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Double-antigen sandwich time-resolved immunofluorometric assay for the detection of anti-HCV

Double-antigen sandwich time-resolved

immunofluorometric assay for the detection of

anti-hepatitis C virus total antibodies with improved specificity and sensitivity

Feng-Bo Wu,13Hai-Qiao Ouyan,1Xiao-Yan Tang 1and Zhen-Xian Zhou 23

Correspondence Feng-Bo Wu

gaoweiwuli@https://www.wendangku.net/doc/d01438488.html,

Research &Development Department,SYM-BIO Life-Science Co.Ltd,Tai-Cang City,Jiang-Su Province 215400,PR China

Clinical Laboratory,Nanjing Second Hospital,Nanjing City 210003,PR China

Received 20December 2007Accepted 24March 2008

Current anti-hepatitis C virus (HCV)antibody screening immunoassays are routinely based on an indirect format.Although their use for anti-HCV antibody detection has achieved a very high specificity and sensitivity,false-positive results are still a problem especially among populations with a low prevalence of HCV infection.One strategy to obviate this problem is to adapt the assay from an indirect format to a double-antigen sandwich one to further improve its specificity.In this study,a double-antigen sandwich time-resolved immunofluorometric assay (DAS-TRIFMA)has been developed to detect total anti-HCV antibodies based on biotin–streptavidin interaction.For comparison,1025samples were analysed by the DAS-TRIFMA and three indirect anti-HCV antibody detection methods.For samples with discordant results,PCR-ELISA and Inno-LIA were employed as supplementary assays to analyse the presence of HCV antibodies.With regard to the 1025clinical samples,the overall concordance between the DAS-TRIFMA and the three indirect methods was 99.41,98.93and 98.93%for Ortho ELISA 3.0,WAT ELISA and I-TRIFMA,respectively.The specificity/sensitivity of the DAS-TRIFMA,Ortho HCV ELISA 3.0,WAT HCV ELISA and I-TRIFMA were 100/99.09,99.34/98.18,99.23/97.27and 99.01%/98.18%,

respectively.The DAS-TRIFMA was able to detect HCV antibodies at a concentration about 1/10of that detectable by indirect methods.From the obtained results and their comparison,it is concluded that the DAS-TRIFMA is a more specific and reliable method for screening anti-HCV antibodies,and weakly positive S/Co values by the DAS-TRIFMA were more predictive of HCV infection than those by indirect methods.

INTRODUCTION

The detection of hepatitis C virus (HCV)RNA has become an increasingly useful tool in the diagnosis of HCV infection and in the management of patients during therapy (Richter,2002;Lok &Gunaratnam,1997;Chevaliez &Pawlotsky,2006).Compared to HCV RNA testing,anti-HCV antibody immunoassays are thought to be more practicable as an initial screening test because of the ease of use,relative cost-effectiveness and low variability.Accordingly,up to the present time anti-HCV antibody immunoassays are still the most commonly used tests to determine past or present exposure to HCV.In addition,studies in recent years have revealed that the detection of anti-HCV antibodies still has a role in the diagnosis of HCV infection,since the possibility of active

HCV infection can not be ruled out in patients who test positive for HCV antibodies but negative for HCV RNA in serum due to low-level undetectable viraemia and inter-mittent viraemia (Carren

?o et al.,2006;Radkowski et al.,2005;Carren

?o,2006).Anti-HCV antibody immunoassays have now progressed to

the third generation.Although these assays have better sensitivity and specificity than their predecessors (Colin et al.,2001;Abdel-Hamid et al.,2002),there is still a high prevalence of false-positive results,especially among immunocompromised patients or populations without liver-related diseases,leading to unnecessary health-care costs and diagnosis puzzles (Ansari &Omrani,2006;Zylberberg &Pol,1996;Hyams et al.,2001;CDC,2000).Immunoassays for detection of viral-specific antibodies have been developed for various viruses.ELISAs for detection of anti-human immunodeficiency virus or anti-Treponema pallidum antibodies have validated that

Abbreviations:DAS-TRIFMA,double-antigen sandwich time-resolved immunofluorometric assay;I-TRIFMA,indirect TRIFMA.

3These authors contributed equally to this work.

Journal of Medical Microbiology (2008),57,947–953DOI 10.1099/jmm.0.47835-0

47835G 2008SGM Printed in Great Britain

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employing a double-antigen sandwich(DAS)format instead of the original indirect format can substantially improve the assay’s specificity(Bu¨rgisser et al.,1996; Schmidt et al.,2000).However,up to now there has been little investigation into the development and usage of a DAS assay for anti-HCV antibody detection.In this study, we developed a DAS time-resolved immunofluorometric assay(DAS-TRIFMA)for detecting total anti-HCV anti-bodies by using biotin as indirect label.With the benefits of the sandwich assay format and the biotin–streptavidin interaction,the DAS-TRIFMA showed obviously improved specificity and at least the same good sensitivity compared to that of two widely used commercial indirect ELISAs. This study indicates that most of the false-positive results in the indirect anti-HCV immunoassays are associated with their indirect assay format,and the DAS immunoassay,as exemplified by the present DAS-TRIFMA,is more reliable for screening for the presence of anti-HCV antibodies. METHODS

Samples.A total of1025samples from domestic hospitals and blood

centres were used for comparison.Seventeen of the1025samples were

from asymptomatic blood donors with S/Co values(S/Co,the ratio of

the assay signal divided by the cut-off value of the assay)of0.8–4.7by indirect anti-HCV ELISAs(Wu-Han Blood Center,Wu-Han City,

China).The remaining1008samples were from patients in the

routine evaluation of the cause of their liver disease.

For hook effect evaluation,a further56sera screened as highly anti-HCV antibody reactive by the Abbott AxSYM HCV(version3.0)were

obtained from the First Affiliated Hospital of Guang-Xi Medical

University.Eight hundred and seventy negative samples used for cut-

off determination were from outpatients and blood centres. Reagents and instruments.C185(molecular mass55kDa)was a

recombinant chimeric antigen condensed with the major HCV epitopes located in the HCV core,NS3,NS4and NS5regions(JKE-L

Biotechnologies).C188was a mixture of recombinant peptides

composed of HCV core,NS3,NS4and NS5fragments(Bite Biological).The low background microtitre plate(8612wells)was

from Nunc.Goat anti-hIgG and monoclonal anti-human IgM(m-chain

specific)antibodies were from Genetimes Technology.Streptavidin,

biotin-cap-NHS,BSA,casein and other chemicals were from Sigma-Aldrich.N1-Benzyl-DTTA-Eu3+[N1-(p-isothiocyanato-benzyl)-diethylene-triamine-N1,N2,N3,N4-tetraacetate-Eu3+),the VICTOR2fluo-

rometer,Plateshake(1296-003)and Platewash(1296-026)were from

Perkin-Elmer.The ELISA reader was Multiskan MK3(Thermo Labsystems).The CP-70MX preparative ultracentrifuge was from Hitachi.

Comparison methods.Two indirect anti-HCV ELISAs used were Ortho HCV ELISA3.0(Ortho-Clinical Diagnostic)and WAT HCV ELISA(Beijing,China).Inno-LIA HCV Score(Innogenetics)and quantitative PCR-ELISA(Hao-Yuan Biotechnologies)were used as a supplementary assay to confirm the anti-HCV antibody detection. PCR-ELISA was a colorimetric microtitre plate based assay for detection of HCV RNA,in which RT-PCR was performed in the first step for amplification of HCV RNA and ELISA was used for amplicon identification.Inno-LIA was performed based on the16h sample incubation procedure.The above assays were carried out strictly according to the manufacturer’s instructions.

In order to obtain direct comparison,an indirect anti-HCV TRIFMA (I-TRIFMA)based on the same solid-phase antigen,C188,as that used in the DAS-TRIFMA was designed as described in the‘I-TRIFMA of anti-HCV antibodies’section.The schematic diagram of the DAS-TRIFMA and indirect methods for anti-HCV antibody detection is shown in Fig.1.

Biotinylation and Eu3+labelling.One millilitre of the recombinant C185was dialysed for12h at room temperature(RT)against PBS buffer(0.1M,pH7.0)containing5mol urea l21,2%(w/v)ethylene glycol and0.01mol dithiothreitol l21.The solution was transferred to a glass bottle and10m l biotin-cap-NHS at50mg ml21in N,N-dimethylformamide was added with continuous stirring.After2h reaction,the mixture was dialysed against the same PBS buffer to remove the unconjugated biotin molecules.The biotinylated antigen (biotin-C185)was centrifuged at4u C for1.5h at35000g in the P40ST rotor of an Ultracentrifuge CP-70MX;the clear supernatant containing the biotinylated C185was stored at4u C. Streptavidin,goat anti-hIgG and monoclonal anti-m-chain of human IgM were labelled with Eu3+using the same protocol as previously described(Wu et al.,1999).

Microwell coating.One hundred microlitres of C188at2m g ml21 in phosphate buffer(0.1M,pH7.0,containing6M urea)was incubated in microwells for12h at RT.The microwells were then washed twice with washing solution(10mM Tris/HCl buffer, containing0.9%NaCl,0.05%NaN3and0.05%Tween20).One hundred and fifty microlitres of0.1M phosphate buffer(pH7.0) containing0.5%casein and10%calf serum was added and incubated for3h to block the coated wells.

