GenWay Biotech, Inc. Human Resistin ELISA. Protein and Antibody Solutions. GenWay. Cat. No.: Human Resistin ELISA (page 1 of 24)

(1)

Human Resistin ELISA (page 1 of 24)

Cat. No.: 40-055-200007

GenWay Biotech, Inc.

Protein and Antibody Solutions

(2)

Human Resistin ELISA (page 2 of 24)

1. INTENDED USE

3

2. STORAGE, EXPIRATION

3

3. SUMMARY

4

4. TEST PRINCIPLE

5

5. PRECAUTIONS

5

6. REAGENTS SUPPLIED

6

7. MATERIALS REQUIRED BUT NOT SUPPLIED

6

8. PREPARATION OF REAGENTS

7

9. PREPARATION OF SAMPLES

8 10. ASSAY PROCEDURE

9

11. _CALCULATIONS

11

12. _{LIMITS OF ASSAY}

11

13. PERFORMANCE CHARACTERISTICS

12 14. DEFINITION OF RESISTIN MASTER CALIBRATOR

14

15. TROUBLESHOOTING AND FAQS

16. REFERENCES

18 Use only the actual version of Product Data Sheet enclosed with the kit

(3)

Human Resistin ELISA (page 3 of 24)

1. Intended Use

2. Storage, Expiration

The Resistin ELISA is a biotin labelled antibody based sandwich enzyme

immunoassay for the quantitative measurement of human resistin in serum,

plasma and tissue culture medium. It is intended for research use only

Features

• The total assay time is less than four hours.

• The kit measures total serum or plasma (homodimeric) resistin.

• Quality Controls are human serum based. No animal sera are used.

Store the kit at 2-8°C. Under these conditions, the kit is stable until the

expiration date (see label on the box).

(4)

3. Summary

Resistin, a product of the RSTN gene, is a peptide hormone belonging to the class of cysteine-rich secreted proteins which is termed the RELM family, and is also described as ADSF (Adipose Tissue-Specific Secretory Factor) and FIZZ3 (Found in Inflammatory Zone). Human resistin contains 108 amino acids as a prepeptide, and its hydrofobic signal peptide is

cleaved before its secretion. Resistin circulates in human blood as a dimeric protein consisting of two 92 amino acid polypeptides, which are disulfide-linked via Cys26.

Resistin may be an important link between obesity and insulin resis-tance. Mouse resistin, specifically produced and secreted by adipocyte, acts on skeletal muscle myocytes, hepatocytes and adipocytes themselves so that it reduces their sensitivity to insulin. Steppan et al. have suggested that resistin suppresses the ability of insulin to stimulate glucose uptake. They have also suggested that resistin is present at elevated levels in blood of obese mice, and is down regulated by fasting and antidiabetic drugs. Way et al., on the other hand, have found that resistin expression is severly suppressed in obesity and is stimulated by several antidiabetic drugs.

Other studies have shown that mouse resistin increases during the differentiation of adipocytes, but it also seems to inhibit adipogenesis. In contrast, the human adipogenic differentiation is likely to be associated with a down regulation of resistin gene expression. Recent studies have shown that human resistin is expressed also in macrophages and may be a novel link between inflammation and insulin resistance.

(5)

Human Resistin ELISA (page 5 of 24)

4. Test Principle

5. Precautions

In GenWay’s Human Resistin ELISA, calibrators or samples are incubated with

a goat polyclonal anti-human resistin antibody coated in microtiter wells.

After one-hour incubation and a washing, biotin-labelled polyclonal

anti-human resistin antibody is added and incubated with captured resistin. After

a thorough wash, streptavidinhorseradish peroxidase conjugate is added.

After one hour incubation and the last washing step, the remaining conjugate

is allowed to react with the substrate H2O2- tetramethylbenzidine. The

reac-tion is stopped by addireac-tion of acidic solureac-tion and absorbance of the resulting

yellow product is measured at 450 nm. The absorbance is proportional to the

concentration of resistin. A standard curve is constructed by plotting

absor-bance values versus resistin concentrations of calibrators, and concentrations

of unknown samples are determined using this standard curve.

