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鏈│鍔戠洅鐢ㄤ簬楂斿瀹氶噺妾㈡脯(c猫)琛€娓�銆佽婕�銆佺祫绻斿嫽婕垮強鐩搁棞(gu膩n)娑查珨妯ｆ湰涓�鏄嗚煵(ch贸ng)涓夐叞鐢樻补锛�TAG锛�鐨勫惈閲�銆�

鏈夋晥鏈燂細6鍊�(g猫)鏈�

淇濆瓨姊濅欢锛�2-8鈩�

鏈│鍔戠洅鍍呬緵绉戠爺浣跨敤锛屼笉寰楃敤浜庤嚚搴婅ê鏂�

瀵�(sh铆)椹�(y脿n)鍘熺悊

瑭﹀姂鐩掗噰鐢ㄩ洐鎶楅珨涓€姝ュぞ蹇冩硶閰惰伅(li谩n)鍏嶇柅鍚搁檮瑭﹂(y脿n)锛�ELISA锛�銆傚線闋�(y霉)鍏堝寘琚槅锜�(ch贸ng)涓夐叞鐢樻补锛�TAG锛夋崟鐛叉姉楂旂殑鍖呰寰瓟涓�锛屼緷娆″姞鍏ユ(bi膩o)鏈�銆佹(bi膩o)婧�(zh菙n)鍝併€丠RP妯�(bi膩o)瑷樼殑妾㈡脯(c猫)鎶楅珨锛岀稉(j墨ng)閬�(gu貌)婧偛骞跺竟搴曟礂婊�銆傜敤搴曠墿TMB椤壊锛孴MB鍦ㄩ亷(gu貌)姘у寲鐗╅叾鐨勫偓鍖栦笅杞�(zhu菐n)鍖栨垚钘�(l谩n)鑹�锛屽苟鍦ㄩ吀鐨勪綔鐢ㄤ笅杞�(zhu菐n)鍖栨垚鏈€绲傜殑榛冭壊銆傞鑹茬殑娣辨泛鍜屾ǎ鍝佷腑鐨勬槅锜�(ch贸ng)涓夐叞鐢樻补锛圱AG锛夊憟姝ｇ浉闂�(gu膩n)銆傜敤閰舵(bi膩o)鍎€鍦�450nm 娉㈤暦(zh菐ng)涓嬫脯(c猫)瀹氬惛鍏夊害锛圤D 鍊硷級锛岃▓(j矛)绠楁ǎ鍝佹績搴︺€�

妯ｆ湰铏曠悊鍙婅姹�

1.  琛€娓�锛�灏囨敹闆嗕簬琛€娓呭垎闆㈢鐨勫叏琛€妯�(bi膩o)鏈湪瀹ゆ韩鏀剧疆2灏忔檪(sh铆)鎴�4鈩�閬�(gu貌)澶�锛岀劧鍚�1000脳g闆㈠績20 鍒嗛悩锛屽彇涓婃竻鍗冲彲锛屾垨?q奴)鈶搁敤閫硿?/span>-20鈩�鎴�-80鈩冧繚瀛�锛屼絾鎳�(y墨ng)閬垮厤鍙嶅京(f霉)鍑嶈瀺銆�
2.  琛€婕�锛�鐢�EDTA鎴栬倽绱犱綔鐐烘姉鍑濆姂閲囬泦妯�(bi膩o)鏈�锛屽苟灏囨(bi膩o)鏈湪閲囬泦鍚庣殑30鍒嗛悩鍏�(n猫i)浜�2-8鈩� 1000脳g闆㈠績15鍒嗛悩锛屽彇涓婃竻鍗冲彲妾㈡脯(c猫)锛屾垨?q奴)鈶搁敤閫硿?/span>-20鈩�鎴�-80鈩冧繚瀛�锛屼絾鎳�(y墨ng)閬垮厤鍙嶅京(f霉)鍑嶈瀺銆�
3.  绲勭箶鍕绘伎锛�鐢ㄩ爯(y霉)鍐风殑PBS (0.01M, pH=7.4)娌栨礂绲勭箶锛屽幓闄ゆ畼鐣欒娑诧紙鍕绘伎涓瑙ｇ殑绱呯窗(x矛)鑳炴渻(hu矛)褰遍熆娓�(c猫)閲忕祼(ji茅)鏋滐級锛岀ū(ch膿ng)閲嶅悗灏囩祫绻斿壀纰庛€傚皣鍓鐨勭祫绻旇垏灏�(du矛)鎳�(y墨ng)楂旂⿳鐨�PBS锛堜竴鑸寜1:9鐨勯噸閲忛珨绌嶆瘮锛屾瘮濡�1g鐨勭祫绻旀ǎ鍝佸皪(du矛)鎳�(y墨ng)9mL鐨�PBS锛屽叿楂旈珨绌嶅彲鏍规摎(j霉)瀵�(sh铆)椹�(y脿n)闇€瑕侀仼鐣�(d膩ng)瑾�(di脿o)鏁�锛屽苟鍋氬ソ瑷橀寗銆傛帹钖﹀湪PBS涓姞鍏ヨ泲鐧介叾鎶戝埗鍔戯級鍔犲叆鐜荤拑鍕绘伎鍣ㄤ腑锛屼簬鍐颁笂鍏呭垎鐮旂（銆傜偤浜嗛€�(j矛n)涓€姝ヨ瑙ｇ祫绻旂窗(x矛)鑳�锛屽彲浠ュ皪(du矛)鍕绘伎娑查€�(j矛n)琛岃秴鑱茬牬纰庯紝鎴栧弽寰�(f霉)鍑嶈瀺銆傛渶鍚庡皣鍕绘伎娑蹭簬5000脳g闆㈠績5~10鍒嗛悩锛屽彇涓婃竻妾㈡脯(c猫)銆�

