two dose Sclerostin antibody in cynomolgus monkeys increases bone formation bone density andstrength
Two Doses of Sclerostin Antibody in Cynomolgus Monkeys Increases Bone Formation,Bone Mineral Density,and Bone Strength
Michael S Ominsky,1Fay Vlasseros,2Jacquelin Jolette,2Susan Y Smith,2Brian Stouch,3
George Doellgast,3Jianhua Gong,1Yongming Gao,1Jin Cao,1Kevin Graham,4Barbara Tipton,4Jill Cai,5 Rohini Deshpande,5Lei Zhou,6Michael D Hale,6Daniel J Lightwood,7Alistair J Henry,7
Andrew G Popplewell,7Adrian R Moore,7Martyn K Robinson,7David L Lacey,1
W Scott Simonet,1and Chris Paszty1
1Metabolic Disorders,Amgen,Inc.,Thousand Oaks,CA,USA
2Bone Research,Charles River Laboratories,Preclinical Services Montreal,Senneville,Quebec,Canada
3Pharmacokinetics and Drug Metabolism,Amgen,Inc.,Thousand Oaks,CA,USA
4Protein Sciences,Amgen,Inc.,Thousand Oaks,CA,USA
5ATO Cell Sciences and Technology,Amgen,Inc.,Thousand Oaks,CA,USA
6Biostatistics,Amgen,Inc.,Thousand Oaks,CA,USA
7UCB-Celltech,Slough,United Kingdom
ABSTRACT
The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal.Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation.More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation,bone mass,and bone strength.To explore the effects of sclerostin inhibition in primates,we administered a humanized sclerostin-neutralizing monoclonal antibody(Scl-AbIV)to gonad-intact female cynomolgus monkeys.Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels(3,10,and30mg/kg),with study termination at2months.Scl-AbIV treatment had clear anabolic effects,with marked dose-dependent increases in bone formation on trabecular, periosteal,endocortical,and intracortical surfaces.Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content(BMC)and/or bone mineral density(BMD)at several skeletal sites(ie, femoral neck,radial metaphysis,and tibial metaphysis).These increases,expressed as percent changes from baseline were11to29 percentage points higher than those found in the vehicle-treated group.Additionally,significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group.Taken together,the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable,such as in fracture healing and osteoporosis.?2010 American Society for Bone and Mineral Research.
KEY WORDS:SCLEROSTIN;ANTIBODY;BONE FORMATION;BONE STRENGTH;PRIMATE
Introduction
B one-related conditions cause significant morbidity world-
wide,particularly among the elderly,and include systemic bone loss(eg,postmenopausal osteoporosis),focal bone loss,and traumatic fractures.(1)Bone is continuously remodeled, with osteoclasts resorbing bone and osteoblasts forming new bone.Various therapeutics are currently used for the treatment of low bone mass,with the vast majority being antiresorptive agents(eg,bisphosphonates)that act by decreasing the rate of
ORIGINAL ARTICLE J BMR
Received in original form September24,2009;revised form November11,2009;accepted December17,2009.Published online December21,2009. Address correspondence to:Chris Paszty,PhD,Metabolic Disorders,M/S14-1-B,One Amgen Center Drive,Amgen,Inc.,Thousands Oaks,CA91320-1799,USA. E-mail:cpaszty@https://www.wendangku.net/doc/035148663.html,
Parts of the manuscript were presented at the28th Annual Meeting of the American Society for Bone and Mineral Research,in Philadelphia,PA,September15–19, 2006
Journal of Bone and Mineral Research,Vol.25,No.5,May2010,pp948–959
DOI:10.1002/jbmr.14
?2010American Society for Bone and Mineral Research
948
osteoclast-mediated bone resorption,thereby preventing further bone loss and skeletal weakening.(2,3)However,because these agents cannot replace bone that has been lost,there has been a long-standing goal to develop therapeutics that can stimulate bone formation to increase bone mass and bone strength.It is thought that such bone-rebuilding anabolics could provide important treatment options not only for low-bone-mass conditions but also for fracture healing and other orthopedic applications.Currently, the only approved bone anabolic agents for osteoporosis are full-length and truncated parathyroid hormone(PTH),both of which are administered by daily subcutaneous injection.(2,3)
The human high-bone-mass genetic disease sclerosteosis is caused by lifelong absence of the osteocyte-secreted protein sclerostin and is characterized by increased osteoblast-mediated bone formation.(4–8)Similar to the human condition,sclerostin knockout mice have robust increases in bone formation,bone mass,and bone strength.(9)In ovariectomized(OVX)rats,a model of postmenopausal osteoporosis,short-term administration of a sclerostin-neutralizing monoclonal antibody(mAb)had strong anabolic effects with marked increases in bone formation on trabecular,periosteal,endocortical,and intracortical surfaces.(10) Bone mass and bone strength were increased to levels that exceeded those of age-matched non-OVX control rats,suggest-ing that antibody-mediated sclerostin inhibition might be a viable approach for the treatment of bone-related disorders. Furthermore,the powerful anabolic response seen in these aged OVX rats showed that sclerostin plays a pivotal role in the regulation of bone formation even during later stages of life when the incidence of bone-related disorders is highest.
In a mouse model of colitis,short-term treatment with a sclerostin mAb increased bone formation and bone strength, thereby countering the effects of accelerated bone loss caused by chronic inflammation.(11)The results of that study showed that inhibition of sclerostin has beneficial effects on bone in the physiologic setting of inflammation-induced bone loss.In rodent models of fracture healing,sclerostin mAb treatment resulted in increased callus density and bone strength at fracture sites and accelerated bone repair.(12)
Nonhuman primates are considered to be the most appropriate species to approximate human bone biology owing to similarities in cortical bone remodeling and response to estrogen with-drawal.(13–15)To further explore the clinical potential of sclerostin inhibition,we treated cynomolgus monkeys with a humanized sclerostin-neutralizing mAb(Scl-AbIV).Our broad aim in this study was to investigate the effects of antibody-mediated sclerostin inhibition in monkeys using a dosing interval and dose levels similar to what generally might be tested in early human trials for antibody therapeutics.To this end,the effects of once-monthly dosing of Scl-AbIV on serum bone turnover biomarkers,bone density, bone histomorphometry,and bone strength were examined in adolescent gonad-intact female monkeys for2months. Materials and Methods
Animals,treatment,and experimental design
Female cynomolgus monkeys(Macaca fascicularis)aged3to5 years(mean3.9years)were received from Primate Products,Inc.(Miami,FL,USA).Animals were socially housed in wall-mounted cages(2to3animals/cage)equipped with an automatic watering system.All animals had access to2050C Certified Global Primate Diet(PMI Nutrition International,Inc.)twice daily, containing0.93%calcium,0.75%phosphorus,and8.0IU of vitamin D/g,as well as daily food supplements including fresh fruit.The animal room environment was controlled,with settings targeted at temperature24?38C,humidity50?20%, photoperiod12hours of light and12hours of dark,and12air changes per hour.
An acclimation period of6weeks was allowed between animal receipt and the start of Scl-AbIV administration.Only animals considered in good health,with minimal skeletal abnormalities (by radiograph),and normal serum/urine chemistry panels were used in the study.Vehicle(PBS,n?4)or a sclerostin-neutralizing monoclonal antibody(Scl-AbIV)at3(n?2),10(n?3),or30mg/ kg(n?3)was administered subcutaneously in a volume of3mL/ kg on days1and29.These groups were balanced by baseline body weight and confirmed to have similar mean baseline lumbar bone mineral density(BMD)and content(BMC)by dual-energy X-ray absorptiometry(DXA).The study was terminated on day61when bones were harvested for analysis.Scl-AbIV is a high-affinity(K D less than15pM against cynomolgus sclerostin) humanized IgG2mAb produced in Chinese hamster ovary(CHO) cells.The study was performed at Charles River Laboratories Montreal,Senneville,Quebec,Canada.Protocols and procedures involving animals were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)accredited facility in accordance with the require-ments and guidelines of the US National Research Council and the Canadian Council on Animal Care and complied with the protocols approved by the Charles River Montreal Institutional Animal Care and Use Committee.All endpoints were collected at Charles River Laboratories Montreal except for the serum ELISAs for Scl-AbIV and Crosslaps,which were performed at Amgen.Inc. Serum levels of sclerostin antibody and biochemical markers of bone turnover
Blood samples were collected prior to dosing on days1and29 and0.5,1,2,4,6,13,20,and27days after each dose.Animals were food-deprived overnight(approximately12hours)prior to blood sampling on all occasions,except for predose on day1and day29and for the0.5day postdose bleeds,where food deprivation was for approximately7hours.Scl-AbIV concentra-tions were measured by sandwich ELISA in all treated serum samples.The bone-formation markers osteocalcin(OC)and intact N-terminal propeptide of type1procollagen(P1NP)were measured in all serum samples by radioimmunoassay(OC:DSL-6900,Diagnostic System Laboratoriescountry;P1NP:Intact P1NP, Orion Diagnostica).The bone-resorption marker serum C-telopeptide(CTx)was quantified in all serum samples using a Serum Crosslaps ELISA(Nordic Biosciences). Histomorphometry
Double fluorochrome labels were administered intravenously on days14and24(25mg/kg of tetracycline)and on days47and57 (8mg/kg of calcein).The L2vertebrae,right proximal tibiae,and
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS Journal of Bone and Mineral Research949
right femoral diaphyses were partially trimmed and fixed overnight in neutral buffered10%formalin,transferred into 70%alcohol,dehydrated,and embedded in methyl methacry-late.For trabecular bone evaluation,sections were prepared from the proximal tibia along the craniofrontal plane and from the body of the second lumbar vertebra(L2)along the median plane.Static parameters were determined from5-m m-thick sections stained with Goldner’s trichrome,whereas dynamic parameters were assessed in7-m m-thick unstained sections. Cortical bone histomorphometric parameters were determined from20-to40-m m-thick ground,unstained sections.The dynamic bone-formation parameters,mineralizing surface(MS; calculated as double-label surfacethalf the single-label sur-face),and bone-formation rate(BFR)were generated for both sets of labels and normalized to bone surface(BS)measured at the study end;mineral apposition rate(MAR)was calculated as the mean separation between each set of labels divided by the label interval(10days).These dynamic parameters were generated on trabecular,periosteal,endocortical,and intracor-tical surfaces.Static endpoints in trabecular regions included bone volume as a percent of total volume(BV/TV),trabecular thickness,osteoclast surface,and osteoblast surface.
Bone densitometry by DXA and pQCT
Prior to scanning,monkeys were first sedated by injection with a cocktail of glycopyrrolate,ketamine hydrochloric acid(HCl),and xylazine,followed by anesthesia with isoflurane gas prior to and during scanning.Areal BMC and BMD of the lumbar spine(L1–L4), right proximal femur(including femoral neck subregion),and right distal radius(one-third and ultradistal)were measured using DXA(Hologic QDR2000t)at baseline(predose1)and on days26and61.The precision[coefficient of variation(CV,%)]of DXA scanning with repositioning ranged from0.8%at the lumbar spine to4.5%at the femoral neck.
Peripheral quantitative computed tomography(pQCT;Stratec XCT Research SA scanner,Software Version.5.40B)was used to measure volumetric bone mineral content(vBMC),volumetric BMD(vBMD),and geometric parameters of the right distal radius and right proximal tibia at baseline and on days26and61. Metaphyseal data were generated as an average from three scans separated by0.5mm beginning at3%of the length for the distal radius and at the tibial-fibular junction for the proximal tibia(contour mode2,peelmode20,trabecular area:30%for radius,40%for tibia).A diaphyseal scan was taken at approximately15%of the bone length for the distal radius and12%for the proximal tibia(peelmode2,cortmode2: threshold0.930cmà1).Nominal voxel size was0.35mm at the proximal tibia and0.2mm at the distal radius.The precision (CV,%)of pQCT scanning with repositioning was0.2%to1.1%at the proximal tibia and0.2%to4.0%at the distal radius.Cross-sectional moment of inertia(CSMI)for in vivo diaphyseal scans was calculated from periosteal and endocortical circumferences based on a circular geometry assumption.
