vlps .
after that , the microspheres were put into luminex systems for detection of the pe signal . therefore ,
strong signals mean more pe - labeled mabs binding , and thus less neutralizing antibodies existed in the serum sample . in order to quantitatively evaluate the high and low levels of neutralizing antibodies ,
serum from african green monkeys hyperimmunized with hpv l1 vlps was reported   to be used as reference standards successfully .
an alternative is to make use of normal human serum spiked with unlabeled type - specific neutralizing monoclonal antibodies as reference standards . in this study
, a 2-plex luminex - based competitive immunoassay was developed to evaluate the efficacy of our hpv-16 and hpv-18 l1 vlp - derived vaccine candidates .
the reference standard was prepared using normal human serum spiked with various amounts of no - fluorescence labeled type - specific neutralizing mabs rather than hpv - positive serum .
the fluorescence - labeled neutralizing mabs were not coupled directly with pe but with biotin first , and an amount of streptavidin - pe was added then for detection , which makes the assay more flexible . in developing the assay , at first ,
the coupling ratio of vlps to microspheres and the coupling ratio of biotin to type - specific neutralizing mabs were optimized , the mixed vlp - coupled microspheres were blended with the mixed biotinylated neutralizing mabs to confirm the lack of cross - neutralizing activity , and finally a 2-plex standard curve for the competitive immunoassay was constructed for neutralizing antibody detection in serum samples .
in this study , the microspheres bio - plex cooh bead 038 and bio - plex cooh bead 053 were purchased from bio - rad corporation , hercules , calif .
the hpv-16 l1 vlp and hpv-18 l1 vlp were produced by yeast protein expression system in the laboratory of shanghai zerun biotechnology co. , ltd .
the neutralizing monoclonal antibodies ( mabs ) h16.v5 for hpv-16 and h18.j4 for hpv-18 were kindly provided by dr .
the luminex 200 system was purchased from luminex corporation , and data was collected and analyzed with luminex is 2.3 and prism 4 .
microspheres were incubated in 96-well 1.2  m pvdf filter microtiter plates ( millipore , billerica , mass ) and rotated in ms 3 digital ( ika , janke & kunkel - str .
the washing steps were carried out in bio - plex pro ii wash station ( bio - rad , hercules , calif ) .
vlps were coupled to microspheres according to the previously published methods with a few modifications [ 13 , 14 ] .
l1 vlp ( vlp-16 ) and hpv-18 l1 vlp ( vlp-18 ) were covalently conjugated to bio - plex cooh bead 038 ( # 38 beads ) and bio - plex cooh bead 053 ( # 53 beads ) , respectively , as follows .
initially carboxylated beads ( 1.25  10 beads / ml ) were fully suspended by vortexing and sonication , and then about 500  l beads were transferred to a 1.5  ml protein lobind tube ( eppendorf ) for coupling .
after centrifuging the tube at 14000  g for 4  min , the supernatant was removed .
then the beads were washed once with 500  l 0.1  m nah2po4 ( ph 6.2 ) by vortexing and sonication , followed by an incubation with 50  l 50  mg / ml sulfo - nhs ( pierce , rockford , il ) , 70  l 0.4  m 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride ( edc ) ( ge , shanghai , china ) , and 380  l pbs ( ph 7.4 ) for activation , where amine - reactive esters of carboxylate groups were formed for labeling .
the activation lasted in darkness for 30  min , at 8001000  rpm , rt . after that , the beads were washed twice with 50  mm mes buffer ( sigma , ph 6.0 ) and pelleted in mes buffer before counting using a hemocytometer .
the hpv l1 vlps were transferred to 50  mm mes buffer ( ph 6.0 ) by ultrafiltration , and then its concentration was determined by bca ( bca protein assay kit , pierce , rockford , ill ) before use . in coupling , 1  10
activated beads and buffer with transferred vlps were mixed in mes buffer to a final volume of 100  l200  l . the mixture was rotated in darkness , at 8001000  rpm , rt , overnight . consequently , the beads were washed twice with pbs plus 0.05% tween-20 ( ph 7.4 ) , followed by blocking in histidine buffer ( 20  mm histidine ( sigma ) , 0.5  m nacl , 1%  bsa , ph 6.2 ) for 30  min in darkness , rt , at 8001000  rpm .
after washing the beads twice with histidine buffer , they were counted by hemocytometer and diluted to 1  10 beads / ml in histidine buffer for preservation in darkness , at 4c . the hpv-16 and hpv-18 neutralizing mabs
h16.v5 and h18.j4 were biotinylated using micro sulfo - nhs - lc - biotinylation kit ( pierce ) . the procedure followed the manufacturer 's instructions . in brief , neutralizing monoclonal mabs were transferred to pbs ( ph 7.27.4 ) and subjected to bca ( bca protein assay kit , pierce ) to determine concentration before biotinylation .
an amount of active sulfo - nhs - lc - biotin for different mole ratios of biotin and mab was then added , and the mixture was rotated at 8001000  rpm in darkness , rt , for 45  min .
finally , the biotinylated beads were put at 20c immediately to stop the reaction and stored before use .
the biotinylation of h16.v5 and h18.j4 was confirmed to determine the optimal mole ratio of biotin and mabs for reaction .