4.  绱�(x矛)鑳炲煿椁�(y菐ng)鐗╀笂娓呮垨鍏跺畠鐢熺墿妯�(bi膩o)鏈細璜�(q菒ng)1000脳g闆㈠績20鍒嗛悩锛屽彇涓婃竻鍗冲彲妾㈡脯(c猫)锛屾垨?q奴)鈶搁敤閫硿?/span>-20鈩冩垨-80鈩冧繚瀛�锛屼絾鎳�(y墨ng)閬垮厤鍙嶅京(f霉)鍑嶈瀺銆�

娉細妯�(bi膩o)鏈憾琛€鏈�(hu矛)褰遍熆鏈€鍚庢娓�(c猫)绲�(ji茅)鏋�锛屽洜姝ゆ憾琛€妯�(bi膩o)鏈笉瀹滈€�(j矛n)琛屾闋�(xi脿ng)妾㈡脯(c猫)銆�

闇€瑕佽€屾湭鎻愪緵鐨勮│鍔戝拰鍣ㄦ潗

1.  閰舵(bi膩o)鍎€锛�450nm锛�

2.  楂樼簿搴﹀姞妯ｅ櫒鍙婃闋細0.5-10uL銆�2-20uL銆�20-200uL銆�200-1000uL

3.  37鈩�鎭掓韩绠�

4.  钂搁ぞ姘存垨鍘婚洟瀛愭按

瑭﹀姂鐩掔祫鎴�

1.  妯�(bi膩o)婧�(zh菙n)鍝佹績搴︿緷娆＄偤锛� 2000銆�1000銆� 500銆�250銆�125銆�62.5 nmol/L

2.  缍�(j墨ng)閬�(gu貌)澶ч噺姝ｅ父妯�(bi膩o)鏈椹�(y脿n)锛屾(bi膩o)鏈殑姝ｅ父婵冨害鍊煎潎鍦ㄨ│鍔戠洅鎻愪緵鐨勬娓�(c猫)鑼冨湇鍏�(n猫i)锛屽(sh铆)椹�(y脿n)閬�(gu貌)绋嬩腑鐩存帴鍙�50渭L妯ｆ湰涓婃ǎ鍗冲彲銆傜暥(d膩ng)鏈夐儴鍒嗘ǎ鏈€艰秴閬�(gu貌)鏈€澶ф(bi膩o)婧�(zh菙n)鍝佹績搴︽檪(sh铆)锛屽彲鐢ㄦǎ鏈█閲嬫恫灏囨(bi膩o)鏈€�(j矛n)琛岄仼鐣�(d膩ng)绋€閲嬪悗鍐嶉€�(j矛n)琛屽(sh铆)椹�(y脿n)銆�

娉ㄦ剰浜嬮爡(xi脿ng)

1.  鍤�(y谩n)鏍兼寜鐓ц(gu墨)瀹氱殑鏅�(sh铆)闁撳拰婧害閫�(j矛n)琛屾韩鑲蹭互淇濊瓑婧�(zh菙n)纰虹祼(ji茅)鏋溿€傛墍鏈夎│鍔戦兘蹇呴爤鍦ㄤ娇鐢ㄥ墠閬�(d谩)鍒板婧�20-25鈩�銆備娇鐢ㄥ悗绔嬪嵆鍐疯棌淇濆瓨瑭﹀姂銆�

2.  娲楁澘涓嶆纰哄彲浠ュ皫(d菐o)鑷翠笉婧�(zh菙n)纰虹殑绲�(ji茅)鏋�銆傚湪鍔犲叆搴曠墿鍓嶇⒑淇濈洝閲忓惛骞插瓟鍏�(n猫i)娑查珨銆傛韩鑲查亷(gu貌)绋嬩腑涓嶈璁撳井瀛斿共鐕ユ帀銆�

3.  娑堥櫎鏉垮簳娈樼暀鐨勬恫楂斿拰鎵嬫寚鍗�锛屽惁鍓囧奖闊�OD鍊笺€�

4.  搴曠墿椤壊娑叉噳(y墨ng)鍛堢劇(w煤)鑹叉垨寰堟泛鐨勯鑹�锛屽凡缍�(j墨ng)璁婅棈(l谩n)鐨勫簳鐗╂恫涓嶈兘浣跨敤銆�

5.  閬垮厤瑭﹀姂鍜屾(bi膩o)鏈殑浜ゅ弶姹℃煋浠ュ厤閫犳垚閷�(cu貌)瑾ょ祼(ji茅)鏋�銆�

6.  鍦ㄥ劜(ch菙)瀛樺拰婧偛鏅�(sh铆)閬垮厤寮�(qi谩ng)鍏夌洿鎺ョ収灏�銆�

7.  骞宠　鑷冲婧悗鍐嶆墦闁�(k膩i)瀵嗗皝琚嬩互闃叉按婊村嚌鑱氬湪鍐锋澘姊濅笂銆�

8.  浠讳綍鍙嶆噳(y墨ng)瑭﹀姂涓嶈兘鎺ヨЦ婕傜櫧婧跺姂鎴栨紓鐧芥憾鍔戞墍鏁ｇ櫦(f膩)鐨勫挤(qi谩ng)鐑堟埃楂�銆備换浣曟紓鐧芥垚鍒嗛兘鏈�(hu矛)鐮村瑭﹀姂鐩掍腑鍙嶆噳(y墨ng)瑭﹀姂鐨勭敓鐗╂椿鎬�銆�

9.  涓嶈兘浣跨敤閬�(gu貌)鏈熺敘(ch菐n)鍝併€�

10.  濡傛灉鍙兘鍌虫挱鐤剧梾锛屾墍鏈夌殑妯ｅ搧閮芥噳(y墨ng)绠＄悊濂�锛屾寜鐓ц(gu墨)瀹氱殑绋嬪簭铏曠悊妯ｅ搧鍜屾娓�(c猫)瑁濈疆銆�

瑭﹀姂婧�(zh菙n)鍌�

瑭﹀姂鐩掑緸鍐疯棌鐠�(hu谩n)澧冧腑鍙栧嚭鎳�(y墨ng)鍦ㄥ婧钩琛″悗鏂瑰彲浣跨敤銆�

20脳娲楁粚绶╂矕娑茬殑绋€閲�?zhu膩n)璧捍s姘存寜1锛�20绋€閲�?zhu膩n)顑�?/span>1浠�20脳娲楁粚绶╂矕娑插姞19浠借捀椁炬按銆�

鎿嶄綔姝ラ

1.  寰炲婧钩琛�20min鍚庣殑閶佺當琚嬩腑鍙栧嚭鎵€闇€鏉挎锛屽墿浣欐澘姊濈敤鑷皝琚嬪瘑灏佹斁鍥�4鈩�銆�

2.  瑷�(sh猫)缃(bi膩o)婧�(zh菙n)鍝佸瓟鍜屾ǎ鏈瓟锛屾(bi膩o)婧�(zh菙n)鍝佸瓟鍚勫姞涓嶅悓婵冨害鐨勬(bi膩o)婧�(zh菙n)鍝�50渭L锛�

3.  妯ｆ湰瀛�涓�鍔�鍏�寰呮脯(c猫)妯ｆ湰50渭L锛涚┖鐧藉瓟涓嶅姞銆�

4.  闄ょ┖鐧藉瓟澶栵紝妯�(bi膩o)婧�(zh菙n)鍝佸瓟鍜屾ǎ鏈瓟涓瘡瀛斿姞鍏ヨ荆鏍归亷(gu貌)姘у寲鐗╅叾锛�HRP锛夋(bi膩o)瑷樼殑妾㈡脯(c猫)鎶楅珨100渭L锛岀敤灏佹澘鑶滃皝浣忓弽鎳�(y墨ng)瀛�锛�37鈩�姘存荡閸嬫垨鎭掓韩绠辨韩鑲�60min銆�

5.  妫勫幓娑查珨锛屽惛姘寸礄涓婃媿骞�锛屾瘡瀛斿姞婊挎礂婊屾恫锛�350渭L锛�锛岄潨缃�1min锛岀敥鍘绘礂婊屾恫锛屽惛姘寸礄涓婃媿骞�锛屽姝ら噸寰�(f霉)娲楁澘5娆★紙涔熷彲鐢ㄦ礂鏉挎(j墨)娲楁澘锛夈€�

6.  姣忓瓟鍔犲叆搴曠墿A銆�B鍚�50渭L锛�37鈩�閬垮厜瀛佃偛15min銆�

7.  姣忓瓟鍔犲叆绲傛娑�50渭L锛�15min鍏�(n猫i)锛屽湪450nm娉㈤暦(zh菐ng)铏曟脯(c猫)瀹氬悇瀛旂殑OD鍊笺€�

瀵�(sh铆)椹�(y脿n)绲�(ji茅)鏋滆▓(j矛)绠�

浠�鎵€娓�(c猫)妯�(bi膩o)婧�(zh菙n)鍝佺殑OD鍊�鐐烘┇鍧愭(bi膩o)锛�妯�(bi膩o)婧�(zh菙n)鍝佺殑婵冨害鍊肩偤绺卞潗妯�(bi膩o)锛屽湪鍧愭(bi膩o)绱欎笂鎴栫敤鐩搁棞(gu膩n)杌熶欢绻埗妯�(bi膩o)婧�(zh菙n)鏇茬窔锛屽苟寰楀埌鐩寸窔鍥炴鏂圭▼锛�灏囨ǎ鍝佺殑OD鍊间唬鍏ユ柟绋嬶紝瑷�(j矛)绠楀嚭妯ｅ搧鐨�婵冨害銆�

瑭﹀姂鐩掓€ц兘

1.  妾㈡脯(c猫)鑼冨湇锛�62.5 nmol/L 鈥� 2000 nmol/L銆�

2.  闈堟晱搴︼細鏈€浣庢娓�(c猫)婵冨害灏忎簬10 nmol/L銆�

3.  鐗圭暟鎬э細涓嶈垏鍏跺畠鍙憾鎬х祼(ji茅)妲�(g貌u)椤�(l猫i)浼肩墿浜ゅ弶鍙嶆噳(y墨ng)銆�

4.  閲嶅京(f霉)鎬э細鏉垮収(n猫i)璁婄暟绯绘暩(sh霉)灏忎簬10% 锛�鏉块枔璁婄暟绯绘暩(sh霉)灏忎簬15% 銆�

瑾�(shu艒)鏄�

鐢变簬鐝�(xi脿n)鏈夋浠跺強绉戝(xu茅)鎶€琛�(sh霉)姘村钩灏氫笉鑳藉皪(du矛)鎵€鏈変緵璨ㄥ晢鎻愪緵鐨勬墍鏈夊師鏂欓€�(j矛n)琛屽叏闈㈢殑閼掑畾鑸囧垎鏋�锛屾湰鐢�(ch菐n)鍝佸彲鑳藉瓨鍦ㄤ竴瀹氱殑璩�(zh矛)閲忔妧琛�(sh霉)棰�(f膿ng)闅�(xi菐n)銆�

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