• For research use only.

• This kit contains components of human origin. These materials were

found non-reactive for HbsAg, HCV antibody and for HIV 1/2 antibody and

antigen. However, these materials should be handled as potentially

infec-tious, as no tests can guarantee the complete absence of infectious agents.

• Avoid contact with the acidic Stop Solution and Substrate Solution which

contains hydrogen peroxide. Wear gloves and eye protection when handling

these reagents. In case of contact with the Stop Solution and the Substrate

Solution wash skin thoroughly with water and seek medical attention, when

necessary.

• Wear gloves and laboratory coats when handling immunodiagnostic

mate-rials.

• The materials must not be pipetted by mouth.

• Do not drink, eat or smoke in the areas where immunodiagnostic

materi-als are being handled.

• Reagents with different lot numbers should not be mixed.

(6)

6. Reagents Supplied

7. Materials Required but Not Supplied

Kit Components

Microtiter Strips, coated with capture polyclonal Anti-Resistin Antibody, sealed

Biotin Labelled Anti-Resistin Antibody, ready to use Streptavidin-HRP Conjugate, ready to use

Resistin Master Calibrator, lyophilized Quality Control High, lyophilized Quality Control Low, lyophilized Dilution Buffer, ready to use Wash Solution Concentrate (10x)

Substrate Solution (TMB), ready to use Stop Solution (0.2 M H2SO4), ready to use Instruction Manual + Certificate of Analysis

Quantity 96 wells 13 ml 13 ml 50 ng 1 vial 1 vial 20 ml 100 ml 13 ml 13 ml 1 pc

• Test tubes for diluting samples

3UHFLVLRQSLSHWWHVWRGHOLYHUǋODQGGLVSRVDEOHWLSV 0XOWLFKDQQHOSLSHWWHǋO

• Microplate reader with 450 nm filter • Microplate shaker (optional)

• Software package facilitating data generation and analysis (optional) • Microtitration plate washer (optional) [Manual washing is possible but not preferable.]

• Glassware (graduated cylinder and bottle for Wash Solution) • Deionized (distilled) water

(7)

8. Preparation of Reagents

Calibrator volume Dilution Buffer Concentration stock --- 50 ng/ml 500 ul of stock 750 µl 20 ng/ml 500 ul of std. 20 ng/ml 500 µl 10 ng/ml 500 ul of std. 10 ng/ml 500 µl 5 ng/ml 500 ul of std. 5 ng/ml 750 µl 2 ng/ml 500 ul of std. 2 ng/ml 500 µl 1 ng/ml

All reagents need to be brought to room temperature prior to the assay.

Assay reagents are supplied ready-to-use, with the exception of Resistin Master Calibrator, Quality Controls and Wash Solution Concentrate (10x).

Preparation of reagents for 1 plate:

• If you do not use the whole plate, return unused strips in the provided alu minium bag with dessicant and seal the bag carefully. Keep the unused strips at 2-8°C, protected from the moisture.

Wash Solution:

Dilute 100 ml of Wash Solution Concentrate with 900 ml of deionized (distilled) water. Stability and storage:

The diluted Wash Solution is stable for one month when stored at 2-8°C.

Human Resistin Calibrators:

Reconstitute Resistin Master Calibrator with 1 ml of Dilution Buffer. The concentration of the human resistin in the stock solution is 50 ng/ml. Prepare Calibrators as follows:

'LOXWHSUHSDUHG&DOLEUDWRUVZLWK'LOXWLRQ%XIIHUSULRUWRXVHLQ(/,6$HJǋO &DOLEUDWRUǋO'LOXWLRQ%XIIHUIRUGXSOLFDWHV

Stability and storage:

The reconstituted and undiluted Calibrators should be frozen at –20°C until next use. Do not store the diluted (1:3) Calibrators.