5000 vlp-16 conjugated # 38 beads ( 25  l , 2  10 beads / ml , coupling ratio = 40  g protein per million beads ) or vlp-18 conjugated # 53 beads ( 25  l , 2  10 beads / ml , coupling ratio = 40  g protein per million beads ) were mixed with 25  l of diluted biotinylated h16.v5 ( biotin / h16.v5 mole ratio = 20/1 , 40/1 , 80/1 , 160/1 , 320/1 , 640/1 , 1280/1 ) or biotinylated h18.j4 ( biotin / h16.v5 mole ratio
= 20/1 , 40/1 , 80/1 , 160/1 , 320/1 , 640/1 , 1280/1 ) and 50  l pbs - bsa to a final volume of 100  l , where the biotinylated mabs were 20-fold diluted , followed by a 2-fold serious dilution in a total of 11 different dilutions
. then the microtiter plate was rotated at 8001000  rpm , in darkness , for 3  h , followed by 3 times of wash in wash station .
after that , 50  l 2.5  g / ml streptavidin - pe was added for incubation at 8001000  rpm , in darkness , for 20  min .
the beads were then washed 3 times and resuspended in 125  l pbs - bsa for reading in luminex 200 system ( luminex corporation , austin , tex ) . after the coupling reaction of hpv l1 vlps to microspheres has been completed , the coupling efficiency was confirmed . in brief , 5000 vlp-16 conjugated # 38 beads ( 25  l , 2  10  beads / ml ) or vlp-18 conjugated # 53 beads ( 25  l , 2  10  beads / ml ) were mixed with 25  l biotinylated h16.v5 ( biotin / h16.v5 mole ratio = 100/1 ) or biotinylated h18.j4 ( biotin / h18.j4 mole ratio = 100/1 ) and 50  l pbs - bsa buffer ( pbs , 1% bsa , ph 7.4 ) to a final volume of 100  l , for incubation in a 96-well prewetted filter plate , in darkness , at 8001000  rpm for 3  h , where the biotinylated mab was 1  :  50 , 1  :  100 , 1  :  1000 diluted in pbs - bsa buffer ( pbs , 1% bsa , ph 7.4 ) . then the beads were washed 3 times in bio - plex pro ii wash station ( bio - rad ) using pbs - bsa buffer . after that , 50  l 2.5  g / ml streptavidin - pe was added , followed by incubation in darkness , at 8001000  rpm for 20  min . after washing the beads again for 3 times with the wash station , 125  l pbs - bsa buffer was added for reading in luminex 200 system ( luminex corporation ) .
the normal human serum spiked with different amounts of the no - biotin labeled neutralizing mabs was used as the standard .
it was prepared as follows : initially the normal human serum ( nhs ) was blended with certain amounts of unlabeled h16.v5 and h18.j4 to a final concentration of 20  g / ml . then , the mixture was 2-fold diluted with nhs to make h16.v5 and h18.j4 in a series of 15 different concentrations .
for each 50  l diluted sample , 5000 vlp-16 conjugated # 38 beads ( coupling ratio = 40  g protein per million beads ) , 5000 vlp-18 conjugated # 53 beads ( coupling ratio = 40  g protein per million beads ) , 500-fold diluted ( final dilution ) biotinylated h16.v5 ( biotin / h16.v5 mole ratio = 320/1 ) , and 200-fold diluted ( final dilution ) biotinylated h18.j4 ( biotin / h18.j4 mole ratio = 640/1 ) were added to a final volume of 100  l , followed by an incubation in 96-well prewetted filter plate , in darkness , at 8001000  rpm for 3  h. after washing in triplicate by wash station using pbs - bsa buffer , the beads were added with 50  l 2.5  g / ml streptavidin - pe and rotated at 8001000  rpm , in darkness , for 20  min for staining .
finally the beads were washed again in triplicates and resuspended in 125  l pbs - bsa buffer before reading in luminex 200 system ( luminex corporation ) .