DAS-TRIFMA of the total anti-HCV antibodies.In the DAS-TRIFMA(Fig.1),25m l undiluted samples and100m l assay buffer (50mM Tris/HCl,pH7.5,containing0.9%NaCl,0.05%NaN3, 0.05%Tween20,0.5%casein and10%calf serum)containing 300ng biotin-C185ml21were added in microwells successively.The anti-HCV antibodies in the sample were allowed to react with the surface antigen and biotin-C185for30min at RT with slow stirring. The plate was washed four times with washing buffer,then100m l assay buffer containing1m g Eu3+-labelled streptavidin ml21was added and stirred for15min.The wells were washed six times.One hundred microlitres of fluorescence enhancement solution was added and stirred for5min to dissociate Eu3+from the surface complex into the solution,where a highly fluorescent complex was formed. The results of the DAS-TRIFMA were interpreted as positive or negative based on the S/Co values:an S/Co￠1represented

anti-HCV Fig.1.Mechanism of the DAS-TRIFMA(a)and typical indirect immunoassays(b)for antibody detection.

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antibody positive;otherwise,negative.This criterion was the same for the other immunoassays in this study.Each sample was measured in duplicate to obviate the fluorescence aberrations that occasionally occurred in the TRIFMA.

I-TRIFMA of anti-HCV antibodies.In the I-TRIFMA,C188-coated strips were the same as those used in the DAS-TRIFMA for direct performance comparison.All samples were diluted1:100with dilution buffer(50mM Tris/HCl buffer,containing0.9%NaCl, 0.05%NaN3,0.05%Tween20,0.1%chloracetamide,0.05% Escherichia coli extract,0.04%EDTA and20%calf serum).One hundred microlitres of the freshly diluted samples was added in duplicate in microwells and stirred for30min at RT.The plate was washed four times.One hundred microlitres of Eu3+-labelled goat anti-hIgG at0.5m g ml21in assay buffer(50mM Tris/HCl buffer, containing0.9%NaCl,0.05%NaN3,0.05%Tween20,20%calf serum,0.5%casein and0.1%fish gelatin)was added and stirred for 30min.The wells were washed six times with washing buffer,and the fluorescence was detected in the same way as that in the DAS-TRIFMA.

Detection of the anti-HCV IgM.To study the possible contribution of the anti-HCV IgM to the response of the DAS-TRIFMA and to estimate the prevalence of anti-HCV IgM in the DAS-TRIFMA positive samples,109positive and267negative samples determined by the DAS-TRIFMA were analysed by the anti-HCV IgM TRIFMA as follows.Samples were diluted1:100with TSA buffer(50mmol Tris/ HCl l21,0.9%NaCl,pH7.75)containing4%goat anti-hIgG serum, 0.5%BSA,0.05%Tween20and0.05%NaN3.After incubation for 30min with slow stirring,the diluted samples were centrifuged at 10000g for10min.Twenty microlitres of the supernatant sample was transferred to the C188-coated microwells,which were pre-filled with100m l TSA buffer containing0.5%BSA,0.1%casein,0.05% Tween20,0.1%chloracetamide,0.05%E.coli extract and0.05% NaN3.The mixture was incubated for1h under continuous stirring. The wells were washed four times.One hundred microlitres of assay buffer containing Eu3+-labelled monoclonal anti-m chain of human IgM at500ng ml21was added and incubated for30min.The wells were washed and the fluorescence was measured in the same way as described above.

The lowest detection limits.To assess the lowest detection limit of the DAS-TRIFMA,one sample prepared by pooling17positive sera was serially diluted and measured by the DAS-TRIFMA,Ortho ELISA and I-TRIFMA simultaneously.The highest dilution rates at which the sample could still be detected as positive were used to evaluate the lowest detection limits of the anti-HCV antibody immunoassays. Precision of the DAS-TRIFMA.The reproducibility of the DAS-TRIFMA was evaluated by assaying three positive sera with different positive levels within one assay or in different assays.Coefficients of variation were calculated based on the S/Co values and the standard deviations(SD).

Hook effect.The susceptibility of the DAS-TRIFMA to the hook effect(high-dose prozone effect)was evaluated by analysing56highly positive anti-HCV sera in their original form and at1:10and1:100 dilutions.

Clinical sample analysis.One thousand and twenty-five clinical samples were analysed by the DAS-TRIFMA,Ortho HCV ELISA3.0, WAT HCV ELISA and I-TRIFMA.Samples with consistent positive/ negative results obtained by the four methods were considered as true anti-HCV antibody positive/negative,and no further study was done. When the assay results were discordant by at least one of above different methods,Inno-LIA and/or HCV RNA analysis was performed.Interpretation of the anti-HCV antibody results of the discordant samples was according to the MMWR recommendations (Alter et al.,2003):(1)a sample was considered negative or positive when the strip immunoblot assay(Inno-LIA)gave negative or positive results;(2)a sample was considered positive when the sample was Inno-LIA-IND(IND,indeterminate)but PCR-positive;(3)a sample was considered anti-HCV antibody indeterminate when the sample was Inno-LIA-IND but PCR-negative.The indeterminate results were not included in the statistical evaluation.All discordant samples and samples with S/Co values,0.30by the DAS-TRIFMA but in the range0.8–1.0by at least one of the three indirect methods were analysed by PCR-ELISA.The cut-off values of the Ortho ELISA 3.0and WAT ELISA were calculated according to the instructions included in the kits.To obtain a maximum specificity and sensitivity, the cut-off values of the DAS-TRIFMA,I-TRIFMA and anti-HCV IgM TRIFMA were determined as the mean plus five standard deviations on the basis of analysis of the870negative samples. RESULTS AND DISCUSSION

The lowest detection limits of the different methods

The lowest detection limit of the anti-HCV antibody immunoassay was studied by assaying one positive sample at different dilutions by the DAS-TRIFMA,I-TRIFMA and Ortho HCV ELISA3.0.The tested positive sample was a pooled serum containing different specificities of anti-HCV antibodies.The maximum dilution of this sample detected as positive by the DAS-TRIFMA was1:12500,and was 1:500and1:2500by the Ortho ELISA and I-TRIFMA, respectively.These data suggested that the DAS-TRIFMA is able to detect HCV antibodies at about a10-times lower concentration than that detectable by the two indirect methods.

Due to the identical coating antigen used in the DAS-TRIFMA and I-TRIFMA,the improved analytical sensitiv-ity of the DAS-TRIFMA over the I-TRIFMA can be ascribed to the following factors:(1)the HCV antigen labelled with biotin under the described conditions allowed well protection of its binding activity;(2)generally more than 4.2biotins were coupled to C185,leading to significant signal amplification(Wu et al.,2002);(3)the small bulk of the biotin molecule makes the biotinylated C185available for binding the target antibodies without serious steric hindrance;(4)the high specificity of the DAS-TRIFMA allowed the use of undiluted sample,while in indirect methods the samples had to be diluted to decrease the interfering molecules that may cause false-positive results,so a low titre of target antibodies in the sample are more likely to be detected by the DAS-TRIFMA than by indirect methods.The enhanced sensitivity of the DAS-TRIFMA may be helpful for detecting the weak antibody response from newly infected patients or patients with suppressed or compromised immunity.

Precision of the DAS-TRIFMA

The reproducibility of the DAS-TRIFMA was studied by assaying three positive anti-HCV sera with S/Co values of Sandwich immunoassay for HCV antibody detection

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1.3, 5.1and16.8,respectively.The within-assay and between-assay coefficients of variation based on S/Co values(n512)were in the range3.97–6.33%and5.31–11.72%,respectively.

Hook effect

In the one-step procedure of the DAS-TRIFMA,the biotinynated and the solid-phase HCV antigen were incubated simultaneously with the HCV antibodies in the samples.A high concentration of HCV antibodies in samples may simultaneously saturate the epitopes of both the biotinylated and solid-phase HCV antigen,leading to a falsely decreased response.To evaluate whether the DAS-TRIFMA suffered this problem in clinical applications,56 highly anti-HCV-positive sera were tested in their original form and at dilutions of1:10and1:100.In this way,the hook effect could be spotted if the fluorescence of the diluted sample was stronger than that of the original one. As shown in Table1,the hook effect was detected in3of the56samples with the highest fluorescence at a dilution of 1:4,1:8and1:2,respectively.No false-negative was caused by the hook effect in the56samples. Immunoassay of1025clinical specimens

One thousand and seven of the1025(98.24%)samples measured by the four immunoassays(DAS-TRIFMA,Ortho HCV ELISA3.0,WAT HCV ELISA and the I-TRIFMA)gave consistent results,including899negative samples and108 positive samples.The concordance between the DAS-TRIFMA and the three indirect methods was99.41% (1019/1025),98.93%(1014/1025)and98.93%(1014/1025) for Ortho ELISA 3.0,WAT ELISA and I-TRIFMA, respectively.Of the1007concordant samples,one sample was negative by all of the four immunoassays but showed an Inno-LIA-IND and PCR-positive result(1.316105copies ml21).The S/Co values were0.87,0.6,0.68and0.38by the DAS-TRIFMA,Ortho HCV ELISA3.0,I-TRIFMA and the WAT HCV ELISA,respectively.

Eighteen of the1025samples showed discordant results and were further studied by Inno-LIA and PCR-ELISA.As shown in Fig.2,15of the18samples were DAS-TRIFMA-negative and3were DAS-TRIFMA-positive.With regard to the15DAS-TRIFMA-negative samples,at least one of the indirect methods gave positive results.Twelve of the15 DAS-TRIFMA-negative samples were validated as negative by Inno-LIA results.Four of the18samples with Inno-LIA-IND and PCR-negative results(samples1,2,8and10in Fig.2)were excluded from the statistical analysis due to their unclear anti-HCV antibody status.Samples12and13 were correctly detected as positive by the DAS-TRIFMA but missed by at least one of the indirect methods.