Biomechanical testing
Lumbar vertebrae(L3–L4)and left femurs were stored at–208C prior to biomechanical testing.Vertebral endplates and spinous processes were removed to obtain a specimen with planoparallel ends using a diamond saw.Bone densitometry scans were performed prior to biomechanical testing in the vertebral midplane and femoral diaphysis by pQCT,as described earlier. Nominal voxel size for pQCT was reduced to0.15mm for the femur and0.2mm for L3–L4vertebral bodies.
Destructive strength testing was performed using an MTS858 Mini Bionix servo-hydraulic test system(MTS Systems Corpora-tion)with data collected using Testworks(Version.3.8A)for Teststar(Version.4.0c)software(MTS Systems Corporation).The femoral diaphysis was tested to failure in three-point bending (displacement rate1mm/s).L3and L4vertebral samples were tested to failure in compression(displacement rate20mm/min), with data reported as an average of both vertebral tests.Peak load was recorded as the maximum of the load-displacement curve,and stiffness was the slope of the linear portion.Energy to failure was calculated as the area under the curve to the breaking point for the three-point bending tests and to peak load for the compression tests.Ultimate stress,modulus,and toughness were calculated from the bending tests based on pQCT-based cortical geometry data,as described previously.(16) Statistical analysis
Results are expressed as the mean?SE.Statistical analyses were conducted using Release9.1of SAS/STAT(SAS Institute Inc.),and each statistical test was conducted at the0.05level of significance.A one-way ANOVA was applied to each data set: change from baseline for biomarker data,percent change from baseline for in vivo DXA/pQCT data,and raw data for histomorphometric and biomechanical endpoints.The Brown and Forsythe variation of Levene’s test(Brown and Forsythe, 1974)was applied to confirm the variance homogeneity among groups.The Dunnett multiple-comparison procedure was used to compare each dose level of Scl-AbIV to the vehicle control group.For biomechanical parameters,baseline lumbar BMC was used as a covariate in the analyses.Linear regression analyses were performed within GraphPad PrismVersion5.01(GraphPad Software Inc.)across all groups.
Results
Effect of sclerostin antibody treatment on biochemical markers of bone turnover
To determine the effects of sclerostin inhibition in nonhuman primates,a sclerostin-neutralizing mAb,Scl-AbIV,was adminis-tered to intact adolescent female cynomolgus monkeys.Once-monthly injections of Scl-AbIV(ie,two injections,day1and day 29)at three dose levels(3,10,and30mg/kg)were given,and the study was terminated at2months(day61).At all doses used, Scl-AbIV did not alter body weight gain or general health.Peak Scl-AbIV concentrations at each dose level were found to occur within the first week following dosing,after which mAb levels declined.For the3mg/kg dose group,Scl-AbIV was at undetectable levels(<10ng/mL)at both the4-week postdosing timepoints(Fig.1A).The bimodal serum Scl-AbIV concentration profile over2months resulted in the observed bimodal increases in the serum bone-formation markers P1NP and osteocalcin.
950Journal of Bone and Mineral Research OMINSKY ET AL.
Changes in serum P1NP peaked 2weeks after the first injection and 1week after the second injection,with significant increases found in all three dose groups (Fig.1B ).The serum osteocalcin profile was shifted about a week later than the P1NP profile,with the highest percent increase being found in the 30mg/kg dose group (Fig.1C ).The peak changes in P1NP and osteocalcin were in excess of 50%above baseline values.By 4weeks after the first and second Scl-AbIV doses,serum P1NP and osteocalcin had returned to baseline levels,consistent with the observed clearance of Scl-AbIV from the circulation over time.No clear effect on the serum bone-resorption marker CTx was found with any dose of Scl-AbIV,although the 10mg/kg dose group was significantly lower than the vehicle control group 4days after the first dose (Fig.1D ).
Histomorphometry
To measure bone formation following each of the two once-monthly administrations of Scl-AbIV (dosed on days 1and 29),newly mineralizing bone was labeled in vivo with tetracycline (days 14and 24)and calcein (days 47and 57).Bones were harvested at study termination on day 61.Histomorphometric analysis was performed at trabecular (L 2vertebra,proximal tibia)and cortical bone sites (femoral diaphysis).Extensive labeling of bone surfaces showed that Scl-AbIV markedly stimulated bone formation in both the trabecular and cortical bone compart-ments.Even with this increased rate of mineralized bone deposition,the newly formed bone was found to be lamellar in appearance,and no osteoid accumulation or marrow fibrosis was observed.
Qualitatively,at the highest dose (30mg/kg),Scl-AbIV treatment resulted in increased trabecular thickness,greater labeled surface,and greater separation between labels com-pared with vehicle controls,in which few regions with all four labels were identifiable (Fig.2A–H ).Quantitatively,Scl-AbIV dose-dependently increased mineralizing surface (MS/BS),mineral apposition rate (MAR),and bone-formation rate (BFR/BS).Specifically,MS/BS was increased significantly at the 30mg/kg dose in the first month of dosing at the L 2vertebra (t345%;Fig.2K )and in the second month of dosing at the proximal tibia (t262%;Fig.2L )compared with vehicle.MAR and BFR/BS were increased significantly at the 30mg/kg dose during both months at both trabecular sites (t28%to 57%for MAR,t124%to 490%for BFR/BS;Fig.2M–P ).The increased bone formation with Scl-AbIV treatment in trabecular bone was associated with significant dose-dependent increases in trabecular thickness (Fig.2J )and nonsignificant but dose-dependent increases in trabecular bone volume (Fig.2I ).Trabecular tunneling was not observed in sections from Scl-AbIV-treated cynomolgus mon-keys,and trabecular number remained unchanged at both trabecular sites (data not shown).Wall thickness was non-significantly greater (20%to 38%)in both the proximal tibia and the L 2vertebra after treatment with 10or 30mg/kg compared with vehicle controls (data not shown).At the end of the study,osteoblast surface on trabecular bone was not significantly different for Scl-AbIV treatment versus control.This was the case for osteoblast surface both at the proximal tibia (4.4%?1.8%for 30mg/kg Scl-AbIV versus 4.2%?1.9%for vehicle)and at the L 2vertebra (0.53%?0.17%for 30mg/kg Scl-AbIV
versus
Fig.1.Increases in serum markers of bone formation (ie,P1NP and osteocalcin)following administration of sclerostin monoclonal antibody Scl-AbIV.Cynomolgus monkeys were injected subcutaneously on days 1and 29with vehicle (VEH)or Scl-AbIV at 3,10,or 30mg/kg,and serum was collected at various time points.(A )Serum concentration of Scl-AbIV.Serum levels,as change from baseline,for (B )P1NP,(C )osteocalcin,and (D )the bone-resorption marker CTx.Data represents mean ?SE;n ?2to 4per group.?p <.05versus vehicle control.
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS
Journal of Bone and Mineral Research
951
Fig.2.Scl-AbIV administration increases trabecular bone volume and bone formation.Trabecular regions in the L2vertebrae and right proximal tibias from vehicle-and Scl-AbIV-treated monkeys were analyzed for static and dynamic histomorphometry.Goldner’s trichrome–stained sections in the top panel(4?objective)illustrate the increases in trabecular bone volume(blue)and trabecular thickness for the30mg/kg Scl-AbIV dosing(B,D)versus vehicle(A,C)in the L2vertebra and proximal tibia at study termination(2months).Fluorescent microscopic images in panels E–H(20?objective)show the surfaces where new bone was forming when the tetracycline(orange;injected on days14and24)and calcein(green;injected on days47and57)labels were https://www.wendangku.net/doc/035148663.html,pared with vehicle-treated controls(E,G),Scl-AbIV(F,H)increased the amount of labeled surfaces as well the distance between labels at both skeletal sites.Quantitative histomorphometric analyses show dose-dependent increases in(I)trabecular bone volume(BV/TV)and(J) trabecular thickness after2months of Scl-AbIV treatment.These changes were associated with dose-dependent increases in(K,L)mineralizing surface (MS/BS),(M,N)mineral apposition rate(MAR),and(O,P)bone-formation rate(BFR/BS)at the L2vertebra and proximal tibia.D14/24?day14and24 tetracycline labels;D47/57?day47and57calcein labels.Data represent mean?SE;n?2to4per group.?p<.05versus vehicle control.
952Journal of Bone and Mineral Research OMINSKY ET AL.
1.52%?0.45%for vehicle).This finding for osteoblast surface was consistent with the declines noted for P1NP and osteocalcin and showed that the osteoblast stimulating effect of Scl-AbIV waned as the antibody was cleared from the circulation.Similar to the results for osteoblast surface,no significant difference was found at study end for osteoclast surface on trabecular bone for Scl-AbIV treatment versus control(data not shown).
The femoral diaphyseal cortex contained more calcein and tetracycline labels on the endocortical and periosteal surfaces after treatment with30mg/kg Scl-AbIV compared with vehicle controls(Fig.3A).Histomorphometric analysis showed dose-dependent increases in MS/BS,MAR,and BFR/BS for both surfaces after each dose,with greater percent increases in MS/BS and BFR/BS found for the endocortical surface.Specifically,at the 30mg/kg dose,there were significant increases in endocortical MS/BS(t706%to748%)and BFR/BS(t895%to1013%;Fig.3C, G),as well as nonsignificant increases in periosteal MS/BS(t72% to122%)and BFR/BS(t171%to233%;Fig.3B,F).Of note,the absolute magnitudes of MS/BS and BFR/BS were similar for the periosteal and endocortical surfaces when compared
across
Fig.3.Scl-AbIV administration increases cortical bone formation at the femur midshaft.(A)Fluorescent microscopy images of the femur midshaft illustrate the Scl-AbIV-mediated increase in tetracycline(orange;days14and24)and calcein labels(green;days47and57)on the endocortical and periosteal surfaces.The white boxes in the whole-cortex images are magnified5?in the panels to the right.Quantitative data for the periosteal surface are in panels B,D and F and for the endocortical surface in C,E and G as noted on the y axes.Quantitative histomorphometric analyses show dose-dependent increases in(B,C)mineralizing surface(MS/BS),(D,E)mineral apposition rate(MAR),and(F,G)bone-formation rate(BFR/BS)at both the periosteal and endocortical surfaces.Data represent mean?SE;n?2to4per group.?p<.05versus vehicle control.
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS Journal of Bone and Mineral Research953
each of the three Scl-AbIV dose levels.The greater treatment-related percent increases found for MS/BS and BFR/BS at the endocortical surface compared with the periosteal surface were due to the relatively low absolute values for these parameters at the endocortical surface in vehicle-treated control animals.For MAR,the dose-dependent increases observed were similar for the periosteal and endocortical surfaces.Analysis of the intracortical region of the femoral diaphysis at2months showed that cortical porosity,as a percentage of bone volume,was unchanged at the3and10mg/kg doses and slightly elevated in the30mg/kg group compared with the low levels found in controls(1.92%?0.04%versus0.96%?0.18%;p<.05).Bone formation on intracortical surfaces was increased significantly during month2at the10and30mg/kg doses(t393%and t465%,respectively;data not shown).
Serum bone-formation-marker concentrations were asso-ciated with local bone-formation rates at trabecular(proximal tibia)and endocortical surfaces(femoral diaphysis).Month 1BFR/BS was significantly and positively correlated with P1NP and osteocalcin on days13and20;month2BFR/BS was significantly and positively correlated with P1NP and osteocalcin on days13,20,35,and42and day48osteocalcin(day42 correlations are shown in Fig.4).