Quality Controls:

5HFRQVWLWXWH4XDOLW\&RQWUROZLWKǋORI'LOXWLRQ%XIIHUPL[WKHPL[WXUHDQGOHWLW reconstitute thoroughly at RT for 30 minutes and mix again.

(8)

9. Preparation of Samples

Dilute reconstituted Quality Control 1:3 prior to use in ELISA with Dilution Buffer, HJǋOVDPSOHǋO'LOXWLRQ%XIIHUZKHQDVVD\LQJVDPSOHVLQVLQJOHWVRU SUHIHUDEO\ǋOVDPSOHǋO'LOXWLRQ%XIIHUIRUGXSOLFDWHV

Stability and storage:

Reconstituted Quality Control is stable until the expiration date (see label on the box) when stored at –20°C.

Dilute serum or plasma samples 1:3 prior to use in ELISA with Dilution %XIIHUHJǋO

VDPSOHǋO'LOXWLRQ%XIIHUZKHQDVVD\LQJVDPSOHVLQVLQJOHWVRUSUHIHU-DEO\ǋO

VDPSOHǋO'LOXWLRQ%XIIHUIRUGXSOLFDWHV Stability and storage:

(9)

10. Assay Procedure

3LSHWǋORIGLOXWHG&DOLEUDWRUV4XDOLW\&RQWUROV'LOXWLRQ%XIIHU %ODQNDQGVDPSOHVSUHIHUDEO\LQGXSOLFDWHVLQWRWKHDSSURSULDWHZHOOV 6HH)LJXUHIRUH[DPSOHRIZRUNVKHHW ,QFXEDWHWKHSODWHDWURRPWHPSHUDWXUHFD&IRUKRXUVKDNLQJ DWFDUSPRQDQRUELWDOPLFURSODWHVKDNHU :DVKWKHZHOOVWLPHVZLWK:DVK6ROXWLRQPOSHUZHOO 3LSHWǋORI%LRWLQ/DEHOOHG$QWL5HVLVWLQ$QWLERG\6ROXWLRQLQWRHDFK ZHOO ,QFXEDWHWKHSODWHDWURRPWHPSHUDWXUHFD&IRUKRXUVKDNLQJ DWFDUSPRQDQRUELWDOPLFURSODWHVKDNHU :DVKWKHZHOOVWLPHVZLWK:DVK6ROXWLRQPOSHUZHOO 3LSHWǋORI6WUHSWDYLGLQ+53&RQMXJDWH ,QFXEDWHWKHSODWHDWURRPWHPSHUDWXUHFD&IRUKRXUVKDNLQJ DWFDUSPRQDQRUELWDOPLFURSODWHVKDNHU :DVKWKHZHOOVWLPHVZLWK:DVK6ROXWLRQPOSHUZHOO 3LSHWǋORI6XEVWUDWH6ROXWLRQ$YRLGH[SRVLQJWKHPLFURWLWHUSODWH WRGLUHFWVXQOLJKW&RYHULQJWKHSODWHZLWKHJDOXPLQLXPIRLOLVUHFRP-PHQGHG ,QFXEDWHWKHSODWHIRUPLQXWHVDWURRPWHPSHUDWXUH7KHLQFXED-WLRQWLPHPD\EHH[WHQGHG>XSWRPLQXWHV@LIWKHUHDFWLRQWHPSHUDWXUH LVEHORZWKDQ&1RVKDNLQJ 6WRSWKHFRORXUGHYHORSPHQWE\DGGLQJǋORI6WRS6ROXWLRQ 'HWHUPLQHWKHDEVRUEDQFHE\UHDGLQJWKHSODWHDWQP7KH DEVRUEDQFHVKRXOGEHUHDGZLWKLQPLQXWHVIROORZLQJVWHS

Note: If the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine resistin concentration of off-scale samples. The readings at 405 nm should not replace the on-off-scale readings at 450 nm.