By excluding the four samples(samples1,2,8and10in Fig.2)that showed unclear anti-HCV antibody status,911 of the1021samples were correctly identified as negative by the DAS-TRIFMA,yielding100%(911/911)specificity. Meanwhile,the specificity of the Ortho HCV ELISA3.0, WAT HCV ELISA and I-TRIFMA was99.34%(905/911), 99.23%(904/911)and99.01%(902/911),respectively.The sensitivity of the DAS-TRIFMA,Ortho HCV ELISA3.0, WAT HCV ELISA and I-TRIFMA was99.09%(109/110), 98.18%(108/110),97.27%(107/110)and98.18%(108/ 110),respectively.These results indicated that the reliability of the DAS-TRIFMA for anti-HCV antibody detection was significantly improved with respect to both specificity and sensitivity when compared to that of the three indirect methods.

As shown in Fig.2,16of the21false-positive events by the three indirect methods had S/Co values less than3.0,while 5had S/Co values greater than3.0,with the highest at 17.92.This result suggested that judging the anti-HCV antibody status based on the S/Co value of an indirect anti-HCV antibody assay may be inadequate,although higher S/Co values in indirect anti-HCV antibody immunoassays is more predictive of true anti-HCV positives(Alter et al., 2003;Dufour et al.,2003a;Ren&Zhuang,2005).

The S/Co values of the1025samples by the DAS-TRIFMA were compared to those of the three indirect methods.As shown in Fig.3(a,b,c),the positive and negative results were clearly separated by the DAS-TRIFMA,whereas these show obvious overlap by the indirect methods.Of the911 negative samples,9,33,49and61samples show S/Co values in the range of0.4–1.0and0,6,7and9samples were wrongly identified as positive by the DAS-TRIFMA, Ortho ELISA,WAT ELISA and I-TRIFMA,respectively. The overlap of the S/Co values between the negative and

Table1.Hook effect of the DAS-TRIFMA observed in three samples from56highly positive sera

The S/Co values of the56sera were in the range22.3–161.7by the DAS-TRIFMA.

Sample Fluorescence for samples at different dilutions(c.p.s.)*

Original1/21/41/81/161/321/64 Sample A9638471669083267937818027641109238700368467896 Sample B7093251181907155892320109801630681976839590878 Sample C578923983092676302439276299342192822135629

*The cut-off of the DAS-TRIFMA was15820c.p.s.in above table.

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positive results by indirect methods makes it difficult to estimate the true HCV status,especially when the S/Co values were around 1.0.Moreover,this phenomenon in indirect methods also makes it difficult to set up a reasonable cut-off value,since high cut-off may miss samples with low titres of target antibodies,while low cut-off may give rise to an unacceptably high ratio of false-positives.Due to the enhanced specificity of the DAS-TRIFMA,the weakly positive results by the DAS-TRIFMA (e.g.S/Co values from 1.0to 2.0),which are prone to be confused by false-positive results by indirect methods,were highly predictive for identifying the existence of HCV antibodies.Taking into account the above findings,it can be expected that further improvement of the analytical sensitivity of the DAS-TRIFMA may be helpful for enhancing its ability to detect trace amounts of HCV antibodies;this work is presently in progress in our laboratory.

Excluding the 18discordant samples,14of the 1025samples totally nonreactive by the DAS-TRIFMA but with S/Co values in the range 0.8–1.0by at least one of the indirect methods were analysed by PCR-ELISA;none were PCR-ELISA-positive.Therefore,the possibility that these samples were from newly infected persons with a low concentration of HCV antibodies was excluded.The causes of the elevated signals in the indirect methods were not studied further.

The amino acid sequence and the purity of the HCV antigen used for assay development are important factors influencing both the specificity and sensitivity of anti-HCV antibody immunoassays.Sharing the same solid-phase antigen,the improved specificity of the DAS-TRIFMA over the I-TRIFMA was mainly associated with its DAS format.The DAS format endows the assay two levels of binding selection;namely,the target antibodies must be recognized

by both the coated antigen and labelled antigen and it is then possible to produce a response.In such a case,if some molecules were nonspecifically attached by the coated HCV antigen,it is possible to choose another HCV antigen lacking this attachment as tracer antigen to prevent the ‘sandwich’formation.As a result,the possibility that interference molecules bridge the coated and the labelled antigen can be decreased to a low level.The labelled second anti-hIgG antibodies used to trace the captured anti-HCV IgG antibodies in the indirect methods recognize not only HCV-specific IgG but all hIgG molecules.Because of the high IgG concentration in human blood (generally more than 5mg ml 21),there is a strong tendency for some of these IgG molecules to be bound to the well surface by direct adsorption or by indirect capture via the surface molecules,and then arouse a signal,giving false-positive results.This problem might be more serious when the samples are from patients with systemic lupus erythema-tosus,portal cirrhosis,rheumatoid arthritis and some infectious diseases due to the very complicated,higher concentration of immunoglobulin components in their blood.To alleviate such a problem,indirect anti-HCV immunoassays usually require a 1:10–1:100-fold sample dilution prior to test.This strategy is effective;however,it does not always work well,and will inevitably be detrimental to the detection sensitivity when the sample contains a very low concentration of target antibodies.The above investigations proved that the DAS format is an effective strategy for improving the specificity of anti-HCV antibody immunoassays;however,other choices exist for the same purpose.For example,the specificity of the Ortho Anti-HCV Chemiluminescence immunoassay (CLIA)is enhanced compared to the Ortho ELISA,as reported by Dufour et al.(2003b).Because both the CLIA and ELISA are indirect format-based,the CLIA specificity improvement can probably be ascribed to factors other than the

assay

Fig. 2.S/Co values of the 18discordant samples obtained by DAS-TRIFMA and three indirect methods.HCV RNA analysis:the concentration of HCV RNA was 7.18?106copies ml ”1for sample 13by PCR-ELISA.No results were obtained for samples 6,7and 11due to the strong PCR inhibition caused by heparin in the plasma.The remaining 14samples were PCR-ELISA-negative.Inno-LIA analysis:samples 1,2,8and 10were Inno-LIA-IND;samples 12and 13were Inno-LIA-positive;the remaining 12samples were Inno-LIA-negative.

Sandwich immunoassay for HCV antibody detection

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format,e.g.the differences in the coating materials,the optimization of the assay components and the setting of cut-off values.

Anti-HCV IgM detection by the TRIFMA

Of 109DAS-TRIFMA-positive samples,17(15.60%)were anti-HCV IgM reactive (mean S/Co 52.782)and 9(8.26%)had S/Co values of 0.6–1.0by the indirect anti-HCV IgM TRIFMA.Of the 17anti-HCV IgM-positive/DAS-TRIFMA-positive samples,the ratio of the mean S/Co by the DAS-TRIFMA to that of the I-TRIFMA was 2.06,and was only 1.25for the remaining 92anti-HCV IgM-negative/DAS-TRIFMA-positive samples.This result sug-gested that anti-HCV IgM in the 17samples might have contributed to the response of the DAS-TRIFMA.In addition,the weakly positive sample 13by the DAS-TRIFMA (S/Co 51.839;Fig.2)was also anti-HCV IgM reactive with S/Co at 1.76.This result suggested that HCV antibodies in this sample were mainly of the IgM class;this hypothesis could explain why it was detected by the DAS-TRIFMA but missed by all three indirect methods with labelled anti-hIgG as tracer.The possibility that the response of the IgM TRIFMA on the 17samples was caused by IgG antibodies rather than IgM was excluded since no significant response was observed with the use of labelled goat anti-hIgG in place of the anti-m second antibodies in the anti-HCV IgM TRIFMA.Based on these observations,it could be rationally deduced that anti-HCV IgM,and perhaps other classes of HCV-specific antibodies which were not investigated in this study,had strengthened the detectability of the target antibodies in the DAS-TRIFMA.The enhanced detectability of the HCV antibod-ies in the DAS-TRIFMA by antibodies other than those of the IgG class is helpful for more sensitive diagnosis of HCV infection.Of the 267DAS-TRIFMA-negative samples,3gave positive results in the anti-HCV IgM TRIFMA.This discrepancy was perhaps also associated with the indirect format of the anti-HCV IgM TRIFMA,in which any IgM molecules in the sample,if non-specifically attached on the solid-phase surface,may cause an elevated signal by the same mechanism as that described above.

In conclusion,a DAS-TRIFMA was developed and evaluated in this study to determine its ability to detect the total antibodies to HCV.The use of biotin as an indirect label allowed efficient antigen labelling and good preservation of the antigen’s immunoreactivity.The DAS-TRIFMA was sensitive,precise and could be completed within 60min.Although the clinical samples studied in this paper were relatively limited,the results of this study had sufficient statistical power to demonstrate the superiority of the DAS-TRIFMA over the indirect methods with respect to specificity and sensitivity.Such improve-ments may be useful for screening for HCV infection and other clinical applications.The DAS-TRIFMA omitted the sample pre-dilution due to its excellent specificity,leading to simplification of the assay procedure and a

Fig. https://www.wendangku.net/doc/d01438488.html,parison of the S/Co values of the 1025clinical samples by the DAS-TRIFMA and three indirect methods.The S/Co span between the two dashed lines parallel to the x -or y -axis was 0.7–1.5.The relatively lower S/Co values for the positive samples obtained by the Ortho anti-HCV ELISA 3.0(b)were partly because of its higher cut-off value,which was generally more than 0.60(OD 492)in our experiments.