all assays were conducted in duplicates . in brief , 5000 vlp-16 conjugated # 38 beads ( coupling ratio = 40  g protein per million beads ) and 5000 vlp-18 conjugated # 53 beads ( coupling ratio = 40  g protein per million beads ) were added with 40-fold diluted ( final dilution ) biotinylated h16.v5 ( biotin / h16.v5 mole ratio = 320/1 ) and 200-fold diluted ( final dilution ) biotinylated h18.j4 ( biotin / h18.j4 mole ratio = 320/1 ) or both .
different ratios ( g / million beads ) of hpv-16 l1 vlp or hpv-18 l1 vlp and # 38 or # 53 microspheres were used for coupling , respectively .
the vlp coupled microspheres were then reacted with the same amount of related neutralizing mabs .
the coupling ratio at which the vlp - conjugated beads can generate the strongest signal was then selected as the optimal .
the coupling reaction for vlp-16 and # 38 beads was saturated when coupling ratio was bigger than 40  g protein per million beads ( figure 1(a ) ) , while for vlp-18 the optimal coupling ratio was 40  g protein per million beads ( figure 1(b ) ) .
another concern for choosing 40  g protein per million beads was to find a robust way for coupling ; more vlps used could ascertain the same quality of coupled beads at each time .
it would be important in qc and clinical trial .   for biotinylation of neutralizing mabs h16.v5 and h18.j4 , different coupling mole ratios of amine reactive biotin and mab were tried , and the biotinylated mabs were then mixed with vlp - conjugated microspheres for reaction .
the optimal coupling mole ratio was the one at which the reaction could generate the strongest signal .
for both h16.v5 and h18.j4 , the optimal coupling ratios of biotin to mabs were 320/1 or higher ( figures 2(a ) and 2(b ) ) , where the biotinylation reaction was saturated at the current detection system .
when changing our system , for example , the added amount and concentration of streptavidin - pe , the optimal coupling ratio for biotinylation may be higher , because there may be not enough streptavidin - pe for staining . before mixing the 2 vlp - conjugated microspheres together for immunoassay ,
studies were carried out to make sure that no cross - reactivity existed between the 2 analytes .
just as predicted , with our produced hpv vaccine candidates , biotinylated h16.v5 could only bound to vlp-16 coupled microspheres for signaling while biotinylated h18.j4 only bound to vlp-18 coupled microspheres ( figure 3 ) .
the results ascertained the establishment of the 2-plex immunoassay with high sensitivity and specificity . in this section ,
the saturated vlp coupled beads plus the saturated biotin coupled mab just generate no cross - reactivity .
the coupling ratios for vlps to microspheres were both 40  g protein per million beads .
not surprisingly , standard curve preparations ( figure 4 ) for the 2-plex luminex - based competitive immunoassay were established successfully with good fit ( r > 0.99 ) for both analytes and wide dynamic range ( 9.8  ng / ml to 5.0  g / ml for vlp-16 neutralizing antibodies , while 39.0  ng / ml to 5.0  g / ml for vlp-18 neutralizing antibodies ) , where the no - biotinylated mabs ( standards ) and biotinylated mabs competitively bind to hpv vlp coupled microspheres .
luminex xmap system is a technology combined with flow cytometry , microspheres , and lasers together , which allows the quantitation of up to 100 different analytes simultaneously using quite small sample volumes in a single reaction .
it has been applied to hini detection , kidney biomarkers detection , human leukocyte antigen genotyping , cytokine detection and quantitation , and so forth . with high sensitivity and specificity .
cervical cancer , as the second prevalent cancer in women in the world , was caused by hpv infections .
some studies have revealed that hpv l1 vlp - based vaccine could effectively induce type - specific neutralizing antibodies to hpvs and thus protect against their invasions . in order to make the vaccine more powerful
, it was often designed to target more than one type of hpvs , for instance , merck 's gardasil aimed at hpv-6 , -11 , -16 , and -18 simultaneously .
as a sensitive , economic and high - throughput method , luminex system was reported [ 13 , 14 ] to evaluate the efficacy of the hpv l1 vlp - based vaccine products .
compared to the traditional elisa- or facs - based method , luminex has many advantages .
it could be used to quantitate type - specific neutralizing antibodies induced by various hpvs simultaneously and thus was more practical for clinical use , where large number of serum samples should be analysed in a short time . here , in order to establish an assay for efficacy determination of our hpv-16 , -18 l1 vlps based vaccine candidate , also for further clinical use , a 2-plex luminex - based competitive immunoassay was also developed .
