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All about Shibayagi's Insulin Assay Kits

About Insulin

What is insulin?

Insulin is a protein hormone secreted from B cells of islet of Langerhans in the pancreas with a molecular weight of about 5,800 and pI 5.4. It is consisted of 2 chains, A and B. It has 3 disulfide bonds formed between A6 and A11, A7 and B7, and A20 and B19. Exists as a dimer molecule in acidic to neutral water solution without Zn ion, and as a hexamer including two Zn ions in neutral solution if Zn ions are present.
Main targets of insulin are liver, muscle, and adipose tissue. Insulin actions in these targets are as follows. In the liver, it promotes glycogenesis, protein synthesis, fatty acid synthesis, carbohydrate utilization, and inhibition of gluconeogenesis. In the muscle, it promotes membrane permeability of carbohydrates, amino acids and K ion, glycogenesis, protein synthesis, while inhibits protein degradation. In the adipose tissue, it promotes membrane permeability of glucose and fatty acid synthesis.

Insulin biosynthesis and structure

A precursor of insulin, called proinsulin with a single polypeptide chain, is first synthesized in the cell, then sulfide bonds are formed, and finally by enzymatic cutting at two sites, active insulin and c-peptide (connecting peptide) are formed as shown in below figure.

wb034238_img01.gif

Species difference of amino acid sequence of insulin

Table 1: Comparison of insulin amino acid sequences of various animals A-chain

Sepecies 1 6 11 16 21
Human GIVEQ CCTSI CSLYQ LENYC N
Chimpanzee GIVEQ CCTSI CSLYQ LENYC N
Crab-eating macaque GIVEQ CCTSI CSLYQ LENYC N
Green monkey GIVEQ CCTSI CSLYQ LENYC N
Squirrel monkey GVVDQ CCTSI CSLYQ LQNYC N
Dog GIVEQ CCTSI CSLYQ LENYC N
Cat GIVEQ CCASV CSLYQ LEHYC N
Cattle GIVEQ CCASV CSLYQ LENYC N
Sheep GIVEQ CCAGV CSLYQ LENYC N
Pig GIVEQ CCTSI CSLYQ LENYC N
Horse GIVEQ CCTGI CSLYQ LENYC N
Rabbit GIVEQ CCTSI CSLYQ LENYC N
Rat I GIVDQ CCTSI CSLYQ LENYC N
Rat II GIVDQ CCTSI CSLYQ LENYC N
Mouse I GIVDQ CCTSI CSLYQ LENYC N
Mouse II GIVDQ CCTSI CSLYQ LENYC N
Fat sand rat GIVEQ CCTGI CSLYQ LENYC N
Guinea pig GIVDQ CCTGT CTRHQ LQSYC N
Golden hamster GIVDQ CCTSI CSLYQ LENYC N
Long-tailed hamster GIVDQ CCTSI CSLYQ LENYC N
Chicken GIVEQ CCHNT CSLYQ LENYC N
Ostrich GIVEQ CCHNT CSLYQ LENYC N
Turkey GIVEQ CCHNT CSLYQ LENYC N
Duck GIVEQ CCENP CSLYQ LENYC N
Hummingbird GIVEQ CCHNT CSLYQ LENYC N
Xenopus I GIVEQ CCHST CSLFQ LESYC N
Xenopus II GIVEQ CCHST CSLFQ LENYC N
Chum salmon GIVEQ CCHKP CNIFD LQNYC N
Tilapia GIVEE CCHKP CTIFD LQNYC N
Carp GIVEQ CCHKP CSIFE LQNYC N