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sensitive detection of the low concentration of target antibodies.It is anticipated that a DAS immunoassay for anti-HCV antibody detection,as exemplified by the present DAS-TRIFMA,will play a important role in future diagnosis of HCV infection in clinical laboratories and blood banks,as well as for different research purposes. ACKNOWLEDGEMENTS

We would like to thank Professor Sun LP,Shanghai Public Sanitation Center,Nanjing85Hospital and the Second Affiliated Hospital of Nanjing Medical University for providing their valuable specimens. This work was supported by the foundation of High Technology Research Program of Jiangsu Province,China(no.BG2007606)and the foundation for Talents in the Six Main Professions of Jiangsu Province,China(no.2006130).

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Carren?o,V.,Pardo,M.,Lo′pez-Alcorocho,J.M.,Rodr?′guez-In?igo,E., Bartolome′,J.&Castillo,I.(2006).Detection of hepatitis C virus (HCV)RNA in the liver of healthy,anti-HCV antibody-positive, serum HCV RNA-negative patients with normal alanine aminotrans-ferase levels.J Infect Dis194,53–60.

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英语教学法主要流派

英语教学法主要流派常见术语：Method, Approach, Design, Procedure, Technique 一、语法翻译法 1、语法翻译法是一种通过学习语法规则和词汇，并且按照规则用本国语和目的语进行互译来教授语言的方法。 2、教学过程阅读/朗读——句子翻译——讲解语法/语言点——书面回答——理解性问题 3、原则和技巧原则：（1）书面语重要，重视写作和阅读（2）熟记语法规则和单词（3）教师的绝对权威（4）本族语的中介作用技巧：（1）书面翻译/互译（2）阅读理解性问答（3）演绎法讲解语法规则（4）填空、背诵、造句、作文等 4、评价：（1）使用方便（2）学习语言知识多于语言技能掌握（3）改良式的语法翻译法：弥补口语的听说训练、重视交际能力的培养、重视调动学生学习的主动性。二、直接法 1、直接法是一种通过实物、图画、动作、表情等手段把外语和其对应的意思直接联系，从而达到直接理解和直接应用的外语教学法。 2、教学步骤用目的语讲解——提问——回答用归纳法教语法：例子——总结规则——巩固性练习——听写练习 3、原则与技巧原则：（1）先听说后书面语言（2）通过有意义的上下文来学习单词和句子（3）禁止使用本族语翻译（4）自我更正来促进语言学习技巧：大声朗读、问答练习、自我更正、会话、填空、听写段落、画图讲解、实物演示等。 4、评价：（1）强调语言实践和运用，有利于听说（2）学习用外语思考，重视语言的交际作用（3）排斥本国语，有时花费很长的时间来解释，或者解释不清楚（4）对教师口语、教学技能要求高三、听说法 1、听说法是一种运用句型操练形式学习外语的方法。其理论基础为结构主义语言学和行为主义心理学。 2、教学步骤听外语对话——模仿——纠正语音语调——逐句背诵——对话练习——看书面材料——语法点归纳——替换/回答练习 3、原则与技巧

军训队列示范教学法教案适用于教官培训

军训队列示范教学法教案适用于教官培训标准化管理部编码-[99968T-6889628-J68568-1689N]

队列教案作业提要-------------------------------10分钟课目:队列目的:通过训练,使大家学会军人的徒手队列动作,养成良好的军人姿态和严格的纪律观念,为以后的学习和生活打好基础。把所学的用于生活实践中。内容:（一）立正（军姿）（二）跨立、（三）稍息、（四）停止间转法。（五）齐步行进与立定、方法:讲解示范,组织练习,观摩评比,小结讲评. 时间:2小时地点:本训练场. 要求:1,严守队列纪律,一切行动听指挥,精神振作,姿态端正. 2,认真听讲解,注意看示范动作,认真练习. 3,相互学习,相互帮助,把所学知识运用到生活实践中去. 4,克服天气带来的不良影响. 作业进程----------------------------------20分钟（一）整理服装、清点人数、向在场首长报告（二）准备教学设施及教案（三）按照训练计划实施训练作业实施--------------------------------30分钟第一个训练内容：立正立正是军人的基本姿态，是队列动作的基础。军人在宣誓、接受命令、进见首长和向首长报告、回答首长问话、升降国旗和军旗、奏国歌和军歌等严肃庄重的时机和场合，均应当自行立正。要领：两脚跟靠拢并齐，两脚尖向外分开约60度；两腿挺直；小腹微收，自然挺胸；上体正直，微向前顷；两肩要平，稍向后张；两臂下垂自然伸直，手指并拢自然微曲，拇指尖贴于食指第二节，中指贴于裤缝头要正，颈要直，口要闭，下颌微收，两眼向前平视。

单个军人队列动作教学教案汇总

单个军人队列动作教学教案作业提要课目：单个军人队列动作教学法。目的：通过训练，使同志们掌握单个军人队列动作的教学方法和组织训练程序，提高同志们的组训能力。内容：齐步与立定；时间：约40 分钟；方法：讲解示范、个人体会、分班（组）练习、评比验收。地点：队列训练场：要求： 1、严格遵守训练场纪律，服从命令，听众指挥，做到令行禁止，培养良好的队列作风； 2、认真听课、观看示范、开动脑筋、刻苦训练； 3、大力发扬军事民主、做到能者为师、互帮互学、共同提高。以上要求同志们能不能做到？（众答：“能”）作业进程教学准备,,,,,,,,,,,,,,,, ,,5 分钟 1、清点人数，整理着装，向首长报告； 1/13 2、宣布教学提要；教学实施,,,,,,,,,,,,,,,,,,,30 分钟一、理论提示：齐步是军人的常用步伐，一般用于队列的整齐行进。其应用范围十分广泛，是所有军事科目训练的基础，作为一名军人必须学会和掌握。其口令下达是：“齐步—走”立“—定”。为了便于训练，请同志们跟我熟悉口令（略）；为了使大家对动作有个直观形象的了解，下面由我体现一遍动作。（教练员体现齐步与立定动作）二、要领讲解：动作要领边讲边做：听到齐步一走”的口令后，左脚向正前方迈出75cm,按照先脚跟后脚掌的顺序着地，同时身体重心前移，右脚照此法动作；上体正直、微向前倾；手指轻轻握拢，拇指贴于食指第二节；两臂自然前后摆动，向前摆臂时，肘部微弯，小臂自然里合，手心向内稍向下，拇指根部对正衣扣线，并与最下方衣扣同高（着夏季作训服时，与第四衣扣同高；着冬季作训服时，与第五衣扣同高；着水兵服时与腰带同高）；离身体25cm。向后摆臂时，手臂自然伸直，手腕前侧距裤缝线约30cm 。行进速度每分钟116 —122 步。听到“立—定”的口令后，左脚向前大半步着地（脚尖向外约30 度），两腿挺直，右脚取捷径迅速靠拢左脚，成立正姿势。齐步行进时要保持良好的军人姿态，做到精神振奋、姿态端正、表情自然、节奏分明，步幅步速准确，有勇往直前的精神。为了便于大家记忆，我把要领归纳为：“走直线、身体稳、摆臂自然、靠脚准。” 为了使大家对动作有个完整的印象，下面我再体现一遍齐步行进。以上讲解的是齐步与立定的动作要领，同志们清楚没有？（众答：“清楚”！）为了检查同志们对齐步与立定要领的掌握情况，我提两个问题： 1、齐步行进时前摆和后摆各距身体多少厘米？ 2/13 2、齐步的步幅和步速各是多少？谁来回答?（众答：“我来回答”！）回答正确，说明同志们在听课时精力比较集中，掌握要领比较快。

队列训练教案范文

队列训练教案范文单人军人队列训练教材课前准备教学准备：1清点人数（即报数） 2着装（顺序不要变，指挥员不稍息）下达科目科目：单个军人队列动作（一个字也不要错）内容：立正，稍息、停止间转法、跨立，立正???（根据所训练内容而定）目的：通过xx的训练，使同志们掌握xx的动作要领及其组织训练方法，打牢队列动作基础。（指挥员敬礼，下达请稍息）时间：大约X小时地点：队列训练场方法：理论提示，讲解示范，分组练习，评比竞赛，小结讲评，考核验收要求：（全体人员自行立正） 1 认真听，仔细看，勤思考，刻苦练 2 精神饱满，姿态端正，严格遵守队列纪律 3 提倡能者为师，互帮互助，共同进步 ??????