In vivo densitometry
Two injections of Scl-AbIV at the30mg/kg dose in adolescent cynomolgus monkeys resulted in significant increases,as percent change from baseline after2months,in areal BMC for the whole body(24%versus6.4%for vehicle)and femoral neck(35.2%versus5.4%for vehicle;Table1).Nonsignificant increases,which were greatest in the high-dose Scl-AbIV group, also were found by DXA at the lumbar spine and distal radius for both areal BMC and areal BMD.pQCT analysis(Table2)showed dose-related increases that attained significance in the30mg/kg dose group at the metaphyses of the distal radius and proximal tibia for both total volumetric bone mineral content(vBMC; radius:19.8%versus0.8%for vehicle;tibia:27.3%versusà1.0% for vehicle)and volumetric BMD(vBMD;radius:14.2%versus 2.6%for vehicle;tibia:18.8%versus2.9%for vehicle).In the trabecular subregion of the metaphysis,dose-related increases in vBMD were seen at both sites,reaching significance at the 30mg/kg dose in the proximal tibia(34.9%versusà1.7%for vehicle).Cortical vBMC after2months was nonsignificantly increased with each Scl-AbIV dose compared with controls for the diaphysis of both radius and tibia.Mean cortical thickness,cortical area,and cross-sectional moment of inertia(CSMI)were increased at the radial and tibial diaphyses,with greater than9%increases above baseline at the tibia after2months in the10and30mg/kg Scl-AbIV groups.For both sites,these changes were associated with consistent,dose-dependent increases in periosteal circum-ference.No consistent treatment-related changes were found for endocortical circumference and cortical vBMD at either site. The increases in the bone-formation markers osteocalcin and P1NP resulting from Scl-AbIV treatment correlated well with the increases in BMC and BMD measured at the end of the study. Because these densitometric changes reflect the cumulative increases in bone formation that occurred throughout the study, we used area under the curve(AUC,baseline-corrected)as a variable to represent the cumulative biomarker response.
The
Fig.4.Serum bone-formation marker levels reflect local bone-formation rates in trabecular and cortical sites.Linear regression analyses were performed across vehicle and Scl-AbIV(3,10,and30mg/kg)treatment groups between the day-42determination of serum levels of bone-formation biomarkers and histomorphometric bone-formation rates(BFR/BS).BFR/BS represents bone-formation rates measured from the calcein labels administered on days47 and57.(A)P1NP versus BFR/BS at the trabecular surface of the proximal tibia.(B)P1NP versus BFR/BS at the endocortical surface of the femoral midshaft.
(C)Osteocalcin versus BFR/BS at the trabecular surface of the proximal tibia.(D)Osteocalcin versus BFR/BS at the endocortical surface of the femoral midshaft.The coefficients of determination(r2)are indicated on each graph;all the dotted regression lines had slopes that were significantly different from zero(p<.05).
954Journal of Bone and Mineral Research OMINSKY ET AL.
regressions for serum osteocalcin are shown in Fig.5.Increases in lumbar spine and femoral neck areal BMD were significantly and positively correlated with serum osteocalcin AUC.Trabecular vBMD at the metaphyses of the proximal tibia and distal radius also were significantly and positively correlated with serum osteocalcin AUC.At purely cortical sites in the tibial and radial diaphyses,cortical vBMC was significantly correlated with serum osteocalcin AUC.Serum P1NP AUC also was significantly correlated with these densitometric changes(r2?0.45–0.61; data not shown).Thus,at both cortical and trabecular sites,by both DXA and pQCT,the increases in BMD and bone mass after Scl-AbIV treatment were correlated with the cumulative increases in the serum bone-formation biomarkers osteocalcin and P1NP.
Bone strength
To assess vertebral bone strength,compression testing of the third and fourth lumbar vertebrae was used to measure peak load,stiffness,and energy to failure,expressed as an average of L3and L4data.Two once-monthly injections of Scl-AbIV at the30mg/kg dose resulted in increases in lumbar vertebral bone strength,with significant increases found for peak load(t97%)and energy to failure(t183%),compared with vehicle-treated controls(Fig.6).A three-point bending test was used to assess bone strength at the femoral diaphysis.Nonsignificant increases at the30mg/kg dose were found for peak load,stiffness,and energy to failure(Table3).Intrinsic(material)properties were derived from the femur test using ex-vivo pQCT geometric data. Two months of Scl-AbIV administration did not significantly alter parameters of intrinsic strength such as ultimate strength,elastic modulus,and toughness(Table3).Consistent with this finding, there was a strong correlation between the femoral diaphysis vBMC and peak load(r2?0.98;Fig.7A).At the lumbar spine,L3–L4peak load also was highly correlated with L3–L4total vBMC across groups(r2?0.92;Fig.7B).At both sites,the slopes and intercepts of the regression lines were statistically similar(p>.4) for the vehicle group alone compared with those for the pooled Scl-AbIV data.
Discussion
We used a high-affinity sclerostin-neutralizing monoclonal antibody(Scl-AbIV)to study,for the first time,the effects of sclerostin inhibition on bone turnover,bone mass,and bone
Table1.Percent Change in DXA BMD and BMC in Cynomolgus Monkeys After2Months of Scl-AbIV Treatment
DXA ENDPOINT
(%Change from Baseline)VEH
Scl-AbIV
3mg/kg10mg/kg30mg/kg
Whole Body BMD 1.6?3.0 4.4?5.410.8?3.29.4?2.8 Whole Body BMC 6.4?3.0 5.8?6.219.2?6.724.0?2.2?Lumbar Spine BMD 1.7?1.89.8?1.4 4.2?3.811.1?3.0 Lumbar Spine BMC 2.8?2.715.0?0.38.1?6.616.5?6.2 Femoral Neck BMD 4.6?1.810.2?10.911.5?5.819.5?3.4 Femoral Neck BMC 5.4?5.117.3?11.610.5?2.835.2?7.2?Ultra-Distal Radius BMD 2.7?4.08.5?0.9 6.2?5.615.1?1.0 Ultra-Distal Radius BMC 5.3?3.79.7?5.511.3?8.319.8?4.4 Data expressed as mean?SE for percent change versus baseline after2months.?Significantly different from VEH;p<.05.
Table2.Percent Change in pQCT Endpoints in Cynomolgus Monkeys After2Months of Scl-AbIV Treatment
pQCT ENDPOINT
(%Change from Baseline)
DISTAL RADIUS PROXIMAL TIBIA
VEH
Scl-AbIV
VEH
Scl-AbIV 3mg/kg10mg/kg30mg/kg3mg/kg10mg/kg30mg/kg
Metaphysis Total Areaà1.7?1.3 2.2?2.6à0.7?2.1 4.7?3.2à3.6?2.1 5.7?9.9 5.8?2.67.0?2.4 Metaphysis Total vBMC0.8?1.6 4.1?5.17.7?1.719.8?7.2?à1.0?3.29.4?15.017.4?5.927.3?6.2?Metaphysis Total vBMD 2.6?2.0 1.8?2.48.5?2.214.2?3.4? 2.9?3.5 3.2?4.510.9?3.718.8?4.7?Metaphysis Trabecular vBMDà3.2?4.013.1?26.021.7?6.834.3?14.4à1.7?4.98.4?18.721.1?6.734.9?8.2?Diaphysis Cortical Area 2.8?1.27.2?8.5 5.0?3.310.0?4.2 1.2?3.211.5?13.512.1?3.012.6?3.7 Diaphysis Cortical vBMC 2.4?0.77.3?7.2 3.3?1.98.8?2.5 2.5?3.29.7?9.611.0?3.813.6?2.8 Diaphysis Cortical vBMDà0.3?0.50.2?1.2à1.6?1.4à0.9?1.6 1.3?0.7à1.2?3.3à1.1?0.9 1.0?1.0 Diaphysis Cortical Thickness 2.1?0.97.3?7.1 1.6?1.1 4.3?2.40.6?3.413.4?14.210.8?4.310.2?2.6 Periosteal Circumference 1.1?0.8 1.6?2.7 3.0?1.9 5.1?1.90.5?0.60.7?1.9 3.0?1.6 3.8?1.1 Endocortical Circumference0.0?1.4à3.5?1.2 4.0?2.5 5.9?2.80.7?1.6à5.2?3.9à1.3?4.2à0.7?0.8 Moment of Inertia 4.6?2.98.7?12.512.3?7.822.2?8.9 2.3?3.59.1?13.516.1?5.918.8?6.1 Data expressed as mean?SE for percent change versus baseline after2months.
?Significantly different from VEH;p<.05.
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS Journal of Bone and Mineral Research955
strength in cynomolgus monkeys over a 2-month time period.Once-monthly antibody administration at three dose levels (3,10,and 30mg/kg)was specifically chosen as the dosing regimen to investigate the anabolic effects of intermittent sclerostin inhibition and to determine the pharmacokinetic and pharmacologic profile of Scl-AbIV in nonhuman primates.Serum levels of Scl-AbIV and bone-formation markers showed a similar bimodal pattern of increase and decline following each of the two once-monthly administrations of antibody.These results demonstrated that antibody-mediated sclerostin inhibition resulted in a rapid increase in anabolic activity that returned toward baseline as the antibody was cleared from the circulation.In addition,the immediate restimulation of this anabolic pathway upon a second administration of antibody demon-strated the reproducibility and durability of the anabolic response.Consistent with the return to baseline levels observed for serum markers of bone formation,the osteoblast surface in the Scl-AbIV treatment groups at study end was not significantly different from that of vehicle-treated controls.Collectively,these data highlight the substantial and rapid modulation of bone formation that can be achieved with pharmacologic regulation of sclerostin activity.
Histomorphometric analysis showed that the key bone-formation indices MS/BS,MAR,and BFR/BS were dose-dependently elevated on trabecular surfaces as well as on the periosteal and endocortical surfaces of cortical bone.These effects were seen during both the first and the second months of the study.In addition,bone formation (BFR/BS)was
dose-dependently
Fig.5.Increases in serum osteocalcin reflect increases in BMD and bone mass at trabecular and cortical sites.Linear regression analyses were performed across groups between bone densitometric endpoints and the bone-formation biomarker serum osteocalcin.The baseline-corrected area under the curve (AUC )calculated from the serum osteocalcin profile (Fig.1C )was positively correlated with the increases in areal BMD at the (A )lumbar spine and (B )femoral neck.Serum osteocalcin AUC also was positively correlated with metaphyseal trabecular vBMD at the (C )proximal tibia and (D )the distal radius,as well as with diaphyseal cortical vBMC at (E )the tibia and (F )the radius.The coefficients of determination (r 2)are indicated on each graph;all the dotted regression lines had slopes that were significantly different from zero (p <.05).
956
Journal of Bone and Mineral Research
OMINSKY ET AL.
increased on intracortical surfaces during month 2.The obser-ved increases in MAR with Scl-AbIV suggest that there was an increase in the amount of bone matrix deposited per active osteoblast.The marked increases in active bone-forming surface (MS/BS)indicated that sclerostin inhibition resulted in increased recruitment,activation,and/or functional longevity of osteoblasts.
Despite the clear increase in anabolic activity with Scl-AbIV,no increase was found in the bone-resorption marker serum CTx,suggesting that with sclerostin inhibition there is an absence of the coupling that typically exists between osteoblast-mediated bone formation and osteoclast-mediated bone resorption during bone remodeling.The lack of increased bone resorption in Scl-AbIV-treated primates is consistent with previous results in sclerostin knockout mice and in OVX rats treated with a sclerostin antibody.(9,10)The imbalance of bone formation versus resorption with sclerostin inhibition may reflect direct activation of bone formation on quiescent surfaces (bone modeling)without prior activation of resorption (bone remodeling).This mode of anabolism may differ from that found in parathyroid hormone (PTH)treatment studies,in which bone-resorption markers in humans (17)and primates (18)were increased 1month after administration.Histomorphometric analyses in humans have demonstrated that the increases in bone-forming surfaces with PTH treatment were primarily through remodeling-based mechanisms in short-(19)and longer-term studies.(20)Examina-tion of the impact of sclerostin inhibition on bone resorption and bone modeling warrants further investigation.
