• About Insulin
• About Shibayagi's insulin assay kit
• Specificity of kits
• Get focused on serial dilution!

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.

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.

Note: Biotin and avidin

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
  
• Standard curve of U-Type assay kit for mouse
  
• Standard curve of S type assay kit for rat
  

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

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

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

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

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

Dilution test

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

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

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

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

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).

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.