B-chain

Sepecies 1 6 11 16 21 26
Human FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
Chimpanzee FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
Crab-eating macaque FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
Green monkey FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
Squirrel monkey FVNQH LCGPH LVEAL YLVCG ERGFF YAPKT
Dog FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Cat FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Cattle FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Sheep FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Pig FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Horse FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
Rabbit FVNQH LCGSH LVEAL YLVCG ERGFF YTPKS
Rat I FVKQH LCGPH LVEAL YLVCG ERGFF YTPKSv
Rat II FVKQH LCGSH LVEAL YLVCG ERGFF YTPMS
Mouse I FVKQH LCGPH LVEAL YLVCG ERGFF YTPKS
Mouse II FVKQH LCGSH LVEAL YLVCG ERGFF YTPMS
Fat sand rat FVNQH LCGSH LVEAL YLVCG ERGFF YTPKF
Guinea pig FVSRH LCGSN LVETL YSVCQ DDGFF YIPKD
Golden hamster FVNQH LCGSH LVEAL YLVCG ERGFF YTPKS
Long-tailed hamster FVNQH LCGSH LVEAL YLVCG ERGFF YTPKS
Chicken AANQH LCGSH LVEAL YLVCG ERGFF YSPKA
Ostrich AANQH LCGSH LVEAL YLVCG ERGFF YSPKA
Turkey AANQH LCGSH LVEAL YLVCG ERGFF YSPKA
Duck AANQH LCGSH LVEAL YLVCG ERGFF YSPKT
Hummingbird AVNQH LCGSH LVEAL YLVCG ERGFF YSPKA
Xenopus I LVNQH LCGSH LVEAL YLVCG DRGFF YYPKV
Xenopus II LANQH LCGSH LVEAL YLVCG DRGFF YYPKI
Chum salmon AAAQH LCGSH LVDAL YLVCG EKGFF YTP
Tilapia VGGPQ HLCGS HLVDA LYLVC GDRGF FYNPR
Carp NAGAP QHLCG SHLVD ALYLV CGPTG FFYNP


C-peptide

Sepecies 1 6 11 16 21 26 31
Human EAEDL QVGQV ELGGG PGAGS LQPLA LEGSL Q
Chimpanzee EAEDL QVGQV ELGGG PGAGS LQPLA LEGSL Q
Crab-eating macaque EAEDP QVGQV ELGGG PGAGS LQPLA LEGSL Q
Green monkey EAEDP QVGQV ELGGG PGAGS LQPLA LEGSL Q
Squirrel monkey EAEDL QVGQV ELGGG SITGS LPPLE GPMQ
Dog EVEDL QVRDV ELAGA PGEGG LQPLA LEGAL Q
Cat EAEDL QGKDA ELGEA PGAGG LQPSA LEAPL Q
Cattle EVEGP QVGAL ELAGG PGAGG LEGPP Q
Sheep EVEGP QVGAL ELAGG PGAGG LEGPP Q
Pig EAENP QAGAV ELGGG LGGLQ ALALE GPPQ
Horse EAEDP QVGEV ELGGG PGLGG LQPLA LAGPQ Q
Rabbit EVEEL QVGQA ELGGG PGAGG LQPSA LELAL Q
Rat I EVEDP QVPQL ELGGG PEAGD LQTLA LEVAR Q
Rat II EVEDP QVAQL ELGGG PGAGD LQTLA LEVAR Q
Mouse I EVEDP QVEQL ELGGS PGDLQ TLALE VARQ
Mouse II EVEDP QVAQL ELGGG PGAGD LQTLA LEVAQ Q
Fat sand rat GVDDP QMPQL ELGGS PGAGD LRALA LEVAR Q
Guinea pig ELEDP QVEQT ELGMG LGAGG LQPLA LEMAL Q
Golden hamster GVEDP QVAQL ELGGG PGADD LQTLA LEVAQ Q
Long-tailed hamster GVEDP QVAQL ELGGG PGADD LQTLA LEVAQ Q
Chicken DVEQP LVSSP LRGEA GVLPF QQEEY EKV
Ostrich DVEQP LVSSP LRGEA GVLPF QQEEY EKV
Turkey DVEQP LVSSP LRGEA GVLPF QQEEY EKV
Duck DVEQP LVNGP LHGEV GELPF QHEEY Q
Hummingbird DAEHP LVNGP LHGEV GDLPF QQEEF EKV
Xenopus I DMEQA LVSGP QDNEL DGMQL QPQEY QKM
Xenopus II DIEQA QVNGP QDNEL DGMQF QPQEY QKM
Chum salmon DVDPL IGFLS PKSAK ENEEY PFKDQ TEMMV
Tilapia DVDPL LGFLP PKAGG AVVQG GENEV TFKDQ MEMMV
Carp DVDPP LGFLP PKSAQ ETEVA DFAFK DHAEV IR



Table 2 Total molecular weight of insulin of various animals

Human 5808.42
Chimpanzee 5808.42
Rat, Mouse I 5804.47
Rat, Mouse II 5787.38
Dog 5778.39
Cat 5757.38
Monkey (common) 5759.40
Pig 5778.39
Cattle 5734.34

Potencies of insulin preparations of various animal species

Potency of an insulin preparation was originally determined by bioassay. For example, 1 rabbit unit is the amount of insulin which lowers the blood sugar level of 24-hours fasting rabbit by 50% within 5 hours after administration. It corresponds to about 3IU. However, whole body bioassay inevitably shows poor precision owing to individual variation with index of precision about 0.15. From such reason international standard preparation has been used. The 4th international standard preparation is a mixture of purified preparations of bovine(52%) and porcine(48%) insulin, and 1mg of the standard is defined as 24IU (=0.04167mg/IU).
Following further purification of insulin, WHO issued 1st International Standard in 1986 which has the potency of 26IU/mg (0.038mg/IU). In the same year, 1st International Standard of bovine insulin, the potency of which is 25.7IU/mg, and Porcine insulin 1st International Standard, 26IU/mg, were provided. One ampoule conteins about 50mg. Before these standards, in 1974, 1 st International Reference Preparation of human insulin for immunoassay was provided as 3 IU/ampoule. Based on the above data, if the biological activity of insulin per molecule is the same among various animal species, potencies of animal insulin might be calculated from their molecular weights. But, so far, we do not have experimental proof about this. As the molecular weights of insulin of various animals are nearly the same, and the differences are within 1%, there may be no critical fault if we think that the general potency of insulin is 26IU/mg.
Rat and mouse have two molecular species of insulin, type 1 and type 2. Amino acid sequences of these molecular species are same between rat and mouse. But as their ratios are different between the two animals, it is recommended to use standard preparation derived from each animals.

About Shibayagi's insulin assay kit

Assay strategy of Shibayagi's kit

A monoclonal anti-insulin antibody is coated on the assay plate to capture insulin in the assay sample. Then another monoclonal insulin antibody to which biotin is labeled is added to bind to another portion of captured insulin, and finally peroxidase-avidin conjugate is added. As avidin binds to biotin firmly, the amount of insulin can be determined from peroxidase activity by adding chromogenic substrate for peroxidase.

wb034238_img02.gif

Note: Biotin and avidin

wb034238_img03.gif

Biotin: A kind of growth factor present in every cells and belongs to vitamin B complex. Also called vitamin H. As it acts as co-factor of various enzymes related to carboxylation reactions, it is also called coenzyme R. The richest sources are liver, kidney, pancreas, yeast and milk. It is important in fatty acid and carbohydrate metabolism, and deficiency causes skin lesion.
Avidin: A basic glycoprotein present in raw egg-white. Produced in oviducts of avians and amphibians. It is composed essentially same single chain 4 subunits each 128 amino acids (N-terminal amino acid is alanine and C-terminal glutamic acid). Molecular weight is about 66,000. It is destructed by heat treatment (cooking) and irradiation.
Avidin binds firmly with biotin and inactivates it. Each subunit can bind one biotin. The dissociation constant, Kd, is 10-15M.Intake of large amounts of egg-white to rats and chicken makes biotin deficient and causes skin lesion and growth retardation. This effect is reversed by biotin administration.

We are utilizing such a specific and strong binding of biotin and avidin in our assay system.

Classification of Shibayagi's insulin assay kits -Classification according to animal species, specificity, sensitivity, and related substance (C-peptide)- Animal species

Our insulin assay kits are for animal species shown below.

Rat insulin ELISA kits: TMB (liquid type), OPD (tablet type)
High sensitivity type (U type), (U-E type)
High specificity type (S type)
Mouse insulin ELISA kits: TMB (liquid type), OPD (tablet type)
High sensitivity type (U type)
High specificity type (S type)
Dog insulin ELISA kit: TMB (liquid type)
Pig insulin ELISA kit: TMB (liquid type)
Monkey insulin ELISA kit: TMB (liquid type)

Besides these, we are providing insulin standard preparation for rabbit and hamster. By using these standard preparations with rat TMB kit, you can measure rabbit and hamster insulin.

Specificity for insulin

Ordinary insulin assay kits: All ELISA kits except for S type
Ordinary assay kits show the affinity to both insulin and proinsulin. Normally, in circulating blood, both proinsulin and processed insulin are secreted with minor ratio of proinsulin to insulin, and secretion of proinsulin is in parallel with insulin. So, in usual studies, it is enough to measure both of them by using ordinary assay kit.

Insulin-specific assay kit: S type (at present for rat insulin)
This type of kit shows a very low cross-reactivity to proinsulin (see data in specificity section), and practically can measure only insulin in the presence of proinsulin. This type of kit is useful when levels of proinsulin change owing physiological conditions. From the difference of assay values between ordinary and specific assay kits we can estimate proinsulin secretion.

From assay sensitivity

Kits of ordinary sensitivity: TMB and OPD
These types of kits are for measurement of insulin in normal state animals. In any species, a good standard curve is obtained in a range of 0.156 to 10ng/ml.

Ultra-sensitive assay kits: U type, U-E type
For rat and mouse, we are providing ultra-sensitive assay kits, U type. This type of kit is improved to be able to measure insulin levels in fasted animals with an excellent precision. The assay ranges are 25-1,500 pg/ml for rat, and 39-2,500 pg/ml for mouse. Because rat U type needs an overnight incubation, we improved the incubation period, and prepared U-E type the assay range of which is 39-2,500 pg/ml. The highly specific assay kit of S type also attained higher sensitivity than ordinary TMB and OPD, the assay range of which is 100 to 10,000 pg/m.

Kit for insulin-related substance: rat and mouse C-peptide ELISA kits
We provide assay kits for C-peptide that has closely related to insulin. Amino acid sequences of C-peptides are to some extent different between rat and mouse. C-peptide is secreted with insulin after processing of proinsulin to insulin and C-peptide. C-peptide has a longer biological half-life than insulin. So, C-peptide is measured in observation of biosynthesis and secretory functions for insulin. C-peptide is also found in urine in considerably high concentration, and the level is parallel to that in plasma, so, can be applied to urinary samples.
Measurement of C-peptide is also useful in estimation of insulin secretion to culture medium of e.g. isolated islets more than measurement of insulin itself, because very often insulin is added to a culture medium to make the measurement of insulin secreted to the medium by cultured cells. As C-peptide is secreted into the medium in equi-molar base, we can estimate insulin secretion by measuring C-peptide. Our C-peptide kit recognizes both C-peptide 1 and 2, the total amount of C-peptide is measured by the kit. Data for the specificity of the C-peptide assay kit is shown in Section 5, gSpecificity of kitsh.

Standard curve and assay range of the kit

You can find standard curve in instruction manual in every kit. Generally, assay range is 0.2-12ng/ml irrespective of animal species. For mouse and rat, we are also providing ultra-sensitive assay kits, U-type. Their standard curves are shown below.

  • Standard curve of U-Type assay kit for rat
    wb034238_img04.gif
  • Standard curve of U-Type assay kit for mouse
    wb034238_img05.gif
  • Standard curve of S type assay kit for rat
    wb034238_img06.gif

Precision, reproducibility, recovery test and dilution test of the kits

Rat (OPD kit)
Precision (Average CV) 2.06 % 3 samples, 5 replicates
Reproducibility (Average CV) 2.87 % 3 samples, 8 replicates, 3 days
Rat (TMB kit)
Precision (Average CV) 1.65 % 3 samples, 8 replicates
Reproducibility (Average CV) 2.81 % 3 samples, 5 replicates, 3 days
Mouse (OPD kit)
Precision (Average CV) 1.58 % 3 samples, 10 replicates
Reproducibility (Average CV) 1.98 % 3 samples, triplicates, 3 days
Mouse (TMB kit)
Precision (Average CV) 1.98 % 3 samples, 10 replicates
Reproducibility (Average CV) 3.91 % 3 samples, triplicates, 3 days
Dog (TMB kit)
Precision (Average CV) 4.32 % 3 samples, triplicates
Reproducibility (Average CV) 4.18 % 3 samples, 4 replicates, 3 days
Monkey (TMB kit)
Precision (Average CV) 3.21 % 3 samples, 10 replicates
Reproducibility (Average CV) 3.09 % 3 samples, 4 replicates, 3 days


Pig (TMB kit)...data in detail
Precision (Within assay variation) n=5

Mean(ng/ml) S.D. CV(%)
0.991 0.0321 3.24
0.482 0.0175 3.63
0.201 0.0099 4.93


Reproducibility (Between assay variation) n=5

Mean(ng/ml) S.D. CV(%)
1.425 0.0562 3.87
0.901 0.0321 3.56
0.346 0.0162 4.68


Recovery test

Sample Added(ng/ml) Found(ng/ml) Recovered(ng/ml) Recovery(%)
1 0 0.343 * *
0.25 0.576 0.233 93.2
0.5 0.802 0.459 91.7
1 0.952 1.295 95.2
2 2.301 1.958 97.9
2 0 0.202 * *
0.25 0.437 0.235 93.8
0.5 0.666 0.464 92.8
1 1.201 0.999 99.9
2 2.240 2.038 101.9


Rat insulin assay kit (U-type)
Precision (Within assay variation) 3 samples

Sample A Sample B Sample C
Assay value A.450nm pg/ml A.450nm pg/ml A.450nm pg/ml
0.165 25.6 0.413 271.4 1.816 1251.7
0.165 25.6 0.416 273.9 1.801 1242.1
0.164 24.1 0.415 273.1 1.801 1242.1
0.164 24.1 0.417 274.8 1.806 1245.3
0.164 24.1 0.419 276.4 1.798 1240.2
mean SD CV(%) 0.164 24.7 0.416 273.9 1.804 1244.3
0.0005 0.808 0.0022 1.88 0.0071 4.5345
0.3 3.3 0.5 0.7 0.4 0.4


Reproducibility (Between assay variation) n=3

Sample A Sample B Sample C
Assay value A.450nm pg/ml A.450nm pg/ml A.450nm pg/ml
0.173 33.2 0.268 232.7 1.182 1003.5
0.192 35.0 0.339 236.9 1.031 1012.0
0.182 33.7 0.302 230.5 1.075 999.1
mean SD CV(%) 0.182 34.0 0.303 233.4 1.096 1004.9
0.0096 0.943 0.0355 3.26 0.0777 6.5060
5.2 2.8 11.7 1.4 7.1 0.6


Recovery test (n=3)

Sample Added(pg/ml) Found(pg/ml) Recovered(pg/ml) Recovery(%)
1 0 54
50 103 49 98.0
150 198 144 96.0
350 393 339 96.9
2 0 432
500 952 520 104
750 1204 772 103
1000 1430 998 99.8

Dilution test
wb034238_img07.gif

Rat insulin assay kit (U-E type)
Precision

Sample ID A
(pg/ml)
B
(pg/ml)
C
(pg/ml)
1 1058 320 91.0
2 1008 308 87.3
3 987 309 91.9
4 1074 298 97.1
5 1065 304 93.7
6 1058 305 92.3
7 1056 313 92.2
8 1070 333 91.2
mean. 1047 311 92.1
SD 31.8 10.9 2.73
CV(%) 3.0 3.5 3.0


Reproducibility
pg/ml, n=3

Sample ID Day 1 Day 2 Day 3 Day 4 Mean. SD CV(%)
F 1221 1234 1254 1244 1238 13.8 1.1
G 365 311 323 310 327 26.0 8.0
H 75.8 71.8 76.3 73.1 74.3 2.14 2.9


Recovery test
pg/ml, n=3

Sample ID Added Found Recovered Recovery(%)
D 0 41.1 - -
51.0 89.1 48.0 94.1
101 143 102 101
148 185 144 97.3
E 0 386 - -
374 741 355 94.9
1120 1504 1118 100
1494 1844 1458 97.6

Dilution test
wb034238_img08.gif

Rat insulin assay kit (S-type)
Precision
ng/ml

Well A B C
1 6.51 2.97 1.11
2 6.39 2.91 1.01
3 5.97 2.89 1.03
4 6.09 2.94 1.02
5 6.01 2.89 1.00
6 5.95 2.84 1.02
7 5.97 2.98 1.02
8 6.29 2.98 1.05
mean. 6.15 2.92 1.03
SD 0.22 0.051 0.034
CV(%) 3.6 1.8 3.3


Reproducibility
ng/ml, n=3

Sample No. Day 0 Day 1 Day 2 Day 3 Mean SD CV(%)
D 5.10 5.03 5.12 5.12 5.09 0.041 0.8
E 0.989 0.965 0.950 0.984 0.972 0.018 1.8
F 0.510 0.513 0.522 0.536 0.520 0.012 2.2


Recovery test
ng/ml, n=3

Sample No. G
Added Found Recovered Recovery(%)
- 1.01 - -
0.19 1.19 0.18 95
0.39 1.38 0.37 95
0.79 1.76 0.75 95
1.97 2.99 1.98 101
Sample No. H
Added Found Recovered Recovery(%)
- 4.24 - -
1.69 5.89 1.65 98
3.39 7.50 3.26 96
5.08 9.10 4.86 96
6.88 10.6 6.37 94

Dilution test
wb034238_img09.gif

Correlation of between assay values obtained by rat S-type and rat T-type kits
wb034238_img10.gif

Mouse (U-type kit)
Precision (Within assay variation), n=5

Sample A B C
N A.450nm pg/ml A.450nm pg/ml A.450nm pg/ml
1 0.970 1380 0.261 270 0.143 101
2 0.956 1363 0.259 266 0.147 109
3 0.956 1363 0.260 268 0.146 107
4 0.959 1367 0.262 272 0.142 99
5 0.948 1353 0.263 274 0.147 109
Mean 0.958 1365 0.261 270 0.145 105
SD 0.0079 9.68 0.0016 3.27 0.0023 4.81
CV (%) 0.8 0.7 0.6 1.2 1.6 4.6


Reproducibility (Between assay variation), n=3

Sample E F G
Day pg/ml pg/ml pg/ml
1 1365 270.0 106.0
2 1326 271.1 105.4
3 1359 269.5 109.3
Mean 1350 270.2 106.9
SD 20.9 0.799 2.11
CV (%) 1.5 0.3 2.0


Recovery test pg/ml, n=2

Sample Added Found Recovered Recovery(%)
1 0 483 - -
150 646 163 109
550 1040 557 101
1350 1823 1340 99.3
2 0 91.8 - -
50 140 48.2 96.4
200 290 198.2 99.1
350 443 351.2 100.3

Correlation of assay values obtained by U-type and standard kits
wb034238_img11.gif

Dilution test
wb034238_img12.gif

Mouse insulin assay kit (S-type)
Precision

Sample No. A B
1 2488 491
2 2477 470
3 2410 457
4 2434 465
5 2433 459
6 2342 442
7 2358 459
8 2390 495
mean. 2417 467
SD 52.3 17.8
CV(%) 2.2 3.8

pg/ml

Reproducibility

Sample No. Day 1 Day 2 Day 3 Day 4 mean SD CV(%)
C 166 156 153 153 157 6.16 3.9
D 628 625 624 624 625 1.89 0.30
E 2505 2409 2427 2549 2473 65.86 2.7

pg/ml, n=3

Recovery test

Sample No. F
Added Found Recovered Recovery(%)
0 1315 - -
324 1632 317 97.8
630 1935 620 98.4
889 2172 857 96.4
1305 2625 1310 100
Sample No. G
Added Found Recovered Recovery(%)
0 512 - -
426 925 413 96.9
559 1073 561 100
662 100 665 101
745 1270 758 102

pg/ml, n=3

Dilution test
wb034238_img13.gif

Correlation between assay values obtained by mouse S-type kit and mouse U-type kit
wb034238_img14.gif

C-Peptide assay kits
Rat C-peptide assay kit (U-type)
Precision

Well A B
1 1015 214
2 1027 222
3 1038 211
4 1043 219
5 1029 232
6 1034 209
7 1039 231
8 1041 224
mean 1033 220
SD 9.60 8.51
CV (%) 0.93 3.86

pg/ml

Reproducibility

Sample Day 1 Day 2 Day 3 Day 4 mean SD CV (%)
C 1499 1435 1491 1458 1471 29.68 2.02
D 599 605 569 559 583 22.55 3.87
E 63.9 58.2 59.9 64.2 61.6 2.98 4.83

pg/ml, n=4

Recovery test
pg/ml

Added Found Recovered Recovery (%)
0.00 180 - -
110 291 111 101
161 332 152 94.5
209 379 199 95.1
Added Found Recovered Recovery (%)
0.00 360 - -
263 616 256 97.1
526 909 549 104
658 1040 680 103

Dilution test
wb034238_img15.gif

Mouse C-peptide assay kit (U-type)
Precision
pg/ml

Well A B
1 976 238
2 969 230
3 965 230
4 1023 235
5 977 231
6 1018 228
7 1038 229
8 995 225
mean 995 231
SD 27.7 4.10
CV (%) 2.78 1.78


Reproducibility
pg/ml, n=4

Sample Day 1 Day 2 Day 3 Day 4 mean SD CV (%)
C 1502 1500 1499 1501 1500 1.12 0.07
D 301 302 301 300 301 0.66 0.22
E 60.9 63.8 62.2 58.8 61.4 2.13 3.46


Recovery test
pg/ml, n=2

Added Found Recovered Recovery (%)
0.00 300 - -
265 551 250 94
398 683 382 96
531 827 527 99
Added Found Recovered Recovery (%)
0.00 58.2 - -
28.9 86.7 28.5 99
38.6 98.2 40.0 104
77.4 139 80.8 104

Dilution test
wb034238_img16.gif

Specificity of kits

Ordinary insulin assay kits

Our ordinary kits show similar tendency as to their specificity as shown below. C-peptide, as far as we tested, does not show cross-reactivity. Heterologous insulin, more or less, shows cross-reactivity.
As to proinsulin, though we could examined only human proinsulin from the availability of standard preparation, we consider that homologous proinsulin may crossreacts. We will show specificity of rat insulin kit in detail.

Kits Rat insulin kit
(All types)
Mouse insulin kit
(All types)
Dog insulin kit Monkey insulin kit Pig insulin kit
Human insulin + + + + +
Human proinsulin + + + + +
Human C-peptide - - - - -
Rat insulin + + + + +
Mouse insulin + + + + +
Rat C-paptide - - - - -
Mouse C-peptide - - - - -
Dog insulin + + + + +
Monkey insulin + + + + +
Pig insulin + + + + +


Rat insulin ELISA kit cross-reaction test

Preparation Cross-reactivity
Rat insulin C-peptide 100%
-
Mouse insulin 102%
Pig insulin 120%
Dog insulin + (See data below*)
Human insulin 185%


*Comment: When purified dog insulin preparation is measured with rat insulin assay kit, the cross-reactivity will change according to its concentration.

10ng.ml 120%
5ng/ml 100%
1ng/ml 120%


Ref. Mouse insulin ELISA kit cross-reactivity test

Mouse insulin 100%
Mouse C-peptide 0%
Rat insulin 98%


Specificity of highly insulin-specific kits (S type) for rat insulin
Influence of rat proinsulin added to the sample

Rat proinsulin added (A) Assay value as rat insulin (B) Influence on assay system
(B-1.00)/A, %
0 1.00 -
1.0 1.01 1.0
2.0 1.03 1.5
4.0 1.07 1.8
8.0 1.19 2.4

unit:ng/ml, n=2

Cross-reactivity

Substance Reactivity(%) Concentration added
Rat Insulin 100
Rat C-peptide Less than lower assay limit 100ng/ml
Rat Proinsulin < 5 100ng/ml
Mouse Insulin 102 10ng/ml
Mouse C-peptide Less than lower assay limit 100ng/ml
Porcine Insulin 120 10ng/ml
Dog Insulin Cross-reacted 10ng/ml
Bovine Insulin Cross-reacted 10ng/ml
Human Insulin 185 10ng/ml
Rabbit Insulin 180 10ng/ml


Specificity of highly insulin-specific kits (S type) for mouse insulin
Influence of mouse proinsulin added to the sample

Sample serum No. I
Mouse proinsulin added Insulin assay value Cross-reactivity (%)
- 106.0 -
100 106.9 0.9
500 112.5 1.3
5000 286.0 3.6

pg/ml, n=2

Cross-reactivity

Substances Reactivity(%) Concentration tested
Mouse Insulin 100
Mouse C-peptide Less than assay limit 50ng/ml
Mouse Proinsulin < 5 50ng/ml
Rat Insulin 98 10ng/ml
Rat C-peptide Less than assay limit 50ng/ml
Porcine Insulin 118 10ng/ml
Dog Insulin Reacted 10ng/ml
Bovine Insulin Reacted 10ng/ml
Human Insulin 185 10ng/ml
Rabbit Insulin 180 10ng/ml


Specificity of rat C- peptide assay kit (U-type)

Animal species Substances Cross-reactivity(%)
Rat C-peptide 100
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Mouse C-peptide 92
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Human C-peptide 90
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Porcine Insulin Less than assay
Bovine Insulin Less than assay limit

*Cross-reactivity was estimated at 15,000 pg/ml

Specificity of mouse C-peptide assay kit (U-type)

Animal species Substances Cross-reactivity(%)
Mouse C-peptideT 100
C-peptideU 100
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Rat C-peptide 89
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Human C-peptide 85
Insulin Less than assay limit
Pro-Insulin Less than assay limit
Porcine Insulin Less than assay limit
Bovine Insulin Less than assay limit

*Cross-reactivity was estimated at 15000 pg/ml

Sample treatment for measurement

Dilution of samples containing high levels of insulin
Dilute samples with assay buffer when insulin contents are too high to measure.

Measurement of insulin in culture cells
After washing cultured cells, homogenize them in proper volume of acid-ethanol medium (a mixture of 7.5ml of HCl and 370ml of ethanol), and stand it for several hours at 4C. Then centrifuge the homogenate, neutralize supernatant fluid with 6M ammonia and centrifuge. The supernatant fluid can be used as assay sample after proper dilution according to its insulin content.

Use of anticoagulants to obtain plasma
Serum sample is useful for insulin measurement. But if you want to use plasma, obtain plasma by using heparin at final concentration of 10mg/ml(=1.2U/ml)~100mg/ml or EDTA-2Na final 1~1.5mg/ml(EDTA-2K final 1.11~1.7mg/ml), or citric acid final
0.8~1.0%.We do not recommend NaF because it is a peroxidase inhibitor.
Addition of anticoagulants may influence assay results. Our experimental results are shown below.
In rat insulin measurement, anticoagulants examined seem to have no considerable effects. When you use anticoagulants, all the samples should be treated in the same way to get same effects if any. Influence of anticoagulants on rat insulin ELISA kit (n=8)

Sample Anticoagulant Absent(ng/ml) Present(ng/ml) Present/absent(%)
1 Heparin
10mcg/ml
0.523
2.001
0.508
2.041
97.1
102
2 EDTA
1 mg/ml
0.725
2.348
0.718
2.252
99.1
95.9
3 Citric acid
0.8%
0.662
2.161
0.633
1.995
95.7
92.3


Stability of insulin in blood
Blood contains insulinase and other protein-degradating enzymes. So, serum or plasma samples should be assayed as soon as possible after sampling. In some samples insulin levels are decreased following storage at room temperature. Samples could be kept for several hours before assay at 4C, and longer than that at lower temperature than -35C. If samples have to be stored for long period, -80C is recommended.

If you are afraid of degradation of insulin during storage
For instance, if you can not measure immediately after sampling, and have to store them in a refrigerator, you can add aprotinin, an inhibitor of insulin degradating enzyme, at a final concentration of 100~500KIU/ml.

Influence of hemolysis
Test was carried out with n=5
Figures in columns are insulin assay values without hemolysis, while lower values are ratios of assay values ratios (with/without hemolysis) expressed by percentage.

Samples Hemoglobin concentration (mg/dl)
0 10 20 40
1 0.521
100%
0.533
102%
0.500
96.0%
0.456
87.5%
2 1.04
100%
0.998
96%
0.989
95.1%
0.925
88.9%
3 3.56
100%
3.35
94.1%
3.39
95.2%
2.56
71.9%


As shown above, with hemoglobin concentration above 40mg/dl, the assay values are lowered.
Hemoglobin concentration and coloration (shown below).

wb034238_img17.jpg
Be careful! Mouse blood is easily hemolysed.

Get focused on serial dilution!

Including ELISA, usually serially diluted standards and samples were measured at the same time, and the samples' assay values are calculated from the obtained calibration curve in assay systems. In ELISA, standard solutions are mostly prepared by serial dilution, where you should add a fixed quantity of dilution buffer to test tubes, then add the same quantity of thick standard solution to the first test tube and mix, and from the mixed solution, let's say, Std. 7, take the same volume with dilution buffer and apply to the next test tube (Std. 6), and mix to make one rank higher serial standard, and so on. Let's think one example: the volume is 100μL and you use a pipette called 100μL of single stroke dispenser (the most common, tip changeable pipette to dispense 100μL with one whole stroke) for both standard and dilution buffer. In the ISO 8655-5 (International Organization for Standardization), this pipette's acceptable limit of maximum random error is defined as 0.3μL, so let's calculate assuming that there's 0.3% variation. As a result, 95% confidence limit (= 2CV) is +/- 0.6% in the highest Std. 7 and +/- 4% in the lowest Std. 1. What if a multi delivery pipette with a syringe type tip is used for the buffer's dispensing? This type of pipette's acceptable limit of maximum random error would be very large, however, let's assume 1%. If single stroke is used (CV 0.3%) for the standard solution, the confidence limit is +/- 9.2% in the Std. 1. If a pipette with CV 1% is used for both would be about +/- 15%, and another with CV 2% would be +/- 30%. In this way, this value gets larger proportionally almost to the pipette's random error.
To prepare serial standard solutions, chose pipette carefully. Never set a small volume using large nominal volume of pipette (e.g. set at 100μL using 1ml piston pipette), and avoid use of multi delivery pipette.

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