（指挥员下达各班带开组织训练）作业实施注意：1在进行所有内容的训练第一步讲解示范时，首先严格按照条令条例内容完整准确的背诵一遍。随后用自己的语言对所规定内容进行解释，边解释边做动作。最后指挥员不讲解，将规定动作做两到三遍示范。2在进行讲评的时候所需注意的细节：讲评前所有人均为稍息指挥员下达稍息，当指挥员发出讲评的口令，全体人员自行立正，然后指挥员敬礼下达请稍息，当指挥员讲评时讲到存在的问题时，所有人自行立正，当指挥员把存在的问题讲完之后，指挥员敬礼并下达请稍息，最后指挥员说讲评完毕时，所有人自行立正。3所有动作要领均按照新颁布的条令条例作为标准。第一个训练内容：立正，稍息综述：立正是军人的基本姿态，是队列动作的基础。军人在宣誓，接受命令，进见首长和向首长报告，回答首长问话，升降国旗和军旗，奏国歌和军歌等严肃庄重的时机和场合，均应当自行立正。稍息是站法的三种动作之一通常与立正互换，是立正动作的一种调节。一动作要领：1立正：两脚跟靠拢并齐，两脚尖向外分开约60度，两腿挺直；小腹微收，自然挺胸；上体正直，微向

教学设计模板及案例62951

信息技术学科教学设计模板（参考）信息技术课程教学设计案例课程名称：信息技术课程教学论

学院及系：教育科学与技术学院教育技术系班级：06级教本二班姓名：赵国杰提交日期：2008年11月20日题目：自己选择一节信息技术课程内容依据系统的教学设计理论和过程模式对其进行教学设计。《计算机硬件组成》教学设计一、前端分析（一）教材内容分析这节课是高中信息技术教材第一册基础知识中的一节，在教材中这一节叫“微型计算机系统”。是对整个计算机硬件系统和软件系统的介绍，它是针对高中学生的知识接受能力，对计算机的本质进行介绍，使学生充分了解计算机的组成和简单的工作原理，以便在学习后续知识时对知识的理解更为深刻。本节课是其中的硬件系统这一部分，主要介绍计算机由哪些硬件组成，及其各部件的功能。（二）学习者特征分析本节课授课对象是高一年级学生，在这之前学生已经对计算机了有一定的了解，他们认识鼠标、键盘等硬件设备，还掌握了常用的应用软件操作。但学生对计算机的系统组成、计算机内部结构认识不是很清晰，经过本课学习之后，对学生进一步了解计算机主机的外观及内部组成，及了解存储设备和输入、输出设备有很大帮助。这个年龄段的学生对电脑有着很强的好奇心，并且对学习电脑有很大的兴趣。学生的计算机水平有差距，水平高的学生和一般学生的认知能力、思维能力的不同会对教学效果有影响，所以学生通过交流互相学习。教学实施规划二、教学目标设计（一）知识与技能： 1．在观察实物及动手实践的基础上使学生对计算机硬件系统有直观的认识，了解计算机的硬件组成，并简单的了解其功能。? ? ? ? ? 2．培养学生自主学习、自主探索、合作学习、观察、以及总结归纳的能力。 3. 培养学生的动手实践能力，实现概念和实物的对接。（二）过程与方法：通过课件演示、学生交流、师生交流、人机交流等形式，培养学生利用信息技术和概括表达的能力。

军人队列训练指导

、中、八课前教学准备：1 清点人数（即报数） 2 整理着装（顺序不要变，指挥员不稍息）下达科目科目：单个军人队列动作（一个字也不要错）内容：立正，稍息、停止间转法、跨立，立正....... （根据所训练内容而定）目的：通过xx 的训练，使同志们掌握xx 的动作要领及其组织训练方法，打牢队列动作基础。（指挥员敬礼，下达请稍息）时间：X小时地点：队列训练场方法：理论提示，讲解示范，分组练习，评比竞赛，小结讲评，考核验收要求：（全体人员自行立正） 1 认真听，仔细看，勤思考，刻苦练 2 精神饱满，姿态端正，严格遵守队列纪律 3 提倡能者为师，互帮互助，共同进步指挥员下达各班带开组织训练）作业实施注意：1在进行所有内容的训练第一步讲解示范时，首先严格按照条令条例内容完整准确的背诵一遍。随后用自己的语言对所规定内容进行解释，边解释边做动作。最后指挥员不讲解，将规定动作做两到三遍示范。2 在进行讲评的时候所需注意的细节：讲评前所有人均为稍息指挥员下达稍息，当指挥员发出讲评的

口令，全体人员自行立正，然后指挥员敬礼下达请稍息，当指挥员讲评时讲到存在的问题时，所有人自行立正，当指挥员把存在的问题讲完之后，指挥员敬礼并下达请稍息，最后指挥员说讲评完毕时，所有人自行立正。3 所有动作要领均按照新颁布的条令条例作为标准。第一个训练内容：立正，稍息综述：立正是军人的基本姿态，是队列动作的基础。军人在宣誓，接受命令，进见首长和向首长报告，回答首长问话，升降国旗和军旗，奏国歌和军歌等严肃庄重的时机和场合，均应当自行立正。稍息是站法的三种动作之一通常与立正互换，是立正动作的一种调节。一动作要领：1 立正：两脚跟靠拢并齐，两脚尖向外分开约60 度，两腿挺直；小腹微收，自然挺胸；上体正直，微向前倾；两肩要平，稍向后张；两臂下垂自然伸直，手指并拢自然微曲，拇指尖贴于食指第二关节，中指贴于裤缝；头要正，颈要直，口要闭，下颌微收，两眼向前平视。 2 稍息：左脚顺脚尖方向伸出约全脚掌的三分之二，两脚自然伸直，上体保持立正姿势，身体重心大部分落于右脚。二动作要领归纳：立正要做到“三挺”“三收” 一“睁”“一闭” 即：挺膝，挺胸，挺颈，收腹，收臀，收下颚，眼要睁，口要闭。稍息要做到：“两快”“两不变”，即：上体保持军姿不变，出脚收脚路线不变，出脚快，收脚快。

教学设计模板及案例

教学设计模板（参考）

信息技术课程教学设计案例《计算机硬件组成》教学设计一、前端分析（一）教材内容分析这节课是高中信息技术教材第一册基础知识中的一节，在教材中这一节叫“微型计算机系统”。是对整个计算机硬件系统和软件系统的介绍，它是针对高中学生的知识接受能力，对计算机的本质进行介绍，使学生充分了解计算机的组成和简单的工作原理，以便在学习后续知识时对知识的理解更为深刻。本节课是其中的硬件系统这一部分，主要介绍计算机由哪些硬件组成，及其各部件的功能。（二）学习者特征分析本节课授课对象是高一年级学生，在这之前学生已经对计算机了有一定的了解，他们认识鼠标、键盘等硬件设备，还掌握了常用的应用软件操作。但学生对计算机的系统组成、计算机内部结构认识不是很清晰，经过本课学习之后，对学生进一步了解计算机主机的外观及内部组成，及了解存储设备和输入、输出设备有很大帮助。这个年龄段的学生对电脑有着很强的好奇心，并且对学习电脑有很大的兴趣。学生的计算机水平有差距，水平高的学生和一般学生的认知能力、思维能力的不同会对教学效果有影响，所以学生通过交流互相学习。教学实施规划二、教学目标设计（一）知识与技能： 1．在观察实物及动手实践的基础上使学生对计算机硬件系统有直观的认识，了解计算机的硬

件组成，并简单的了解其功能。 2．培养学生自主学习、自主探索、合作学习、观察、以及总结归纳的能力。 3. 培养学生的动手实践能力，实现概念和实物的对接。（二）过程与方法：通过课件演示、学生交流、师生交流、人机交流等形式，培养学生利用信息技术和概括表达的能力。（三）情感与价值观： 1．让学生在自主解决问题的过程中培养成就感，为今后学会自主学习打下良好的基础。2．通过小组协作活动，培养学生合作学习的意识、竞争参与意识和研究探索的精神，从而调动学生的积极性，激发学生对计算机硬件的兴趣。三、教学内容设计教学重点：计算机的硬件系统由几大部分组成，分别包括哪些硬件，基本功能是什么？确定依据：根据高中生现有的接受能力以及应考要求，当给出硬件实物或图片时学生能指出名称和它们的基本作用。教学难点：存储设备和运算设备都包括那些硬件以及它们的功能。确定依据：这两大部件包括的硬件较多，又是计算机的核心部件，但由于这些部件大多集中于主机箱内部，学生平时很难见到学生主机箱内部部件，所以不太容易掌握，故为本节的难点。四、教学策略分析（一）教学方法 1. 任务驱动法让学生在具体任务的驱动下进行学习，在完成任务的过程中掌握应掌握的知识点。本节课的教

单个军人队列动作教学法(新颖)

单个军人队列动作教学法科目：单个军人队列动作目的：通过训练是同志熟练掌握单个军人的的动作要领，达到步调一致、整齐划一，为以后的生活和训练打下良好的基础。内容：1、立正、稍息、跨立2、停止间转法3、行进与立定4、行进间转法5、步伐变换6、蹲下、起立7、脱帽、戴帽、夹帽8、敬礼9、操枪要求：1、严格训练严格要求，做错动作打报告。 2、克服天气带来的不良影响。 3、互帮互助，共同提高。一：立正、稍息、跨立理论提示：立正是军人的基本姿态，是队列动作的基础。军人在宣誓，接受命令，进见首长和向首长报告，回答首长问话，升降国旗和军旗，奏国歌和军歌等严肃庄重的时机和场合，均应当自行立正。稍息是站法的三种动作之一通常与立正互换，是立正动作的一种调节。跨立即跨步站立，其主要用于军体操执勤和舰艇上分区列队等时机和场合，可以与立正互换。口令:立正, 稍息，跨立动作要领：当听到立正的口令以后，两脚跟迅速靠拢并齐，两脚尖向外分开约60度，两脚挺直，小腹微收，自然挺胸，上体正直，微向前倾；两肩要平稍向后张;两臂下垂自然伸直，四指并拢

自然微曲，拇指尖贴于食指第二关节，中指贴于裤缝；头要正，颈要直，下颌微收，两眼向前平视稍高。稍息的动作要领是：当听到稍息的口令以后，左脚顺脚尖方向伸出约全脚掌的三分之二，两脚自然伸直，上体保持立正姿势，身体重心大部分落于右脚。稍息过久可以自行换脚当听到跨立的口令以后，左脚向左跨出约一脚之长，两脚挺直，上体保持立正姿势，身体重心落于两脚之间。两手后背，左手握右手腕，拇指跟部与外腰带下沿（内腰带上沿）同高；右手手指并拢自然弯曲，手心向后。达到的标准及要求：立正是军人的基本姿态，俗称军姿训练，是部队的形象工程,良好的军姿是我军威武之师,文明之师的体现;同时队列训练也是一项比较枯燥乏味的训练项目,作为一名教练员,要善于启发诱导,调动大家的训练积极性,把训练的着眼点放在塑造良好形象,培育坚毅性格上来.使大家养成良好的站立习惯. 立正时我们要做到“三挺，三收，一顶，一睁，一垂，一贴；运好一口气，用好八股劲”稍息时要做到“两快两稳一准确” 三挺:挺膝,挺胸,挺颈三收:收小腹,收臀部.收下颌一顶:胸部向前上方顶一睁:眼睛睁大有神一垂:双臂自然下垂一贴:双手贴于大腿外侧中央部位运好一口气:站立时,深吸一口气,用半口气保持挺胸,用半口气喘息用好八股劲:两脚向前的扒力和脚后跟的蹬力膝盖向后的压力和向内旋转的夹力小腹向上的收力和臀部向前的收力胸部向前上方的挺力

队列训练教学法示教作业教(学)案

批准人：年月日队列训练教学法示教作业（教案）编写人： XXX XXX年X月X日

教学提要课目：队列训练教学法示教作业目的：使学员掌握队列训练教学的方法、特点和要领，做到“会讲解示范、会组织练习、会纠正动作、会做思想、工作”的“四会要求”；提高组织队列训练的能力。内容：一、教学准备二、教学实施三、课终讲评时间：三课时地点：队列训练场方法：分段讲解示范，分班轮流充当教练员练习要求：队列教学法示教作业是使学员掌握队列训练方法的重要途径，因此要求在训练中做到：一、严格遵守队列纪律，牢记自己的队列位置，做到令行禁止；二、精神振奋、着装整齐、仪容严整，理论熟悉、语言清晰、声音宏亮；三、认真听讲看示范，仔细体会教学要领，切实是队列教学方法练习一次、提高一次

教学保障：（略）教学进程教学准备：一、集合队伍，清点人数、整理着装、调整队列至适当位置；（需要时向在场最高首长报告）二、宣布课目、目的、内容、时间、地点、方法、要求等。教学实施：理论提示：队列训练教学法是队列训练的重要组成部份，是教练员按照《队列条令》对学员进行队列训练的途径，通常队列训练教学法分教学准备、教学实施、课终讲评三个阶段。一、教学准备阶段教学准备，是教练员在领受教学任务后，所进行的与教学相关的各项准备工作的统称。分两个部分（一）课前准备包括熟悉训练大纲和教材，收集和阅读相关资料，熟悉了解受训对象，选定教学方法，培养示范分队和人员，进行试讲、示教，组织教学保障等等（二）、授课准备 1、集合队伍，清点人数、整理着装、调整队列至适当位置；（需要时向在场最高首长报告）

教学案例模板,例文

如何撰写教学案例一、什么是教学案例教学案例是真实而又典型且含有问题的事件。简单地说，一个教学案例就是一个包含有疑难问题的实际情境的描述，是一个教学实践过程中的故事，描述的是教学过程中“意料之外，情理之中的事”。这可以从以下几个层次来理解：教学案例是事件：教学案例是对教学过程中的一个实际情境的描述。它讲述的是一个故事，叙述的是这个教学故事的产生、发展的历程，它是对教学现象的动态性的把握。教学案例是含有问题的事件：事件只是案例的基本素材，并不是所有的教学事件都可以成为案例。能够成为案例的事件，必须包含有问题或疑难情境在内，并且也可能包含有解决问题的方法在内。正因为这一点，案例才成为一种独特的研究成果的表现形式。案例是真实而又典型的事件：案例必须是有典型意义的，它必须能给读者带来一定的启示和体会。案例与故事之间的根本区别是：故事是可以杜撰的，而案例是不能杜撰和抄袭的，它所反映的是真是发生的事件，是教学事件的真实再现。是对“当前”课堂中真实发生的实践情景的描述。它不能用“摇摆椅子上杜撰的事实来替代”，也不能从抽象的、概括化的理论中演绎的事实来替代。二、案例与其他体例的区别 1、案例与论文的区别：从文体和表达方式上来看，论文是以说理为目的，以议论为主；而案例

则以记录为目的，以记叙为主，兼有议论和说明。也就是说，案例是讲一个故事，是通过故事说明道理。从写作的思路和思维方式上来看，论文写作一般是一种演绎思维，思维的方式是从抽象到具体，而案例写作是一种归纳思维，思维的方式是从具体到抽象。 2、案例与教案、教学设计、教学实录的区别：教案、教学设计都是事先设想的教学思路，是对准备实施的教学措施的简要说明；案例则是对已发生的教育过程的反映。一个写在教之前，一个写在教之后；一个是预期，一个是结果。案例与教学实录的体例比较相近，它们的区别也体现了案例的特点和价值。同样是对教育情境的描述，教学实录是有闻必录，而案例是有选择的。选择什么内容，取决于案例撰写的目的和功能。三、案例的基本组成元素从文章结构上看，案例一般包含以下几个基本的组成元素：（1）背景案例需要向读者交代故事发生的有关情况：时间、地点、人物、事情的起因等。如介绍一堂课，就有必要说明这堂课是在什么背景情况下上的，是一所重点学校还是普通学校，是有经验的优秀教师还是年青的新教师，是经过准备的“公开课”还是平时的“家常课”，等等。背景介绍并不需要面面俱到，重要的是说明故事的发生是否有什么特别的原因或条件。（2）主题案例要有一个主题。写案例首先要考虑我这个案例想反映什么问题，是

主要外语教学法儿流派

主要外语教学流派一、外语教学法的概念外语教学法是一个多元化、多层次的概念。作为低层次概念是指讲练个别的、具体的语音、词汇、语法知识项目进行听、说、读、写技能训练和培养听、说、读、写交际能力及发展智力的方式方法；高层次概是指研究外语教学的目的任务、教学内容、要求、教学原则、教学过程、教学形式、教学方式方法和评价等整个体系。外语教学早已存在,已有几千年的历史,但将它作为一种科学的方法体系加以研究并逐步形成各种流派,却只是近百年来的事。一百多年来,外语教学法产生了多种流派,各流派都经历了一个产生、发展的历程。外语教学法各流派的产生和发展的过程并不是一个新流派完全替代一个老流派的过程,而是各自有着一个纵横交叉的发展历程。一方面它们有着历史发展的先后顺序,另一方面它们却又处于一个相互联系、取长补短、长期并存的局面之中。从纵的方面来看,八个主要流派的产生和发展的顺序是语法翻译法、直接法、自觉对比法、听说法、视听法、自觉实践法、认知法和功能法。从横的方面来看,各种外语教学法流派并不因为产生新的流派而自行消失,它们是相互取长补短,相互促进,并不断完善自身的教学方法体系。二、国外教学流派的分类及发展在外语教学法发展史上约有24 种外语教学法流派得到过较为广泛的传播。我们将其中发挥过较大作用或至今仍有影响力的外语教学法流派就其教学主张中的某些共同点分成四大流派来评述。即自觉法、直接法, 强化法和综合法。自觉法诸流派包括最早产生的语法翻译法、自觉对比法, 自觉实践法和认知法等。自觉法最显著的特点是, 它强调学习是一种思维活动, 要求学生自觉地理解语言规则。 1、语法翻译法是外语教学法历史上最早诞生的教学法。从18 世纪开始, 欧洲学校开设外语课, 由于没有现成的外语教学法可供借鉴, 他们就自然而然地沿用了当时教古典语的方法。他们教学的方法是把希腊文或拉丁文译成本国文, 或把本国语译成希腊或拉丁语。希腊语和拉丁语都有详尽的文法, 所以文法教学成为课堂上的重要内容。背诵文法被认为是学习外语的捷径。这样做的优点是利用文法和学生的理解力以提高外语教学效果。缺点是忽视口语教学, 不利于交际能力的培养；过分强调语法在教学中的作用；教学方式单一, 课堂气氛沉闷。 2、自觉对比法是50 年代前苏联在外语教学中推行的一种教学法。它的主要特点有: 强调语法规则与自觉学习的重要性；外语与母语的对比是教新课的重要环节；以阅读为中心, 在阅读的基础上开展听、说、写的训练；贯彻由简而繁, 循序渐进的教学原则。 3、60 年代出现的自觉实践法和认知法是各自针对自觉对比法和听说法的缺陷提出来的。自觉实践法是为矫正自觉对比法的弊病而吸收其它外语教学法流派的优点提出的一种折衷法。从直接法那里, 它吸取了“言语实践”这一精华, 从语法翻译法那里继承了“学习外语从自觉开始”这一合理内核, 从听说法中吸取句型操练的思想, 从视听法中吸取情景性原则。它的最大特点是突出外语教学的言语实践性原则,同时又保留了前苏联教育学的重要传统之一---自觉性。作为听说法的对立面, 认识法把语言学习看作是智力活动。在外语教学里, 认知法强调理解, 强调学习语法, 反对单纯依靠机械操练培养语言习惯, 认同使用母语和翻译。在美国, 赞同认识法的人自称为现代语法翻译法流派。