although the reported method represents an important reference for this study , some modifications were tried to make the assay more flexible in the practical use . when developing the immunoassay , first of all , the amount of vlp needed for coupling with microsphere should be determined to generate constant and strong signals . for the 2-plex method ,
2 different beads were prepared for hpv-16 l1 vlp and hpv-18 l2 vlp , respectively .
interestingly , the optimal coupling ratio for vlp-18 was 40  g / million beads , while for vlp-16 the coupling would be saturated if the ratio was bigger than 40  g / million beads , which may be due to the different neutralizing epitopes existed in vlp-16 and vlp-18 .
on the other hand , in this step , it was also found that low - binding tube was very essential for coupling of high quality ( data not shown ) , while the conventional tube may absorb microspheres during the reaction and thus lower the recovery .
the amount of biotin needed for biotinylation of neutralizing mabs should also be optimized . in the reported method ,
the signal dye pe was bound directly to mabs , which we thought was inflexible for the assay 's practical use . sometimes , it is difficult to obtain a stable pe labeled mab or other molecules when several new analytes are to be added to this multiplex assay .
in contrast , biotin labeling technology is highly developed , and it is simple to obtain stable biotinylated mab .
in fact , biotinylated mab plus streptavidin - pe was commonly used for signaling in most elisa , luminex , or other immunoassay - based systems . in this study ,
just as predicted , with our produced hpv-16 and hpv-18 l1 vlps based vaccine candidate , no cross - activity was found between h16.v5 and h18.j4 , which were well - characterized , strong type - specific hpv-16 and hpv-18 neutralizers .
after that , the 2-plex standard curve ( std ) of the competitive immunoassay for quantifying antibodies neutralizing hpv-16 and hpv-18 simultaneously was then tried to be constructed . unlike the published paper from merck , normal human serum spiked with no biotin labeled neutralizing mabs was used for standards , instead of vlp challenged monkey serum .
the major reason for that is the difficulty in preparing enough well - characterized and standardized serum for the assay . in this study ,
the 2-plex std for hpv-16 and hpv-18 was well established with high sensitivity and wide dynamic range . later
the assay was tried to measure the induced neutralizing antibodies in vlp challenged mice serum . it could be distinguished with different antibody levels for hpv-16 and hpv-18 simultaneously , of samples derived from mice challenged with our developed vlp vaccine candidate of different doses ( data not shown ) .
therefore , the 2-plex luminex - based assay provided a fast , sensitive , and effective approach to evaluate the efficacy of our hpv l1 vlp - based vaccine candidate . at present , the dilution buffer used for assay was normal human serum for clinical sample use .
when applying the assay to monkey or mouse serum samples , the dilution buffer should better use normal monkey serum or normal mouse serum accordingly because it is commonly used as the dilution matrix or blank .
the published paper suggests the use of antibody - depleted human serum ( adhs ) as dilution buffer because of its reliable and stable resources , which we thought could also be tried in our developed systems in the future .
in addition ,  the unit  used for characterizing the level of neutralizing antibodies in serum samples should be defined . in doing this
, large number of serum samples with neutralizing antibodies of low , medium , and high levels should be applied to the same std curve , to further validate the assay .
a 2-plex luminex - based competitive immunoassay was developed to evaluate the efficacy of our hpv l1 vlp - based vaccine candidate in inducing neutralizing antibodies against hpv-16 and hpv-18 .
the assay was convenient , sensitive , effective , and requiring only small amount of samples .
recently , additional studies from our laboratory are underway to further validate the assay and also determine the method limitation with plenty of serum samples .
the correlations of the assay to other characterization method for hpv l1 vlp - based vaccines like pseudoneutralization assay   and ivrp   were also investigated . moving forward ,
as researches on the assay become more comprehensive , the 2-plex luminex assay will be routinely applied for evaluating and quantifying neutralizing antibodies in clinical use .
 human papillomavirus ( hpv ) l1 virus - like particles ( vlps ) were proven an effective vaccine candidate to prevent against hpv-16 and -18 infections . in order to evaluate the potency of our produced hpv-16 and -18 l1 vlps - based vaccine candidates , also to quantify neutralizing antibodies induced by them , 
 a 2-plex luminex - based competitive immunoassay was developed . 
 unlike the published paper , the no - biotin conjugated neutralizing mabs spiked normal human serum ( nhs ) was used for standard curve preparation , while phycoerythrin ( pe ) was not labeled directly to neutralizing mabs for signaling . 
 after the coupling optimization of vlps to microspheres and the neutralizing mabs biotinylation , the 2-plex standard curve was prepared with good fit and high dynamic range . 
 in addition , no cross - reactivity was also confirmed . 
 the 2-plex luminex - based immunoassay represents good potential not only for vaccine candidate 's evaluation but also for its further clinical use . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions