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妯ｆ湰铏曠悊鍙婅姹�
1. 琛�娓咃細灏囨敹闆嗕簬琛�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鈩冮亷澶滐紝鐒跺悗1000×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鈩冩垨-80鈩冧繚瀛橈紝浣嗘噳(y墨ng)閬垮厤鍙嶅京(f霉)鍑嶈瀺銆�
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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)褰遍熆娓噺绲�(ji茅)鏋滐級锛岀ū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)鏁达紝骞跺仛濂借閷勩�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)鑳烇紝鍙互灏�(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×g闆㈠績5~10鍒嗛悩锛屽彇涓婃竻妾㈡脯銆�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×g闆㈠績20鍒嗛悩锛屽彇涓婃竻鍗冲彲妾㈡脯锛屾垨?q奴)鈶搁敤閫硿?/span>-20鈩冩垨-80鈩冧繚瀛橈紝浣嗘噳(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�鍚庢娓祼(ji茅)鏋滐紝鍥犳婧惰妯�(bi膩o)鏈笉瀹滈��(j矛n)琛屾闋�(xi脿ng)妾㈡脯銆�
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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�
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瑭﹀姂鐩掔祫鎴�
鍚嶇ū
96瀛旈厤缃�
48瀛旈厤缃�
鍌欐敞
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12瀛�×8姊�
12瀛�×4姊�
鐒�
妯�(bi膩o)婧�(zh菙n)鍝�
0.3mL*6绠�
0.3mL*6绠�
鐒�
妯ｆ湰绋�閲嬫恫
6mL
3mL
鐒�
妾㈡脯鎶楅珨-HRP
10mL
5mL
鐒�
20×娲楁粚绶╂矕娑�
25mL
15mL
鎸夎鏄庢浉閫�(j矛n)琛岀█閲�
搴曠墿A
6mL
3mL
鐒�
搴曠墿B
6mL
3mL
鐒�
绲傛娑�
6mL
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鐒�
灏佹澘鑶�
2寮�
2寮�
鐒�
瑾槑鏇�
1浠�
1浠�
鐒�
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1鍊�(g猫)
1鍊�(g猫)
鐒�
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1. 妯�(bi膩o)婧�(zh菙n)鍝佹績搴︿緷娆＄偤锛�20銆�10銆�5銆�2.5銆�1.25銆�0.625 ng/mL
2. 缍�(j墨ng)閬庡ぇ閲忔甯告(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寖鍦嶅収(n猫i)锛屽(sh铆)椹�(y脿n)閬庣▼涓洿鎺ュ彇50μL妯ｆ湰涓婃ǎ鍗冲彲銆傜暥(d膩ng)鏈夐儴鍒嗘ǎ鏈�艰秴閬庢渶澶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)鏋溿�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帮紝鍚﹀墖褰遍熆OD鍊笺��
4. 搴曠墿椤壊娑叉噳(y墨ng)鍛堢劇鑹叉垨寰堟泛鐨勯鑹诧紝宸茬稉(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)鏋溿��
6. 鍦ㄥ劜(ch菙)瀛樺拰婧偛鏅�(sh铆)閬垮厤寮�(qi谩ng)鍏夌洿鎺ョ収灏勩��
7. 骞宠 鑷冲婧悗鍐嶆墦闁嬪瘑灏佽浠ラ槻姘存淮鍑濊仛鍦ㄥ喎鏉挎涓娿��
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)鐑堟埃楂斻�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с��
9. 涓嶈兘浣跨敤閬庢湡鐢�(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＄悊濂斤紝鎸夌収瑕�(gu墨)瀹氱殑绋嬪簭铏曠悊妯ｅ搧鍜屾娓缃�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×娲楁粚绶╂矕娑茬殑绋�閲嬶細钂搁ぞ姘存寜1锛�20绋�閲嬶紝鍗�1浠�20×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�50μL锛�
3. 妯ｆ湰瀛斾腑鍔犲叆寰呮脯妯ｆ湰50μL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姘у寲鐗╅叾锛�HRP锛夋(bi膩o)瑷樼殑妾㈡脯鎶楅珨100μL锛岀敤灏佹澘鑶滃皝浣忓弽鎳�(y墨ng)瀛旓紝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诧紝姣忓瓟鍔犳豢娲楁粚娑诧紙350μL锛夛紝闈滅疆1min锛岀敥鍘绘礂婊屾恫锛屽惛姘寸礄涓婃媿骞诧紝濡傛閲嶅京(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�50μL锛�37鈩�閬垮厜瀛佃偛15min銆�
7. 姣忓瓟鍔犲叆绲傛娑�50μL锛�15min鍏�(n猫i)锛屽湪450nm娉㈤暦铏曟脯瀹氬悇瀛旂殑OD鍊笺��
瀵�(sh铆)椹�(y脿n)绲�(ji茅)鏋滆▓(j矛)绠�
浠ユ墍娓(bi膩o)婧�(zh菙n)鍝佺殑OD鍊肩偤姗潗妯�(bi膩o)锛屾(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▼锛屽皣妯ｅ搧鐨�OD鍊间唬鍏ユ柟绋嬶紝瑷�(j矛)绠楀嚭妯ｅ搧鐨勬績搴︺��
瑭﹀姂鐩掓�ц兘
1. 妾㈡脯鑼冨湇锛�0.625 ng/mL – 20 ng/mL銆�
2. 闈堟晱搴︼細zui浣�妾㈡脯婵冨害灏忎簬0.1 ng/mL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)椤炰技鐗╀氦鍙夊弽鎳�(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% 锛屾澘闁撹畩鐣扮郴鏁�(sh霉)灏忎簬15% 銆�
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