队列训练教案教学提纲

授课人：xxx 科目：队列动作内容：立正，稍息、停止间转法，跨立、立正，齐步行进与立定目的：通过的训练，使同志们掌握以上的动作要领及其组织训练方法，打牢队列动作基础。时间：大约45分钟地点：北操场要求：1 认真听，仔细看，勤思考，刻苦练 2 精神饱满，姿态端正，严格遵守队列纪律第一个内容：立正，稍息综述：立正是军人的基本姿态，是队列动作的基础。军人在面见领导和向领导报告，回答领导问话等严肃的时机和场合，均应当自行立正。稍息是站法的三种动作之一通常与立正互换，是立正动作的一种调节。一动作要领： 1立正：两脚跟靠拢并齐，两脚尖向外分开约60度，两腿挺直；小腹微收，自然挺胸；上体正直，微向前倾；两肩要平，稍向后张；两臂下垂自然伸直，手指并拢自然微曲，拇指尖贴于食指第二关节，中指贴于裤缝；头要正，颈要直，口要闭，下颌微收，两眼向前平视。 2 稍息：左脚顺脚尖方向伸出约全脚掌的三分之二，两脚自然伸直，上体保持立正姿势，身体重心大部分落于右脚。

二动作要领归纳：立正要做到“三挺”“三收”“一睁”“一闭” 即：挺膝，挺胸，挺颈，收腹，收臀，收下颚，眼要睁，口要闭。稍息要做到：“两快”“两不变”，即：上体保持军姿不变，出脚收脚路线不变，出脚快，收脚快。三组织练习四常犯毛病及纠正方法： 1 立正时上体方向不正，两肩不平纠正方法：立正时，身体向左或者向右摆动，两肩均匀。 2 稍息时身体下塌纠正方法：稍息时上体始终保持军姿姿势。第二个内容：停止间转法一动作要领：（以向右转为例）以右脚跟为轴，右脚跟和左脚掌前半部同时用力，使身体协调一致的向右转90度，身体重心落于右脚，左脚取捷径迅速靠拢右脚，成立正姿势。转动和靠脚时，两腿挺直，上体保持立正姿势。向左转按照向右转的动作要领向左转动90度，向后转按照向右转的动作要领向后转动180度，半面向右转按照向右转的动作要领向右转动45度，半面向左转按照向左转的动作要领向左转动45度。二动作归纳：达到的标准和要求：转体时要保持好良好的军姿，动作迅速，节奏分明，做到“快，稳，准，狠” 快：转体要快、靠脚要快稳：做动作时上体要稳、节奏要稳准：转体位置准确、靠脚位置准确

浅谈现行几种主要英语教学方法

浅谈现行几种主要英语教学方法——我的英语教学体会泸溪一中杨峰目前，我国中学英语教学使用的方法较多，但从影响的范围和发展的方向及推广的价值上看，听说法、交际法、折衷法及张思忠16字教学法当属主流。从方法本身来看，它们之间是一种扬弃的关系，即交际法是对听说法的扬弃，折衷法和张思忠16字教学法又是对听说法、交际法等的扬弃。这里，将对上述几种方法进行若干比较，以供在英语教学实践中借鉴和运用。（一）听说法第二次世界大战后期在美国军队中形成并获得成功，五十年代初在美国流行，六十年代中叶发展到顶峰，流传到世界各国，蜚声世界英语教学界。其特点是： 1、注重语言结构、形式，意思为次，机械记忆为主 2、语言学习项目不必语境化； 3、语言学习的目标就是掌握语音、句型、单词，因此，大量使用机械练习； 4、句型操练为教学中使用的主要技巧； 5、发音要求近似本族语人的标准； 6、课堂上禁止使用语法解释，禁止使用母语，禁止在初期水平上使用翻译； 7、只有在长期严格的句型操练和大量机械练习之后，方可进行交际活动； 8、先听说后读写，即在较为熟练地掌握了口语之后方可从事阅读与写作； 9、通过教授语言结构（即句型的系统教学）来掌握英语的语言系统；

10、以语言能力的形成为教学所追求的最大目标； 11、以语言项目的难易来安排学习顺序； 12、教师为中心决定内容，控制学生并使学生严格纳入此法的训练轨道，尽力避免语言错误； 13、语言的准确性是追求的主要目标； 14、学生学习状态被动，实践量少，实践时间短，实践范围窄； 15、方法机械、单调，学生易厌倦。（二）交际法七十年代产生于西欧，是当代社会的政治、经济和文化交流的需要，特别是欧洲共同体成立以来，各种专业英语人才的需要越来越迫切，于是，这种培养语言交际能力的教学方法便产生了，并很快在世界上流行开来。与听说法相比，它的特点是： 1、意思是最重要的；注重意义，围绕交际功能使用或练习结构； 2、语境化是教学的基本前提； 3、语言学习目标就是学会有效地进行交际； 4、句型操练属辅助训练手段； 5、发音以可以理解为标准； 6、依据年龄、兴趣等条件，只要有助于学习者，可以解释语法，母语在适当的场合可以适当使用，翻译可根据学生需要使用；

项目教学法教案

应用项目教学法进行《AutoCAD》教学教案套二厅室内平面综合布置图装修设计一、项目要求：根据客户提供的一张房型平面图，利用AutoCAD软件，完成室内平面综合布置图装修设计，制作作品。整个设计过程分为以下九个子项目：（1）套二厅墙体框架的绘制。（2）套二厅门窗的绘制。（3）套二厅客厅家具的设计与布置。（4）套二厅餐厅家具的设计与布置。（5）套二厅厨房家具的设计与布置。（6）套二厅卫生间的设计与布置。（7）小孩房家具的设计与布置。（8）主人房家具的设计与布置。（9）标注、图纸说明框及打印。二、教学目标知识技能：学生在实际工作的情境中，综合使用绘图命令，掌握平面布置图设计的图形绘制技巧。过程与方法：通过教师、同学及网络的帮助，感受实际工作中平面布置图设计的一般工作流程，学会表达解决问题的过程和方法。情感、态度、价值观：培养学生实际操作能力，以及与同伴合作交流意识和能力。三、项目分析：（展示一张最终平面布置效果为例子）本项目是将AutoCAD软件与具体的室内设计工作结合起来，设计一张实际工作中的平面布置图，让学生从亲身的感受中说、做、学，优化教学过程，改进学习方式，并倡导学生主动参与学习和同学交流合作，用不同的方式来学习知识。通过自己的讨论交流进行探索和实现问题的解决，形成一定的知识解决模型，并

最终解决实际生活问题，从而能够与行业零距离接轨。重点：绘图命令的综合使用以及平面布置图设计的一般工作流程。（用课件演示一般工作流程）难点：灵活运用各种知识，设计出更加人性化的方案。突破重点、难点：①学生在老师的引导下完成项目。 ②教师帮助个别学生提高水平。四、教学策略分析 1．学习者分析学生学习该项目之前已经掌握了绘图的基本命令，能够熟练使用绘图工具。 2．教学理念和教学方式教学是师生之间、学生之间交往互动与共同发展的过程。计算机教学，要紧密联系学生的生活实际。采用项目教学法学习，教师可以利用网络的优势，成为知识传播者、问题情境的创设者、尝试点拨的引导者、知识反馈的调整者。学生是学习的主人，在教师的帮助下，小组合作交流中，利用动手操作探索，发现新知识，自主学习。教学评价方式多样化，包括师生评价、学生评价、小组评价等多种方式。在课堂上利用明确无误的工作表结果对学生的学习和练习作出评价，让每个学生都能体验到成功的乐趣。采用项目教学法，让学生把分散知识的各知识点综合起来，应用于实际的行业工作中。五、教学准备 50台计算机联网，有电子教室软件。六、时间安排（总课时：9课时）任务1套二厅墙体框架的绘制：1课时任务2套二厅门窗的绘制：1课时任务3套二厅客厅家具的设计与布置：1课时任务4套二厅餐厅家具的设计与布置：1课时任务5套二厅厨房家具的设计与布置：1课时任务6套二厅卫生间的设计与布置：1课时任务7小孩房家具的设计与布置：1课时任务8主人房家具的设计与布置：1课时任务9标注、图纸说明框及打印：1课时七、项目实施

军人队列动作教学法示范作业

作业准备 1. 整理着装，清点人数； 2. 向在场的首长报告； 3.宣布作业提要课目：单个军人队列动作目的：通过训练，使同志们熟练掌握单个军人队列动作要领，培养军人良好形象，为今后的执勤、训练打下良好的基础。内容：齐步行进、立定方法：理论提示，讲解示范，组织练习，小结讲评，考核验收。时间：约40分钟场地：队列训练场训练要求：1、严格要求，严格训练。 2、遵守队列纪律，认真体会动作要领。 3、树立吃苦精神，培养顽强作风。作业实施理论提示:齐步是军人行进的常用步伐，一般用于部队的整齐行进。口令：齐步——走立——定在未讲动作要领之前，先将动作演示一遍，动作要领：当听到“走”的动令后，左脚向正前方迈出约75厘米，按照先脚跟后脚掌的顺序着地，同时重心前移，右脚照此法动作；上体正直，微向前倾，手指轻轻握拢，拇指贴于食指第二

