ELISAELISA

1

2

Ag Ab Ag-Ab Ag AB:

Ka [Ag-AB] [Ag][Ab][ g ] [ g][ ]

Kd Ka

Ka M-1

Kd M

KdKd

3

Kd: R

HH

4

100

5

ELISAELISA

ELISA

6

Kd 10-15M

7

Biotin 244.3.

B H

Coenzyme R

lysine -lysine (biocytin)

8

Avidin68 00068,000

4 128 N4 128 NC

Avidin

11

Kd 10-15M

9

ELISA

10

11

12

RIA

RIA

CV 2~3CV 2 3

13

ELISA

2

14

RIA ELISA

STD

0.1 0.063 0.1004 0.06426 0.1047 4.28

0.25 0.105 0.2496 0.1071 0.2572 3.04

0.5 0.199 0.5018 2.034 0.5124 2.11

1 0.426 0.9977 0.4345 1.0159 1.82

2.5 1.173 2.5119 1.1965 2.5612 1.96

5 2.206 4.9683 2.25 5.0902 2.49

10 3.558 10.0479 3.629 10.4286 3.79

15

16

KdKd

17

ELISA

1000010000

ELISA

18

Std. 1 Std. 2 Std. 3 Std. 4 Std. 5 Std. 6 Std. 7 Std. 8 Std. 9

S lidSolidAb Kd 10pM 20 40 80 160 320 640 1280 2560

9.901 19.802 39.60 79.20 158.39 316.73 633.24 1265.51 2526.210000 100 99.0% 99.0 99.0 99.0 99.0 99.0 98.9 98.9 98.7

10000 3009.708

97.1%19.416

97.138.8397.1

77.6597.1

155.2797.0

310.3997.0

620.1696.9

1237.6396.7

2462.0296.210000 300 97.1% 97.1 97.1 97.1 97.0 97.0 96.9 96.7 96.2

10000 10009.090

90.9%18.179

90.936.35

90.972.68

90.9145.26

90.8290.12

90.7578.59

90.51150.05

89.82266.86

88.5

10000 30007.691

76.9%15.379

76.930.75

76.961.45

76.8122.73

76.7244.74

76.5486.56

76.0961.04

75.11869.97

73.0

4 999 9 995 19 98 39 92 79 68 158 72 314 88 619 54 1198 4110000 10000

4.99950.0%

9.99550.0

19.9850.0

39.9249.9

79.6849.8

158.7249.6

314.8849.2

619.5448.4

1198.4146.8

19

20

Kd 1000pM

21

22

5000pM 10000pM

23

24

25

ELISAELISA

26

ELISAELISA

27

ng/ml

ng/ml ng/ml (%) (%) ng/mlg g

10 0.9672 9.7 90.3 9.035 0.8273 16.55 83.45 4.17

2.5 0.588 23.52 76.48 1.9122.51.25 0.4844 38.75 61.25 0.7656

0.625 0.2868 45.89 54.11 0.33810 313 0.1444 46.21 53.79 0.16810.3130.156 0.086 55.13 44.87 0.07

28

50ng/ml10,000pM

10,000pMKd 1000pM

29

0.1244Ln(x)+0.075

30

31

32

2

33

122

T10000pM 1000pM 0.156 10ng/ml

27~1724pM10ng/ml pM

34

35

Edge Edgeg g

36

Blank absorbance obtained by 2operators

m0(

620)nm

Abs.

450

Well pos ition A 1 to H1

S tandard curves obtained by 2 operators

nm50

(62

0)Abs

45

Insulin concentration, pg/ml37

ELISA

38

IgG F(ab’)2, Fab’

Fc heavy chain

F(ab’)22 Fab’

39

Washing repetition and blank absorbance(Mean & SD)(Mean & SD)

) nm

450(

620)

banc

e Ab

srob

Assay kit: Mouse insulin ELISA KIT (U-type)Difference of mean absorbance: p<0.065 Difference of variance:p<0.1

40

Assay kit: Rat TSH ELISA KITDifference of mean absorbance: p<0.03 Difference of variance: p<0.25p p

41

Biotin-avidin

D galactosidase-D-galactosidase4-methylumbelliferyl- -D-galactoside2 it h l D l t id2-nitrophenyl- -D-galactoside

Horseradish peroxidaseo se ad s pe o dase3-(4-hydroxyphenyl)propionic acid

42

ELISA RIAELISA RIA

pM

pM

43

44

45

ELISA

46

90 l

No. C

10 l

No. CNo. C

47

No. C ng/ml

x0.9 0.1 ng/ml

48

49