Bone densitometry showed that the robust increases in bone formation with Scl-AbIV treatment resulted in significant increases in BMC and/or BMD at several skeletal sites (ie,femoral neck,radial metaphysis,and tibial metaphysis).These increases,expressed as percent changes from baseline,were 11to 29percentage points higher than those found in the vehicle-treated group after only 2months.In addition,nonsignificant increases were found in BMC and BMD at the lumbar spine and in BMC at both the radial and tibial diaphyses.The largest densitometric increases were found in the highest-dose group (30mg/kg),but of note,even in the lowest-dose group (3mg/kg),BMD and BMC almost always were numerically increased relative to vehicle controls.
Despite the small study size and its short duration,the rapid increases in bone mass that were achieved were large enough to be translated into significant increases in bone strength.In
the
Fig.6.Scl-AbIV administration increases bone strength at lumbar ver-tebrae.The bone-strength parameters (A )peak load,(B )stiffness,and (C )energy to failure were generated from destructive compression tests of trimmed third lumbar (L 3)and fourth lumbar (L 4)vertebral bodies.Data represent mean ?SE of the averaged L 3–L 4results;n ?2to 4per group.?
p <.05versus vehicle control.
Table 3.Femoral Diaphysis Bending Strength in Cynomolgus Monkeys After 2Months of Scl-AbIV Treatment
FEMORAL DIAPHYSIS STRENGTH ENDPOINT VEH Scl-AbIV
3mg/kg 10mg/kg 30mg/kg Peak Load (N)1008?102917?1211005?811285?241Stiffness (N/mm)
888?97838?106873?841040?192Energy to Failure (N ?
mm)3600?28225233190?7434994?904Ultimate Strength (MPa)200?14210?2199?8216?10Elastic Modulus (MPa)8932?97910122?399
8929?2188751?616Toughness (MPa)
9.10?0.50
8.48
8.53?1.89
12.69?1.62
Data expressed as mean ?SE.
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS
Journal of Bone and Mineral Research
957
highest-dose group,significant increases in peak load and energy to failure were found at the lumbar vertebrae,whereas nonsignificant increases in bone strength were found at the femoral diaphysis,a purely cortical site.Linear regression analysis revealed very strong correlations between bone mass and bone strength at the femoral diaphysis and at the lumbar vertebrae for the Scl-AbIV and vehicle groups.The similarities between regression lines for the vehicle group alone compared with the pooled Scl-AbIV treatment groups indicated that inhibition of sclerostin preserved the normal relationship between bone mass and bone strength and did not result in changes in the fundamental material properties of bone.Rather,the Scl-AbIV-mediated increases in bone strength were a direct result of the marked increases in bone mass.
Serum biomarkers reflect dynamic changes in bone formation and bone resorption that occur throughout the skeleton,whereas histomorphometry provides insight into the specific compartments where these bone turnover changes occur.In this study,positive correlations were found between serum bone-formation biomarker levels and local bone-formation rates (BFR/BS)on trabecular and cortical bone surfaces.Positive correlations also were found between increases in serum bone-formation biomarkers and the increases seen in BMD and bone mass.These results indicate that in the setting of short-term sclerostin inhibition,increased serum markers of bone formation reflect the anabolic effects on both trabecular and cortical bone surfaces,thus validating their importance and use in future studies.In conclusion,we have shown for the first time that administration of a sclerostin-neutralizing monoclonal antibody in cynomolgus monkeys produced a strong anabolic response in both trabecular and cortical bone,each of which play an important role in overall skeletal strength.Serum bone-formation markers rapidly increased and then returned to baseline,concordant with the rise and fall in serum sclerostin antibody levels.This finding highlights the dynamic nature of the powerful anabolic axis that sclerostin controls.Furthermore,the significant increases in bone formation,bone mass,and bone strength found in this study underscore sclerostin’s pivotal role in negatively regulating the anabolic output of the osteoblast lineage in primates.In this regard,a report from a recent study in humans indicated that administration of a sclerostin-neutralizing monoclonal antibody in healthy postmenopausal women
resulted in dose-dependent increases in biochemical markers of bone formation.(21)Finally,the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable,such as in fracture healing and osteoporosis.
Disclosures
MSO,BS,JG,YG,JC,KG,BT,JC,RD,LZ,MDH,DLL,WSS,and CP are employees of Amgen,Inc.,and have received stock and stock options from Amgen,Inc.GD (Amgen,Inc.)recently passed away.DJL,AJH,AGP,ARM,and MKR are employees of UCB-Celltech and have received stock and stock options from UCB-Celltech.FV,JJ,and SYS received funding from Amgen,Inc.,and UCB-Celltech for this study.
Acknowledgments
This research was funded by Amgen,Inc.,and UCB-Celltech.The authors thank Daniel Burns (Amgen,Inc.)and Thomas Gruetzner (Charles River Laboratories)for biomarker measurements,as well as the imaging,histomorphometry,and biomechanics technical teams at Charles River Laboratories.
References
1.Stepnick LS.The frequency of bone disease.In:MacGowan JA,Noonan AS,Elderkin AL,eds.Bone Health and Osteoporosis:A Report of Surgeon General .Washington,DC:USDHHS;2004:68–87.
2.Shoback D.Update in osteoporosis and metabolic bone disorders.J Clin Endocrinol Metab.2007;92:747–75
3.3.Deal C.Potential new drug targets for osteoporosis.Nat Clin Pract Rheumatol.2009;5:20–27.
4.Hamersma H,Gardner J,Beighton P.The natural history of scler-osteosis.Clin Genet.2003;63:192–197.
5.Brunkow ME,Gardner JC,Van Ness J,et al.Bone dysplasia scleros-teosis results from loss of the SOST gene product,a novel cystine knot-containing protein.Am J Hum Genet.2001;68:577–589.
6.Balemans W,Ebeling M,Patel N,et al.Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST).Hum Mol Genet.
2001;10:537–543.
Fig.7.Bone mass and bone strength remain tightly correlated after 2months of sclerostin inhibition.Linear regression analyses were performed across vehicle and Scl-AbIV (3,10,and 30mg/kg)treatment groups between bone mass (pQCT vBMC)and peak load at the (A )femoral diaphysis and (B )L 3–L 4vertebrae.The coefficients of determination (r 2)are indicated on each graph;both the dotted regression lines had slopes that were significantly different from zero (p <.0001).
958
Journal of Bone and Mineral Research
OMINSKY ET AL.
7.Baron R,Rawadi G.Targeting the Wnt/b-catenin pathway to regulate
bone formation in the adult skeleton.Endocrinology.2007;148:2635–2643.
8.Piters E,Boudin E,Van Hul W.Wnt signaling:a win for bone.Arch
Biochem Biophys.2008;473:112–116.
9.Li X,Ominsky MS,Niu QT,et al.Targeted deletion of the sclerostin
gene in mice results in increased bone formation and bone strength.
J Bone Miner Res.2008;23:860–869.
10.Li X,Ominsky MS,Warmington KS,et al.Sclerostin antibody treatment
increases bone formation,bone mass and bone strength in a rat model of postmenopausal osteoporosis.J Bone Miner Res.2009;24:578–588.
11.Eddleston A,Marenzana M,Moore AR,et al.A short treatment with an
antibody to sclerostin can inhibit bone loss in an ongoing model of colitis.J Bone Miner Res.2009;24:1662–1671.
12.Ke HZ,Xiang JJ,Li X,Simonet WS,Paszty C,Li G.Inhibition of
sclerostin by systemic treatment with a sclerostin monoclonal anti-body enhances fracture healing in mice and rats.Abstract and Oral presented at55th Annual Orthopaedic Research Society Meeting February22–25,2009;Las Vegas,NV,2009.
13.Brommage R.Perspectives on using nonhuman primates to under-
stand the etiology and treatment of postmenopausal osteoporosis.
J Musculoskelet Neuronal Interact.2001;1:307–325.
14.Jerome CP,Peterson PE.Nonhuman primate models in skeletal
research.Bone.2001;29:1–6.15.Smith SY,Jolette J,Turner CH.Skeletal health:primate model of
postmenopausal osteoporosis.Am J Primatol.2009;71:1–14.
16.Turner CH,Burr DB.Basic biomechanical measurements of bone:a
tutorial.Bone.1993;14:595–608.
17.Hodsman AB,Hanley DA,Ettinger MP,et al.Efficacy and safety of
human parathyroid hormone-(1–84)in increasing bone mineral density in postmenopausal osteoporosis.J Clin Endocrinol Metab.
2003;88:5212–5220.
18.Fox J,Miller MA,Newman MK,Turner CH,Recker RR,Smith SY.
Treatment of skeletally mature ovariectomized rhesus monkeys with PTH(1–84)for16months increases bone formation and density and improves trabecular architecture and biomechanical properties at the lumbar spine.J Bone Miner Res.2007;22:260–273.
19.Lindsay R,Cosman F,Zhou H,et al.A novel tetracycline labeling
schedule for longitudinal evaluation of the short-term effects of anabolic therapy with a single iliac crest bone biopsy:early actions of teriparatide.J Bone Miner Res.2006;21:366–373.
20.Ma YL,Zeng Q,Donley DW,et al.Teriparatide increases bone
formation in modeling and remodeling osteons and enhances IGF-II immunoreactivity in postmenopausal women with osteoporo-sis.J Bone Miner Res.2006;21:855–864.
21.Padhi D,Stouch B,Jang G,et al.Anti-sclerostin antibody increases
markers of bone formation in healthy postmenopausal women.
J Bone Miner Res.2007;22:S1.S37.