节，两臂前后自然摆动，向前摆臂进，肘部弯曲，小臂自然里合，手心向内稍向下，拇指根部对正衣扣线，并与最下方衣扣同高，（着夏季作训服时，与第四衣扣同高，着冬季作训服时，与第五衣扣同高）离身体约25厘米，向后摆臂进，手臂自然伸直，手腕前侧距裤缝线约30厘米，行进速度每分种116——122步。听到“立定”口令，左脚再向前大半步着地，两脚挺直，右脚取捷径靠拢左脚，成立正姿势。根据平时的训练经验，齐步要领可归纳为：（1）脚跟先着地，脚腕稍用力，膝盖向后压。身体向前移。（2）走直线，身体稳，摆臂自然，靠脚准。齐步行进应重点突出三点： 1、上体保持军姿不变 2、腿臂要协调 3、三到位（脚跟到位、身体重心到位、手到位）（一）组织练习训练步骤：第一步：摆臂练习第二步：腿部练习第三步：立定练习第四步：连贯练习第一步：摆臂练习目的：结合前面所讲的动作要领，主要通过下面的练习，让同志

项目教学法教案模板演示教学

项目一我的网店铺设一、主要研究点项目教学理念、小组合作学习、探究学习方法。二、项目教学设计方案的指导思想和设计理念本项目《开办我的网上店铺》设计思路以“ 立足网络营销，实施项目教学，提升创业技能”的理念为指导，以网上店铺的开设与运营作为切入点整合《电子商务概论》课程有关教学内容，按照项目导向、任务驱动的思路重构课程内容体系，通过自主开办网上店铺全方位提升学生创业技能。项目实施过程中，采取理论与实践一体化教学，实现教、学、做的有机结合。教师通过案例说明问题，对实际操作进行示范指导，为学生营造学习情境、创造学习条件和激发学习热情，引导学生在实践中感悟、思考和创新，培养创业意识、竞争与合作意识。学生以小组为单位，采取协作学习方式，选择合适的电子商务交易平台，选择熟悉并且感兴趣的商品，结合目前国内外的网络营销环境，进行网上商店的策划、构建及运营，解决在运营过程中遇到的交易信息安全、资金结算、物流配送、网络促销、货源渠道等实际问题，通过对模拟情境的操纵、观察和思考取得学习成果，并在与之相似的真实情境体验中促进知识的广泛迁移，体验学习的成就感。三、项目书 1．项目名称：《开办我的网上店铺》 2．项目实施的实训条件：安装有电子商务师实验室软件、连接到Internet的网络机房 3．项目实施对象分析本项目实施对象是商贸类专业中专二年级学生，他们具备一定的商务基础知识，且自主学习能力较强，有一定的探索、创新精神。部分学生有过网上购物的经验，对网上开店有着浓厚的兴趣，学习积极性很高。经过一段时期的学习，学生掌握了一定的电子商务理论基础，对商务师实验室软件的运用也已初步了解，因此组织教学中尽可能发挥学生的这些优势，进一步激发学习积极性，培养创新能力、分析问题和解决问题的能力。 4．项目实施目标： ①学生8-10人为一学习小组，在协作过程中着重培养团队合作能力及个人独立解决问题的能力。 ②通过教师的指导，各学习小组讨论交流，合作探究，培养学生的学习能力、分析

队列训练方案教学提纲

队列动作训练方案作业提要课目：队列动作训练目的：通过训练，使同志们学会队列动作要领，达到统一，规范。训练问题：一、单个军人队列动作方法：理论讲解、动作示范、组织练习、时间：14课时地点：队列训练场要求：1、认真听讲、勤于思考、勇于提问、互帮互学、共同提高： 2、严格要求、严格训练、养成良好的姿态和严谨的作风： 3、严格遵守训练场纪律第一训练问题:单个军人队列动作一立正\跨立\稍息 (一)立正严肃庄重的时机和场合,均应按口令或自行立正. 口令:立正动作要领:听到“立正”的口令,两脚跟靠拢并齐，两脚

尖向外分开约60度；两脚挺直，小腹微收，自然挺胸；上体正直，微向前倾；两肩要平，稍向后张；两臂自然下垂，拇指尖贴于食指第二节，中指贴于裤缝；头要正，颈要直，口要闭，下颌微收，两眼向前平视。常犯毛病及纠正方法： 1、两脚跟未并齐。纠正方法：划一横线，两脚跟站字横线上反复体会练习。 2、两脚分开的中心线与脚跟线不垂直。纠正方法：教练员对授课对象对正，矫正其两脚位置或在地面上画“T”字线，反复练习。 3、两脚分开大于或小于60度，纠正方法：用角度尺矫正后，进行练习。 4、两脚未夹紧，有空隙。纠正方法：两脚靠拢，裆部夹紧。 5、腰挺不直。纠正方法：向前上方挺胸，以胸带腰。 6、上体方向不正。纠正方法：教练员对正操练者，使其鼻尖对正衣扣线，肩线与足线平行。 7、挺胸蹶臂部。纠正方法：裆部夹紧向上收腹收臂，形成一股夹力。 8、头歪。纠正方法：指出歪的方向，使其体会纠正。（二）跨立跨立主要用于固定岗位，执勤等场合，可与立正互换。

口令：跨立动作要领：听到“跨立”的口令，左脚向左跨出约一脚之长，两腿挺直，上体保持立正姿势，身体重心落于两脚之间。两手后背，左手握右手腕，拇指根部与外腰带下沿（内腰带上沿）同高；右手手指并拢自然弯曲，手心向后。常犯毛病及纠正方法： 1、跨出动作慢。纠正方法：腿挺直，脚腕用力，脚跟提起迅速跨出。 2、跨出距离过大或过小。纠正方法：在左脚侧一脚之长处划一标记，体会炼收。 3、跨出时弯腿。纠正方法：两腿挺直，出腿同时膝盖后压。（三）稍息稍息是列队动作中一种休息和调整姿势的动作，可与立正互换。口令：稍息动作要领：听到“稍息”的口令，左脚顺脚尖方向伸出约全脚的三分之二，两腿自然伸直，上体保持立正姿势，身体重心大部分落于右脚。若稍息过久，可自行换脚。动作标准及要求：稍息时，左脚跟略提起，脚腕用力，前脚掌擦地迅速伸出，方向要正，距离要准确，两脚自然伸直，体重大部分落于右脚，身体不能晃动，上体保持立正姿势，

队列训练教案

队列动作教案作业提要课目:单个军人队列动作内容:一、立正,稍息,跨立二、停止间转法三、行进与立定四、步法变换五、行进间转法六、脱帽、載帽七、敬礼八、坐下、蹲下、起立标准:姿态端正,精神振奋, 军容严整,动作协调方法:理论提示,讲做示范,组织练习,小结讲评时间: *小时地点:队列训练场要求: 1、严格遵守队列纪律,做到令行禁止

2、严禁嘻笑打闹,做错动作打报告器材保障: (略 ) 作业进程一、作业准备: 1、带队入场,整理着装,清点人数,向在场首长报告 2、宣布作业提要二、作业实施同志们,大家都从电视中领略过国旗护卫队的飒爽英姿吧,他们那端正的姿势,严整的军容,振奋的精神,协调一致的动作都来源于队列训练,大家要成为一名标准的军人,必须从队列训练入手。 (一)、立正、跨立、稍息 <一 >立正立正是军人的基本姿势,是队列的基础。军人在宣誓、接受命令、进见首长和向首长报告、回答首长问话、升降国旗、军旗、奏国歌和军歌等严肅庄重的时机和场合,均应自行立正。 1、口令:立正 2、要领:两脚跟靠拢并齐,两脚向外分开约 60度,两腿挺直;小腹微收,自然挺胸,上体正直,微向前倾,两肩要平,稍向后张,两臂下垂自然伸直,手指并拢自然微曲,拇指尖贴于食指第二节,中指贴于裤缝,头要正颈要直,口要闭,下颌微收,两眼向前平视。

3、立正的动作标准与要求: (1)两脚跟未靠齐。纠正的方法:划一条直线,两脚跟站在横线上反复练习。 (2)方向不正,两脚分开的中心线与脚跟线不垂直。纠正的方法:在地面画“T“字线,反复练习。 (3)两腿未夹紧,有空隙。纠正的方法:两腿靠拢,裆部夹紧。 (4)腰挺不直,纠正的方法:向前上方挺胸,以胸带腰。 (5)头歪,纠正的方法:指出歪的方向,使其体会纠正。 (6)面部不自然,纠正的方法:面部的肌肉放松。 <二>跨立跨立主要用于军体操.执勤和舰艇上分区列队等场合,可以与立正互换。 1.口令:跨立 2.要领:左脚向左跨出约一脚之长,两腿挺直,上体保持立正姿势, 身体重心落于两脚之间,两手后背,左手握右手腕,拇指根部与外腰带下沿(内腰带上沿)同高,右手手指并拢自然弯曲,手心向后。 3.标准与要求:跨立出脚背手快,动作协调,跨脚距离准确。 4.常犯的毛病及纠正的方法:(1)上体不稳,挺肚子,蹶臀部。

外语教学法(B)

江西省2013 年 5 月份高等教育自学考试外语教学法试卷（B）准考证号：姓名：一、Multiple Choices: (2’×15=30) Directions: In this section, you are given 15 questions beneath each of which are four choices marked A, B, C, and D. Y ou are to make the best choice either to complete the incomplete statement or to answer the question. One point is given to each correct choice. 1. Which of the following is NOT emphasized by traditional linguists? A. Correctness. B. The purity of a language. C. Literary excellence. D. Communication. 2. _______ the first language is used in the teaching of the second language in the Grammar-Translation Method. A. A lot of B. A little of C. Little of D. Not any 3. According to the Direct Method, every language has _______ structure. A. similar B. its own C. co-related D. the same 4. The Direct Method _______ the similarities between the first language acquisition and second language learning.