SCLEROSTIN MAB INCREASES BONE FORMATION IN MONKEYS Journal of Bone and Mineral Research959
单克隆抗体的制备及应用
单克隆抗体的制备及应用 单克隆抗体是由淋巴细胞杂交瘤产生的、只针对复合抗原分子上某一单个抗原决定簇。单克隆抗体技术(monoclonal antibody technique)：一种免疫学技术，将产生抗体的单个B淋巴细胞同骨髓肿瘤细胞杂交，获得既能产生抗体，又能无限增殖的杂种细胞，并以此生产抗体。是仅由一种类型的细胞制造出来的抗体，对应于多克隆抗体、多株抗体——由多种类型的细胞制造出来的一种抗体。 1 单克隆抗体的优点与局限性： 单克隆抗体的优点：(1)杂交瘤可以在体外“永久”地存活并传代，只要不发生细胞株的基因突变，就可以不断地生产高特异性、高均一性的抗体。(2)可以用相对不纯的抗原，获得大量高度特异的、均一的抗体。(3)由于可能得到“无限量”的均一性抗体，所以适用于以标记抗体为特点的免疫学分析方法，如IRMA和ELISA等。(4)由于单克隆抗体的高特异性和单一生物学功能，可用于体内的放射免疫显像和免疫导向治疗。 总体来说，即：高特异性、高纯度、重复性好、敏感性强、成本低和可大量生产等。 单克隆抗体的局限性：(1)单克隆抗体固有的亲和性和局限的生物活性限制了它的应用范围。由于单克隆抗体不能进行沉淀和凝集反应，所以很多检测方法不能用单克隆抗体完成。 (2)单克隆抗体的反应强度不如多克隆抗体。(3)制备技术复杂，而且费时费工，所以单克隆抗体的价格也较高。 2 单克隆抗体的制备： 单克隆抗体的制备原理：应用细胞杂交技术使骨髓瘤细胞与免疫的淋巴细胞二者合二为一，得到杂种的骨髓瘤细胞。这种杂种细胞继承两种亲代细胞的特性，它既具有B淋巴细胞合成专一抗体的特性，也有骨髓瘤细胞能在体外培养增殖永存的特性，用这种来源于单个融合细胞培养增殖的细胞群，可制备抗一种抗原决定簇的特异单克隆抗体。 单克隆抗体的制备过程：抗原准备、动物的选择与免疫、细胞融合、选择杂交瘤细胞及抗体检测、杂交瘤的克隆化、杂交瘤细胞的冻存与复苏、单克隆抗体的纯化等步骤。 抗原准备 抗原，是指能够刺激机体产生（特异性）免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体外结合，发生免疫效应（特异性反应）的物质。抗原的基本特性有两种，一是诱导免疫应答的能力，也就是免疫原性，二是与免疫应答的产物发生反应，也就是抗原性。很多物质都可以成为抗原，抗原的具体分类可以参见抗原，在进行单克隆抗体制备过程中，很多物质都可以成为抗原，在常规的科研实验中，科研者经常选用每只小鼠/大鼠每次注射10~50ug 重组蛋白、偶联多肽、偶联小分子等作为抗原产生特异性的单克隆抗体。 动物的选择与免疫
干货 I 细胞复苏、冻存、传代之实验操作及注意事项
细胞培养之实验操作及注意事项｜细胞复苏/冻存/传代 一、细胞培养前期准备工作 细胞培养仪器设备： ●细胞培养通风橱（即生物安全柜） ●培养箱（通用推荐：湿式二氧化碳培养箱） ●离心机 ●医用冰箱（4℃）和冰柜（-20℃）、生物超低温冰箱（-80℃）、液氮冷冻罐●倒置显微镜 其他用品：细胞培养容器（培养瓶、培养皿、多孔板等），吸管和移液器，培养基、血清和试剂 注意事项: ●无菌工作区域：细胞培养实验室的主要要求是需要维持细胞培养操作工作区 域处于无菌状态，最简单经济的方式就是使用细胞培养通风橱。 ●无菌试剂和培养基：商品化试剂和培养基经过严格的质量控制以确保无菌， 注意操作过程中不要被污染，其他试剂和溶液需要选择适当的灭菌方法（例如：高压蒸汽、无菌过滤等）进行灭菌。 ●无菌操作：要求操作人员在实验开始前对工作区域和双手操作部分进行75% 酒精消毒；尽量使用一次性塑料吸管进行单次操作，避免交叉污染；试剂瓶和培养用具使用时方可开盖，用后要第一时间盖上，暴露于环境中的时间尽可能要短。 ●整体的细胞实验环境也弥足重要，需要定期对实验室环境进行较为彻底的清
扫消毒和杀菌，包括实验用仪器和室内地面、桌面等。 二、细胞的复苏与冻存 细胞复苏： 在细胞状态不足以满足实验需求或者出现细胞污染的情况下，需要进行细胞复苏：a)从超低温冰箱或者液氮中取出自保存或商品化购买的冻存细胞后，快速转移 至37℃水浴条件下轻轻转动融化（一分钟内）。 b)75%酒精擦拭冻存管外部后，转移至通风橱中。 c)加入适量新鲜的完全培养基混合，约800-1000rpm下离心5-10分钟，实 际离心速度时间取决于细胞种类和细胞状态。 d)新鲜培养基重悬后接种于相应的培养器皿中，做好标记，37℃，5%CO2条 件下培养。 细胞冻存： 为现有细胞系做细胞储备，需要对代数相对靠前的细胞进行扩增培养和细胞冻存： a)准备细胞冻存液，置于2℃-8℃下备用。 b)观察待冻存的细胞，密度达到80%-90%左右，将贴壁/悬浮细胞分别按照传 代方法收集。 c)使用血球计数板对细胞密度进行大致估算后，计算出冻存溶液的体积，从而 保证单支冻存细胞有足够的细胞量用于复苏。 d)约800-1000rpm下离心5-10分钟，无菌条件下小心弃去培养基，不要搅 动细胞沉淀。 e)用合适体积的预冷后冻存液重新混悬细胞沉淀，分装至冻存管中，做好标记
间接免疫荧光法检测抗核抗体的结果分析
间接免疫荧光法检测抗核抗体的结果分析 目的探讨间接免疫荧光法（IIFA）检测抗核抗体在自身免疫性疾病（AID）患者中的结果应用分析。方法分别采用IIFA和胶乳法检测50例自身免疫性疾病患者血清样本抗核抗体（ANA）。结果IIFA法检测血清ANA的灵敏度为76%，胶乳法检测血清ANA的灵敏度为58%，差异有统计学意义（P＜0.01）。结论IIFA 法检测ANA的敏感度明显高于胶乳法，提高了临床的阳性检出率。IIFA法用于ANA的筛查，阳性标本在进行滴度检测，有助于临床诊断。 标签：抗核抗体；间接免疫荧光法；胶乳法 [Abstract] Objective To discuss the results of indirect immunofluorescence in detecting the antinuclear antibodies in patients with autoimmune diseases. Methods The antinuclear antibodies of serum samples of 50 cases of patients with autoimmune diseases were detected respectively by the IIFA and emulsion method. Results The difference in the sensitivity degree of ANA detected by IIFA method and emulsion method had statistical significance（76% vs 58%）（P＜0.01）. Conclusion The sensitivity degree of ANA detected by IIFA method is obviously higher than that detected by the emulsion method，which improves the positive detection rate in clinic，the application of IIFA method for ANA screening and the application of positive specimens for titer detection contribute to clinical diagnosis. [Key words] Antinuclear antibodies；Indirect immunofluorescence；Emulsion method 抗核抗体（ANA）包括细胞核的自身抗体和细胞质内的核酸和核蛋白的所有成分的自身抗体，是指哺乳动物的细胞中以细胞核为靶抗原的自身抗体的总称[1]。ANA在系统性红斑狼疮、干燥综合征、硬皮病、慢性活动性肝炎等自身免疫疾病中均呈不同程度的阳性。因此在自身免疫性疾病的临床诊断、鉴别以及评价预后中，ANA检测已成为重要的筛查指标[2]。临床上ANA检测方法较多，现阶段最常用、最有效的检测方法是IIFA法。由于不同检测方法检测灵敏性、准确性不同，往往影响到检测结果，使检测结果存在一定的差异，尤其在ANA 呈弱阳性的标本中，不同检测方法的检测成为争议的焦点。为进一步确认IIFA 法和乳胶法检测ANA的效果，进而客观评价该两种检测方法差异性，该文2015年10—12月间选取该院确诊或疑似为AID的患者50例作为观察对象，同时采用IIFA法和乳胶法检测，报道如下。 1 资料与方法 1.1 一般资料 所有标本均来自2015年10—12月到该院确诊或疑似AID的门诊和住院患者50例，男性15例，女性35例，年龄15～65岁。
HIV初筛实验室相关工作制度样本
HIV初筛实验室工作制度 1、本实验操作人员必要经省艾滋病初检技术培训学习合格后方可持证上岗。 2、严格遵守样本采集、保存制度；样品运送解决制度；遵守实验室操作、检 验成果报告解决和保密、实验室污染物废弃物以及工作人员防护消毒隔离制度。 3、积极参加上级实验室业务培训和技术考核。 4、必要参加省临检中心室间质量评价工作。 5、未尽事宜按科室实验室按科室工作制度和有关管理条例执行。
HIV抗体检测程序及其流程 1、血液标本验收合格后，用初筛试剂进行检测，如呈阴性反映，则作HIV抗体阴性报告； 2、初筛检测成果呈阳性反映标本，须进行重复检测。复检时用两种不同初筛检测试剂复测； 3、如两种试剂复检成果均呈阴性反映，则作HIV抗体阴性报告；如均呈阳性反映，或有一份阳性，该标本需送上级实验室加以进一步证明。送检时应将重新采集该受检者血液标本和原有血液标本一并送检。 4、检测成果鉴定和解决 对HIV抗体阳性者应做好征询、保密和报告工作。对HIV抗体阴性者，如近期有高危行为如性乱史、吸毒史、受血史，或有急性流感样症状等状况，为排除窗口期也许，建议每3个月复查一次，持续2次。对HIV抗体可疑对象要做好征询和随访工作。5、、反馈与报告程序 （一）初筛检测中发现HIV抗体阳性反映标本，应尽快(城区普通规定在48小时内，农村规定在96小时内)将血样连同原始实验资料（涉及厂家批号、试剂种类、有效期，如ELISA实验应附上阴性、阳性对照值，Cut off值及样品OD值）和送检化验单送卫生行政部门指定初筛中心实验室，再转送确认实验室，或直接送至HIV抗体确认实验室。送检化验单必要由初筛实验室一名直接实验操作人员和一名中级技术职称以上负责人员签名。初筛实验室不得向受检者宣布初检阳性反映成果； （二）做好标本收集与检测登记工作，每月5日按统一表格向卫生行政部门指定HIV抗体初筛中心实验室报告检测状况，如无HIV抗体中心实验室，则直接向省级HIV抗体确认中心报告。
单克隆抗体制备中筛选杂交瘤细胞的原理
单克隆抗体制备过程中筛选杂交瘤细胞的原理和方法单克隆抗体制备过程中，有两次筛选过程，第一次是选出杂交瘤细胞（用选择培养基），第二次是进一步选出能产生我们需要的抗体的杂交瘤细胞。 第一次筛选的原理和方法： 细胞融合后，杂交瘤细胞的选择性培养是第一次筛选的关键。普遍采用的HAT选择性培养液是在普通的动物细胞培养液中家次黄嘌呤、氨基蝶呤和胸腺嘧啶核苷酸。其一居室细胞中的DNA合成油两条途径： 一条途径是生物合成途径（“D途径”），即由氨基酸及其其他小分子化合物合成氨基酸，为DNA分子的合成提供原料。再此合成过程中，叶酸作为重要的辅酶参与这一过程，而HAT培养液中氨基蝶呤是一种叶酸的拮抗物，可以阻断DNA合成的D途径。 另一条途径是应急途径（“S途径”），她是利用次黄嘌呤——鸟嘌呤磷酸核苷转移酶和胸腺嘧啶核苷激酶催化次黄嘌呤和胸腺嘧啶生成相应的核苷酸，两种酶缺一不可。 因此，在HAT培养液中，未融合的效应B 细胞核两个效应B细胞融合的D途径被氨基蝶呤阻断，随S途径正常，但因缺乏在体外培养液中增殖的能力，一般10天左右会死亡。对于骨髓瘤细胞以及自身融合细胞而言，由于通常采用的骨髓瘤细胞是次黄嘌呤-鸟嘌呤磷酸核苷转移酶缺陷型细胞，因此自身没有S途径，且D途径又被氨基蝶呤阻断，所有在HAT培养液中也不能增殖而很快死亡。只有骨髓瘤细胞与效应B细胞相互融合形成的杂交瘤细胞，既具有效应B细胞的S途径，又具有骨髓瘤细胞在体外培养液中长期增殖的特性，因此能在HAT培养液中选择性存活下来，并不断增殖。 第二次筛选的原理和方法： 在单克隆抗体的生产过程中，由于效应B细胞的特异性是不同的，经HAT培养液第一次筛选出的杂交瘤细胞产生的抗体存在差异，必须对杂交瘤细胞进行第二次筛选，
杂交瘤细胞制备
杂交瘤细胞的制备 1. 骨髓瘤细胞的准备 选择生长状态良好的细胞，浑圆透亮，大小均一，边缘清晰，排列整齐，呈半致密分布。弃上清，以不完全培养基洗涤一次后，用10mL不完全培养基将骨髓瘤细胞（SP2/0）轻轻吹下。 2. 脾淋巴细胞的准备 a、取加强免疫后3天的小鼠，摘除眼球采血供分离阳性血清。 b、颈脱位将小鼠致死，用75%酒精消毒体表5min，随即放入超净台内小鼠解剖板上，左侧卧位，用7号针头固定四肢。 c、无菌打开腹腔取出脾脏，用基础培养基洗涤，并仔细去掉周围附着的结缔组织。 d、随后将脾脏转移到另一个盛有DMEM的平皿中。以弯头针头压住脾脏，用小针头在脾脏上插孔，并用镊子挤压，使脾细胞充分释放，制成脾细胞悬液。 3. 饲养细胞的制备 取一只健康的ICR小鼠，摘眼球采血，颈脱臼处死，体表消毒和固定后，从大腿上剪开皮肤，暴露腹膜，酒精棉球消毒腹膜。用10mL注射器，12#针头，注射5-10mL HAT培养基到腹腔，右手固定注射器，左手持酒精棉球轻轻按摩腹部，抽回腹腔内液体，注入已准备好的容器中。 4. 细胞融合 a、将上述制备的骨髓瘤细胞与脾细胞混合于一支50mL的带盖的离心管中，1000rpm离心10min，上清要充分吸净，以免影响PEG的作用。 b、将融合管置于手掌中，轻轻振荡底部，务使两种细胞充分混匀。 c、用1mL吸管将预热的PEG在45~60s内缓慢加到融合管中，边加边轻轻摇匀。 d、立即滴加37℃预热的DMEM，使PEG稀释而失去作用，具体加法是用吸管在第一分钟内加1mL预热的不完全培养基，第二分钟内加2mL，第三分钟加8mL （遵照先慢后快的原则），37℃静置10min，1000rpm离心10min，弃上清。 e、加入5mL的HAT培养基，轻轻悬浮沉淀细胞，再加入适量的腹腔巨噬细胞，最后补加HAT至50mL左右。 f、分装于96孔细胞培养板，然后将培养板置37℃，5%CO2培养箱内培养。 g、5d后用HAT培养基换出一半培养基。 h、观察杂交瘤细胞的生长情况，待其细胞培养上清变黄或克隆分布至孔底面积的1/10以上时，吸取适量细胞上清进行抗体检测。
免疫组化和免疫荧光的区别[参照材料]
请问你曾经被IHC、ICC和IF所困扰过吗？ 作者：北京义翘神州 在实验中，有没有一种感觉，就是对免疫组化(IHC)，免疫细胞(ICC)，以及免疫荧光(IF)傻傻分不清，那么今天我们就来讨论一下这三者的区别，先贴图上来以便有个大致的区分。 从图中我们可以看出，免疫检测技术可以根据报告标签的不同，分为免疫化学和免疫荧光两类，而根据样品类型不同，可以分为组织和细胞检测技术。因此，才会延伸出三个相近的概念。 为了更好的区分这三个概念，我们还可以根据他们的英文词根来分析一下，他们的英文名分别是免疫组织化学Immunohistochemistry (IHC)，免疫细胞化学Immunocytochemistry (ICC)，免疫荧光Immunofluorescence (IF)。 词根分析： Immuno-指的是免疫技术（例如，抗体和抗原的结合） Histo-指的是组织（细胞以及它周围的细胞外基质） Cyto-指的是细胞（不包含细胞外的基质） Chemistry-在这里指的是化学检测方法（例如，颜色的变化）
Fluorescence-对被激发的荧光团的检测 通过对词根的解释，相信你在心中不再迷茫了吧。 这三个应用技术都属于免疫技术，都是将抗原与抗体的结合可视化，即通过一定的方法可以直接看到实验结果。而常用的方法就是化学显色和荧光显色。如果报告标签是酶促的，就是免疫化学，如果是荧光的，就是免疫荧光。 其实我们纠结免疫荧光的一个原因还在于免疫荧光的英文名字Immunofluorescence (IF)，但若是写成免疫组织荧光(IHF)或是免疫细胞荧光(ICF)，那我们是不是就豁然开朗了。虽然这两个词汇在英文文献中应用的还不是很广泛，但绝对不是我自己杜撰的哦，已经有些学者在使用了，规范化应该也只是时间的问题。 可能文字描述还是有些晦涩难懂，那我们就直接上图吧。先不看下面答案，仅从几张图片，你能明白哪张图代表哪种应用吗？ A免疫组织化学：兔EGFR单克隆抗体（10001-R043-50）—人胎盘
HIV的实验室诊断
HIV实验室诊断 一、HIV病毒是带有包膜的RNA逆转录病毒，在我国流行的是HIV-1。 二、HIV实验室诊断 人体感染HIV后，血液中最先出现HIV抗原，然后很快消失直到疾病后期才重新出现。几周后出现IgM抗体并很快消失，此后,IgG抗体出现并一直存在。因此，HIV感染的实验室诊断以抗体检测为主，病毒及相关抗原的检测为辅。 抗体检测分为初筛试验和确证试验两种，初筛试验为阳性的血清必须进一步确证，确证实验为阳性的方可报告为HIV感染阳性。 多聚酶链式反应(PCR)主要用于检测血浆中HIV的RNA含量，目前主要用于预测母亲将HIV传染给胎儿的可能性以及新生儿的HIV感染状况。此外，尚可用于判断病人的预后及监测抗病毒治疗的效果。 （一）HIV抗体的初筛检测 初筛试验的要求是敏感性高，理论上要求达到100%，尽可能避免漏掉可能阳性的对象，相对来说，对特异性要求不是太严，允许有少量假阳性，这些假阳性可以通过重复试验和确证试验排除。 HIV抗体初筛检测的方法很多，如酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)、明胶颗粒凝集实验(gelatine particle agglutination assay, PA)、乳胶凝集实验(latex agglutination assay, LA)、各种快速检测实验(rapid tests)等。 1．酶联免疫吸附实验检测HIV1/2 型抗体，酶联免疫吸附实验（ELISA）是最常见的HIV抗体检测方法，它具有准确性高、价格低廉、判断结果有客观标准、结果便于记录和保存等优点，适合于大批量标本的检测，是献血员筛选和临床诊断最常用的方法。 3．胶体金法检测HIV1/2抗体实验 胶体金法检测HIV抗体是一种不需要任何仪器设备的血清／血浆检测法，它利用免疫层析分析原理来快速检测血清／血浆中是否含有HIV抗体，从而用于判断人体是否受到HIV1型／HIV2型病毒感染。 注意：由于样本中HIV抗体滴度的不同，测试区（T）内的红色条带会显现出不同深浅的颜色。但是，本试剂盒的测试结果不能做为判定样本中抗体滴度高低的依据。 (二)、初筛试验每一次检测阴性者可报告阴性，若第一次检测阳性，则需进行复测，复测时（最好用不同类型的试剂）可同时测定两孔，判定方法如下：
1单克隆抗体药物----科普知识
1 单克隆抗体药物----科普知识 单克隆抗体药物 2009-10-19 15:47 1986年，美国FDA批准了第一个单克隆抗体药物上市，距今已经整整20年了。截止到现在，全世界共有21个治疗用抗体药物被批准上市，实现300亿美元的销售额，在国际，也在国内形成了抗体药物开发热潮。巨大的市场前景和现存的技术问题及壁垒并存的现实不可避免地引发抗体药物新一轮技术革命。而其结果又将毫无疑问地改变抗体药物的市场格局。抗体药物的研究开发能否真能成为中国生物技术药物开启国际市场大门的新钥匙？什么是我们首选的切入点，又如何形成我们自己的特色和竞争优势？回顾国际抗体药物20年风雨飘摇的发展经历，总结其中的经验教训无疑会给我们一些有益的启示。 1986年，美国FDA批准上市了第一个抗体药物Orthoclone，用于治疗移植物抗宿主病（GVHD），翻开了生物医药历史崭新的一页。时隔8年，美国才批准了第二个抗体药物上市。进入21世纪，抗体药物研发上市的速度明显加快。20年后的今天，全球共批准上市21个抗体药物。进入临床验证的数量也直线上升，从上个世纪80年代的70个，到90年代新增140个，以及2000年至2005年6月又增加的130个，预计2010年将再增 240个【1】，显示抗体药物研究异常活跃。目前共有150余个抗体药物正在临床评估中，其中16个已进入III期临床【2】。 抗体药物研发进展迅速及竞争激烈的主要原因是1）抗体药物具有高度特异性，是靶向治疗的基础，在这一方面远优于小分子药物；2）虽然在世界范围内20年仅仅批准上市了21个抗体药物，事实上其淘汰率仍明显低于小分子药物，临床转化率以及批准成功率都较高，以治疗癌症的抗体药物为例，其批准成功率为29％；3）抗体药物即使在专利保护到期后，由于其生产条件的复杂性，也很难受到通用名药价格的威胁；4）更为重要的是已上市的抗体药物具有很高的市场回报率，大大刺激了投资热情。目前，上市抗体药物中已盈利的有8个，其中4个已经成为年销售额10亿美元以上的“重磅弹”，5个销售总额超过30亿美元【3】。预计2005-2010年抗体药物销售的平均增长率为20％，年销售额将超过300亿美元，市场潜力巨大。 但具有讽刺意味的是，从药物经济学的角度看，抗体药物并非很好的药物候选者。首先，单克隆抗体是大分子糖蛋白，结构复杂、不利储存、不能口服、进入体内5-7天才能到达靶位置。其次，抗体药物研发费用较高，达10－18亿美元，高于小分子药物平均研发费的9亿美元。第三，目前抗体药物的单剂用量大，药物的质量标准高，生产成本高昂，导致价格昂贵，以致被喻为“经济负作用”。以治疗肿瘤的抗体药物Avastin为例，单个病人年度费用高达5万美元【4】。然而，正在形成的巨大市场是抗体药物研发的不竭驱动力。 我国在单克隆抗体技术起步非常早，80年代曾出现过研究热潮，但产业化成就还微不足道。目前，受国际抗体研发进展的影响，又出现了新一轮的“抗体热”，北京、上海、广州等纷纷成立了由研究机构、企业和投资商的联合抗体研发中心和公司。面对国际抗体药物竞争的新态势，我国抗体药物产业是否遇到了真正的机遇？我们选择的切入点是什么，又如何形成自己的特色和竞争优势？抗体药物的研究开发能否成为中国生物技术药物开启国际市场大门的新钥匙？回顾国际抗体药物20年风雨飘摇的发展经历，总结其中的经验教训无疑会给我们一些有益的启示，这是本文的主要目的。 一、上市抗体药物的发展现状 从第一个抗体药物上市至今20年内，全球共批准了21个抗体药物，其中美国18个（包括9个被欧盟
单克隆抗体制备中筛选杂交瘤细胞的原理
单克隆抗体制备中筛选杂交瘤细胞的原理 标准化管理部编码-[99968T-6889628-J68568-1689N]
单克隆抗体制备过程中筛选杂交瘤细胞的原理和方法 单克隆抗体制备过程中，有两次筛选过程，第一次是选出杂交瘤细胞（用选择培养基），第二次是进一步选出能产生我们需要的抗体的杂交瘤细胞。 第一次筛选的原理和方法： 细胞融合后，杂交瘤细胞的选择性培养是第一次筛选的关键。普遍采用的HAT选择性培养液是在普通的动物细胞培养液中家次黄嘌呤、氨基蝶呤和胸腺嘧啶核苷酸。其一居室细胞中的DNA合成油两条途径： 一条途径是生物合成途径（“D途径”），即由氨基酸及其其他小分子化合物合成氨基酸，为DNA分子的合成提供原料。再此合成过程中，叶酸作为重要的辅酶参与这一过程，而HAT培养液中氨基蝶呤是一种叶酸的拮抗物，可以阻断DNA合成的D途径。 另一条途径是应急途径（“S途径”），她是利用次黄嘌呤——鸟嘌呤磷酸核苷转移酶和胸腺嘧啶核苷激酶催化次黄嘌呤和胸腺嘧啶生成相应的核苷酸，两种酶缺一不可。 因此，在HAT培养液中，未融合的效应B细胞核两个效应B细胞融合的D途径被氨基蝶呤阻断，随S途径正常，但因缺乏在体外培养液中增殖的能力，一般10天左右会死亡。对于骨髓瘤细胞以及自身融合细胞而言，由于通常采用的骨髓瘤细胞是次黄嘌呤-鸟嘌呤磷酸核苷转移酶缺陷型细胞，因此自身没有S途径，且D途径又被氨基蝶呤阻断，所有在HAT培养液中也不能增殖而很快死亡。只有骨髓瘤细胞与效应B细胞相互融合形成的杂交瘤细胞，既具有效应B细胞的S途径，又具有骨髓瘤细胞在体外培养液中长期增殖的特性，因此能在HAT培养液中选择性存活下来，并不断增殖。 第二次筛选的原理和方法： 在单克隆抗体的生产过程中，由于效应B细胞的特异性是不同的，经HAT培养液第一次筛选出的杂交瘤细胞产生的抗体存在差异，必须对杂交瘤细胞进行第二次筛选，选出能产生特定抗体的杂交瘤细胞。二次筛选通常采用有限稀释克隆细胞的方法，将杂交瘤细胞多倍稀释，接种在多孔的细胞培养板上，是每孔细胞不超过一个，通过培养让其增殖，然后检测各孔上清液中的细胞分泌的抗体，上清液可与特定抗原结合的培养孔为阳性孔。阳性孔中的细胞还不能保证是来自单个细胞，继续进行有限
杂交瘤细胞培养及其注意事项
杂交瘤细胞培养及其注意事项 单克隆抗体的应用是一项迅速发展的技术。本文阐述了单克隆抗体制备前杂交瘤细胞的培养条件和应用，包括细胞的培养、培养基的选择，融合时期的选择，从而指导和优化实验室中杂交瘤的培养，促进融合细胞的融合率和后继单克隆抗体筛选实验的顺利进行。 标签：杂交瘤；细胞培养；单克隆抗体 随着杂交瘤细胞株不断的建立和生产单克隆抗体的日益广泛应用，杂交瘤细胞的培养和优化不断得到改善，但是国内实验室设备和条件有限，多数的单克隆抗体的筛选还是应用基础的免疫抗原包被，结合Elisa方法联合有限稀释法来筛选单克隆抗体。为了获得特异性较强的杂交瘤细胞株，融合前后细胞的培养以及杂交瘤的培养和储存，仍是基础实验室中必须注意和重视的技术。 1杂交瘤的起源和生产 1.1杂交瘤所用的骨髓瘤细胞系1966年Petingel等在体外培养小鼠浆细胞成功。Milstein等1972年由P3中用20μg 8-氮杂鸟嘌呤（8-azaguanine）筛选出缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）的新细胞系，称之为P3-X63-Ag8。由于X63本身分泌IgG1，形成的杂交瘤除分泌B细胞的特异性抗体外，同时分泌X63产生的抗体，因其抗体不纯，现多改用不分泌型骨髓瘤细胞系。国内现已引进NS-1，SP2/0，FO，X63-Ag8-653等小鼠骨髓瘤细胞系。最常使用的是SP2/0及NS-1[1]。 1.2人骨髓瘤细胞系为产生稳定的人免疫球蛋白的杂交瘤，需要人的缺某种酶的骨髓瘤细胞系。国外已筛选了一些人骨髓瘤细胞，但融合率不高，还没有推广使用。目前人一人杂交瘤技术仍存在着杂交瘤在不同培养基中的生长速度问题。细胞生长缓慢，抗体分泌力弱及效价较低，且难于克隆化等困难[2]，限制了人骨髓瘤细胞的应用。 1.3淋巴细胞和骨髓瘤细胞的关系B淋巴细胞容易与浆细胞瘤融合，而T细胞则需要同胸腺淋巴肉瘤融合（Kohler，et al. 1977；Schwaber，1977）。脾脏是大量T或B细胞的来源，与小鼠骨髓瘤细胞融合时，最常用的是小鼠的脾细胞。按照Burnet的细胞系选择学说，一个B淋巴细胞只能产生一种抗体，其”后代”也只能产生此种抗体，因为抗体是由DNA上的基因所决定，基因是可以遗传的。在正常情况下，小鼠脾中含有能产生各种不同抗体的B细胞，一只纯种小鼠体内可有（1～5）×107种不同的抗体，即有（1～5）×107种不同的B细胞。为了提高获得某种杂交瘤的机会，就需要设法增加小鼠体内分泌该种抗体的B细胞的数量。最常用的方法是用特定抗原多次免疫小鼠，使之产生特异性抗体。通过将免疫小鼠的脾细胞和小鼠骨髓瘤细胞的融合，然后经过多次筛选，从而得到特异性的杂交瘤细胞。这样所获得的特异性的杂交瘤细胞在既具有脾细胞分泌抗体的能力的同时，又具有小鼠骨髓瘤细胞永生的特性，从而可以无限制地提供
免疫荧光检测
免疫荧光技术（检测抗核抗体（ANA）的实验方案） 荧光免疫技术是以荧光物质标记的特异性抗体或抗原作为标准试剂，用于相应抗原或抗体的分析鉴定和定量测定。荧光免疫技术包括荧光抗体染色技术和荧光免疫测定两大类。荧光抗体染色技术是用荧光抗体对细胞、组织切片或其他标本中的抗原或抗体进行鉴定和定位检测，可在荧光显微镜下直接观察结果，称为荧光免疫显微技术，或是应用流式细胞仪进行自动分析检测，称为流式荧光免疫技术。荧光免疫测定主要有时间分辨荧光免疫测定和荧光偏振免疫测定等。本次实验以荧光免疫显微技术检测抗核抗体(ANA)为例进行实习。 抗核抗体(antinuclear antibody，ANA)又称抗核酸抗原抗体，是一组将自身真核细胞的各种成分脱氧核糖核蛋白(DNP)、DNA、可提取的核抗原(ENA)和RNA等作为靶抗原的自身抗体的总称，能与所有动物的细胞核发生反应，主要存在于血清中，也可存在于胸水、关节滑膜液和尿液中。 抗核抗体实验原理 以小鼠肝细胞或某些培养细胞(如Hep-2)作抗原片，将病人血清加到抗原片上。如果血清中含有ANA，就会与细胞核成分特异性结合。加入荧光素标记的抗人IgG抗体又可与ANA结合，在荧光显微镜下可见细胞核部位呈现荧光。 试剂与器材 1．抗原片现多用商品试剂。如需自行制备，方法如下： (1)肝印片制备：取4-8周龄小鼠，断颈杀死后，剖腹取肝。将肝脏剪成平面块，用生理盐水洗去血细胞，用滤纸吸干渗出的浆液。将切面轻压于载玻片上，使其在载玻片上留下薄层肝细胞。冷风吹干，乙醇固定，冰箱可保存1周。
(2)Hep-2细胞抗原片制备：Hep-2细胞是建株的人喉癌上皮细胞。经适宜培养在载玻片上形成单层细胞抗原片，用洗涤洗去培养基。干燥后，用无水乙醇固定。 (3)肝切片制备：取小鼠肝组织作冰冻切片，厚4μl。-30℃保存备用。 2．异硫氰酸荧光素(FITC)标记的抗人IgG抗体(FITC-抗人IgG抗体)有商品供应，临用时按效价稀释。 3．L PBS 4．缓冲甘油取甘油9份加PBS 1份。 5.待测血清、阳性和阴性对照血清临床标本筛选获得。 6．器材荧光显微镜、孵箱、有盖湿盒、染色缸、吸管、试管等 操作方法 1．准备：检查加样板，生物载片恢复室温，标记。 2．稀释：PBS-Tween缓冲液稀释血清，设阴阳性对照。 3．加样：加样板放于泡沫塑料板上，加25μl稀释后血清，至加样板的每一反应区，避免气泡。加完所有标本后开始温育。 4．温育：将生物薄片盖于加样板的凹槽里，反应开始，室温温育30分钟。 5．冲洗：用烧杯盛PBS-Tween缓冲液流水冲洗生物薄片，然后立即将其浸入盛有PBS-Tween缓冲液的小杯中至少1分钟。不必混摇。 6．加样：滴加20μl荧光素标记的抗人球蛋白（结合物）至一洁净加样板的反应区，完全加完方可继续温育。荧光素标记的抗人球蛋白用前需混匀并以PBS-Tween缓冲液稀释。
单克隆抗体制备与应用
单克隆抗体制备与应用 姓名：王志豪学号：10073485 班级：工优070 关键词：单克隆抗体，人抗体，杂交瘤细胞 摘要：1975年德国学者Kohler和英国学者Milstein成功地将骨髓瘤细胞和产生抗体的B淋巴细胞融合为杂交瘤细胞,其分泌的抗体是由识别一种抗原决定簇 的细胞克隆所产生的均一性抗体,称之为单克隆抗体。从鼠源单抗之后,单抗历经了鼠源性抗体、嵌合抗体、人源化抗体、人源性抗体4个发展阶段。随着分子生物学和细胞生物学的发展,单抗理论几乎应用到生物学研究的每一个区域。 1975 年, Kohler 和Milstein 创立了杂交瘤技术制备单克隆 抗体,此后单克隆抗体迅速广泛地应用于生物学和医学的各个领域。单克隆抗体可用于分析抗原的细微结构及检验抗原抗体未知的结构 关系;生产出针对复杂生物混合物中的特定分子的抗体,可用于分离、分析及纯化该特定分子抗原;其试剂可用于临床诊断和治疗,或用于 以单抗为弹头的“生物导弹”药物等。但单克隆抗体技术自问世以来,在临床治疗方面进展缓慢,主要原因是目前单克隆抗体大多是鼠源性的,而鼠源单抗应用于人体治疗时存在诸多问题:鼠源单抗在人体中 常不能有效激活补体和Fc 受体相关的效应系统;被人体免疫系统所 识别,产生人抗鼠抗体(HAMA) 反应;且在人体循环系统中很快被清除。因此,在保持对特异抗原表位的高亲和力的基础上人源化和全人化的改造,减少异源抗体的免疫原性成为单抗研究的重点。此外,传统杂交瘤技术还存在制备周期较长,成本较高,杂交瘤细胞不稳定抗性会丢
失等缺陷。近年来,随着分子生物学技术的发展,出现了嵌合单克隆抗体和由转基因小鼠、噬菌体展示技术、核糖体展示技术及共价展示技术所制备的单克隆抗体。这些技术可有效解决传统杂交瘤技术所存在的问题,为单克隆抗体的应用提供更广阔的空间。 1994 年, 美国Cell Genesys 公司和Genpharm公司宣布转基因小鼠作为生产全人抗体的载体问世。这项技术是将人抗体基因微位点转入小鼠体内,产生能分泌人抗体的转基因小鼠。其前提是人的抗体基因片段在小鼠体内进行重排并表达,并且这些片段能与小鼠细胞的信号机制相互作用,即在抗原刺激后,这些片段可被选择、表达并活化B 细胞分泌人抗体。这些转基因小鼠的不足之处在于转移基因片段较小,仅30kb 左右,因此这种抗体库在面对抗原多样性时,其抗体应答显得单薄而不足。此后,Green 等人利用基因打靶技术将编码人抗体轻重链的基因片段大约18Mb 的DNA 全部转到自身抗体基因位点已被灭活的小鼠基因组中,再经过繁育筛选,建立了稳定的转基因小鼠品系。这样得到的转基因小鼠对特异的抗原能产生高亲和力的人抗体。用传统的杂交瘤技术,将表达特异抗体的转基因小鼠B 细胞和骨髓瘤细胞融合,获得杂交瘤细胞系,产生人源抗体。利用转基因小鼠技术已获得了一系列抗IL8 、TNFα以及EGFR 的人单克隆抗体,这些细胞因子在肿瘤或其他疾病中起着重要的作用,因此其单克隆抗体作为导向剂具有重要的临床治疗意义。目前生产的单抗大多是鼠源性的，但其在临床应用方面还存在着很大的弊端，主要是鼠源单抗与NK 等免疫细胞表面Fc 段受体亲和力弱,产生的抗体依赖性细胞介导的细胞毒
单克隆抗体制备过程中经过两次筛选
单克隆抗体制备过程中经过两次筛选 单克隆抗体制备过程中，总共有两次筛选，第一次筛选出杂交瘤细胞，第二次筛选出能产生特异性抗体的杂交瘤细胞，两次筛选的原理和方法是不相同的。 第一次筛选的原理与方法：细胞融合后，杂交瘤细胞的选择性培养是第一次筛选的关键。普遍采用的HAT选择性培养液是在普通的动物细胞培养液中加入次黄嘌呤（H）、氨基喋呤（A）和胸腺嘧啶核苷酸（T）。其依据是细胞中的DNA合成有两条途径：一条途径是生物合成途径（“D途径”），即由氨基酸及其他小分子化合物合成核苷酸，为DNA分子的合成提供原料。在此合成过程中，叶酸作为重要的辅酶参与这一过程，而HAT培养液中氨基喋呤是一种叶酸的拮抗物，可以阻断DNA合成的“D途径”。另一条途径是应急途径或补救途径（“S途径”），它是利用次黄嘌呤—鸟嘌呤磷酸核苷转移酶（HGPRT）和胸腺嘧啶核苷激酶（TK）催化次黄嘌呤和胸腺嘧啶核苷生成相应的核苷酸，两种酶缺一不可。因此，在HAT培养液中，未融合的效应B细胞和两个效应B细胞融合的“D途径”被氨基喋呤阻断，虽“S途径”正常，但因缺乏在体外培养液中增殖的能力，一般10d左右会死亡。对于骨髓瘤细胞以及自身融合细胞而言，由于通常采用的骨髓瘤细胞是次黄嘌呤—鸟嘌呤磷酸核苷转移酶缺陷型（HGPRT）细胞，因此自身没有“S途径”，且“D途径”又被氨基喋呤阻断，所以在HA T培养液中也不能增殖而很快死亡。惟有骨髓瘤细胞与效应B细胞相互融合形成的杂交瘤细胞，既具有效应B细胞的“S途径”，又具有骨髓瘤细胞在体外培养液中长期增殖的特性，因此能在HA T培养液中选择性存活下来，并不断增殖。 第二次筛选的原理和方法：在实际免疫过程中，由于采用连续注射抗原的方法，且一种抗原决定簇刺激机体形成相对应的一种效应B淋巴细胞，因此，从小鼠脾脏中取出的效应B淋巴细胞的特异性是不同的，经HA T培养液筛选的杂交瘤细胞特异性也存在差异，所以必须从杂交瘤细胞群中筛选出能产生针对某一预定抗原快定簇的特异性杂交瘤细胞。通常采用有限稀释克隆细胞的方法，将杂交瘤细胞多倍稀释，接种在多孔的细胞培养板上，使每一孔含一个或几个杂交瘤细胞（理论上30%的孔中细胞数为0时，才能保证有些孔中是单个细胞），再由这些单细胞克隆生长，最终选出分泌预定特异抗体的杂交细胞株进行扩大培养。因此，单克隆抗体制备过程中，两次筛选的原理和方法是不相同的。 单克隆抗体制备的基本原理与过程 原理： B淋巴细胞在抗原的刺激下，能够分化、增殖形成具有针对这种抗原分泌特异性抗体的能力。B细胞的这种能力和量是有限的，不可能持续分化增殖下去，因此产生免疫球蛋白的能力也是极其微小的。将这种B细胞与非分泌型的骨髓瘤细胞融合形成杂交瘤细胞，再进一步克隆化，这种克隆化的杂交瘤细胞是既具有瘤的无限生长的能力，又具有产生特异性抗体的B淋巴细胞的能力，将这种克隆化的杂交瘤细胞进行培养或注入小鼠体内即可获得大量的高效价、单一的特异性抗体。这种技术即称为单克隆抗体技术。 过程： 1）免疫脾细胞的制备制备单克隆抗体的动物多采用纯系Balb/c小鼠。免疫的方法取决于所用抗原的性质。免疫方法同一般血清的制备，也可采用脾内直接免疫法。 2）骨髓瘤细胞的培养与筛选在融合前，骨髓瘤细胞应经过含8-AG的培养基筛选，防止细胞发生突变恢复HGPRT 的活性（恢复HGPRT的活性的细胞不能在含8-AG的培养基中存活）。骨髓瘤细胞用10%小牛血清的培养液在细胞培养瓶中培养，融合前24h换液一次，使骨髓瘤细胞处于对数生长期。 3）细胞融合的关键: 1技术上的误差常常导致融合的失败。例如，供者淋巴细胞没有查到免疫应答。这必然要失败的。 2融合试验最大的失败原因是污染，融合成功的关键是提供一个干净的环境，以及适宜的无菌操作技术。 4）阳性克隆的筛选应尽早进行。通常在融合后10天作第一次检测，过早容易出现假阳性。检测方法应灵敏、准确、而且简便快速。具体应用的方法应根据抗原的性质，以及所需单克隆抗体的功能进行选择。常用的方法有RIA法、ELISA法和免疫荧光法等。其中ELISA法最简便，RIA法最准确。阳性克隆的筛选应进行多次，均阳性时才确定为阳性克隆进行扩增。 5）克隆化克隆化的目的是为了获得单一细胞系的群体。克隆化应尽早进行并反复筛选。这是因为初期的杂交瘤细胞是不稳定的，有丢失染色体的倾向。反复克隆化后可获得稳定的杂交瘤细胞株。克隆化的方法很多，而最常用的是有限稀释法。 （1）显微操作法：在显微镜下取单细胞，然后进行单细胞培养。这种方法操作复杂，效率低，故不常用。 （2）有限稀释法：将对数生长期的杂交瘤细胞用培养液作一定的稀释后，按每孔1个细胞接种在培养皿中，细胞增值后成为单克隆细胞系。第一次克隆化时加一定量的饲养细胞。由于第一次克隆化生长的细胞不能保证单克隆化，所以为获得稳定的单克隆细胞株需经2～3次的再克隆才成。应该注意的是，每次克隆化过程中所有有意义的细胞都
杂交瘤技术的实验原理及其操作演示(4学时)
教案 课程名称细胞生物学实验授课题目（章、节）实验八：杂交瘤技术的实验原理及其操作演示授课老师刘庆平授课对象2002级生物工程授课时间第十三周教学方法理论教学选用教具多媒体教学实验内容提要： 杂交瘤技术的实验原理及其操作演示（4学时） 第一节实验介绍150分钟 （一）、实验原理； （二）、实验目的； （三）、实验用品； （四）、实验方法和过程； （五）、实验结果分析； 第二节．实验多媒体示教30分钟
教学重点、难点及基本要求： 重点及难点： 杂交瘤获得成功的基本要素。 1、动物免疫 2、细胞融合 3、杂交瘤细胞的融合 基本要求： 1. 掌握杂交瘤技术的基本原理和基本操作方法 2. 能运用杂交瘤技术来制备自己需要的单克隆抗体 教研室主任意见： 单元教学小结（经验效果、问题、改进措施、信息反馈等） 本教案以讲授一个单元（2-4学时）一次实验（实习）为单位填写。填写要用钢笔。字迹要清晰、工整。按表项目逐一填写。
实验二杂交瘤技术的实验原理及其操作演示 （多媒体教学演示4学时） 杂交瘤技术是1975年Kohler和Milstein用于制备单克隆抗体而创建的一项重要技术，被誉为“免疫学上的一次革命”。此技术被广泛用于各种单克隆抗体的制备。抗体是由B淋巴细胞分泌的，一个B淋巴细胞只能分泌一种抗体。把B淋巴细胞和骨髓细胞融合，即可形成在体外长期存活并分泌的杂交瘤细胞，如果把单个杂交瘤细胞克隆化，扩增传代，其分泌的抗体即为高度纯一的单克隆抗体。单克隆抗体具有高度专一性，一种单克隆抗体只能结合一种特定的抗原决定簇。正是由于其这种高度专一性，因此被广泛用于疾病的论断和治疗，生物大分子的鉴定、定位和分离纯化，以及一些细胞器，特定细胞或病毒的鉴定、定位和分离等方面, 具有极其远大的应用前景, 因此，用于制备单克隆抗体的杂交瘤技术也变得越来越重要，应用范围越来越广。 实验目的 1. 掌握杂交瘤技术的基本原理和基本操作方法 2. 能运用杂交瘤技术来制备自己需要的单克隆抗体 实验原理 杂交瘤技术的建立基于以下三种关键技术。 一、动物免疫 动物体内的B淋巴细胞在特定外来抗原的刺激下，可以大量增殖变成浆细胞以分泌针对于该抗原的抗体。脾内不同的B淋巴细胞克隆可分泌针对不同抗原的抗体。当受到特定外来抗原刺激时，相应的B淋巴细胞克隆便大量增殖以分泌相应的特异性抗体。动物免疫的作用就是用特定外为抗原对动物进行一次或多次免疫，以刺激能分泌针对于该抗原抗体的B淋巴细胞大量增殖，从而得到大量产生专一的B淋巴细胞。 二、细胞融合 B淋巴细胞受外来抗原刺激后可以分泌抗体，但它在体外存活很短时间(最多两周)后即死亡；而骨髓瘤细胞不分泌任何免疫球蛋白，却能在体外长期存活。如果能将这两种细胞的特性结合起来，我们就能得到既能分泌抗体又能在体外长期存活的细胞。 脾脏是动物体内B淋巴细胞集中的最大免疫器官, 取出脾细胞(B淋巴细胞)和骨髓瘤细胞融合后，能产生五种细胞类型；未融合的脾细胞和骨髓瘤细胞, 自身融合的脾细胞和骨髓瘤细胞，以及脾细胞和骨髓瘤细胞融合形成的杂交瘤细胞。其中杂交瘤才是我们需要的，因此就要设法将此杂交瘤细胞从上述细胞混合液中挑选出来。
单克隆抗体的制备
单克隆抗体的制备 摘要:单克隆抗体技术是现代生命科学研究的重要工具,在基因和蛋白质的结构和功能研究方面有着不可或缺的作用。近年来,随着分子生物学技术的发展,出现了嵌合单克隆抗体和由转基因小鼠、噬菌体展示技术、核糖体展示技术及共价展示技术所产生的单克隆抗体。这些技术将有效解决单克隆抗体的鼠源性等问题。下面主要讲述制备单抗的实验过程。 关键词：抗体，单克隆，肿瘤，细胞融合，淋巴细胞 现代生物技术制药工业始于1971年，现已创造出35个重要治疗药物，全球大约有2500多家公司，主要产品有重组蛋白质药品、重组疫苗和诊断、治疗用的单克隆机体三大类。我国自80年代在采用现代生物技术改造传统生物技术制药产业方面已取得初步成果。但我国生物技术诊断试剂、酶工程、动植物细胞工程医药产品、现代生物技术支撑技术、后处理技术和制剂技术等方面与国外还存在差距。 1．国外现代生物技术产业发展的现状 自1971年Cetus公司成立至今，现代生物技术制药工业已走完了二十五年的路程，创造出35个重要的治疗药物，目前已在治疗癌症、多发性硬化症、贫血、发育不良，糖尿病、肝炎、心力衰竭、血友病、囊性纤维变性和一些罕见的遗传性疾病中取得良好效果。在医药工业中，传统生物技术（包括近代生物技术）已为人类提供了许多重要药品，在保障人类生命健康和推动社会进步中发挥了巨大作用；现代生物技术以其特有的高新技术又为人类提供了传统生物技术难以获得的极微量的珍贵药品。由于这一系列现代生物技术新型药物的出现，使过去无法治疗的疑难疾病得到了治疗。同时，应用现代生物技术DNA重组，细胞融合以及细胞大规模培养等现代生物技术发展和提高传统生物技术的生产水平，为抗生素、氨基酸、维生素以及基体激素等药品的生产，构建了高产新菌株，创造新工艺，提高生产能力，降低生产成本，促进生产发展。