(10)

Human Resistin ELISA (page 10 of 24) Figure 1: Example of work sheet.

A B C D E F G H strip 1+ Calibrator 50 Calibrator 20 Calibrator 10 Calibrator 5 Calibrator 2 Calibrator 1 Blank QC High strip 3 + 4 QC Low Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 strip 5+ 6 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 strip 7+ 8 Sample 16 Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 Sample 23 strip 9+10 Sample 24 Sample 25 Sample 26 Sample 27 Sample 28 Sample 29 Sample 30 Sample 31 strip 11+ 12 Sample 32 Sample 33 Sample 34 Sample 35 Sample 36 Sample 37 Sample 38 Sample 39

(11)

11. Calculations

12. Limits of Assay

Human Resistin ELISA Standard Curve 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 1 10 100 Concentration of Hu Resistin (ng/ml) A b so rb a n c e (4 5 0 n m )

Most microtiter plate readers perform automatic calculations of analyte concentration. The calibration curve is constructed by plotting the absor-bance (Y) of calibrators versus log of the known concentration (X) of cali-brators, using the four-parameter function.

Figure 2: Standard Curve for Human Resistin is plotted using the four-parameter function as a proportion of Human Resistin concentration and absorbance at 450 nm.

Alternatively, the logit log function can be used to linearize the calibration curve (i.e. logit of absorbance (Y) is plotted versus log of the known concen-tration (X) of calibrators).

Samples, Quality Controls and Calibrators are diluted 1:3 prior to analysis, so there is no need to account for this dilution.

Results exceeding resistin level of 50 ng/ml should be repeated with diluted samples. Dilution factors need to be taken into consideration in calculating the resistin concentration.

(12)

• Precision

Intra-assay(Within-Run) (n=8) Sample Mean (ng/ml) SD (ng/ml) CV (%) 1 7.53 0.21 2.8 2 11.35 0.39 3.4

Typical analytical data obtained with GenWay’s Human Resistin ELISA are presented in this chapter.

For actual Standard curve and Quality Controls values see the Certificate of Analysis.

• Sensitivity

The limit of detection (defined as human resistin concentration giving absor-bance higher than mean absorabsor-bance of blank* plus three standard deviations of the absorbance of blank: Ablank + 3xSDblank) is defined as follows:

Analytical Limit of Detection is calculated from the real resistin values in wells and is 0.033ng/ml

Assay Sensitivity takes the dilution of samples into consideration and is calcu-lated according tothe formula:

Assay Sensitivity = Analytical Limit of Detection x sample dilution = 0.033ng/ml x 3 = 0.1ng/ml

*Dilution Buffer is pipetted into blank wells.

• Specificity

The antibodies in Human Resistin ELISA kit are highly specific for human resistin with no detectable crossreactivities to human leptin, leptin receptor, adiponectin, TNF-alfa, RELM-beta, , A-FABP and E-FABP at 100 ng/ml and IL-6, AGRP and ASP (C3adesArg) at 2 ng/ml.

No signal was found to the following animal sera: mouse, rabbit, goat, rat, sheep, chicken, hamster and bovine. However, we have found strong crossre-activity to pig, horse and monkey serum. For details please contact us at sales@genwaybio.com.

Human Resistin ELISA uses a unique Dilution Buffer, which suppresses any potential false positive results caused by the presence of rheumatoid factor in samples.

(13)

Human Resistin ELISA (page 13 of 24) Sample Mean (ng/ml) SD (ng/ml) CV (%) 1 6.46 0.33 5.1 2 13.35 0.93 6.9 •

Spiking Recovery

Sample Observed (ng/ml) Expected (ng/ml) Recovery O/E (%) 1 5.55 8.99 13.35 25.34 - 10.55 15.55 25.55 - 85.2 85.9 99.2 2 7.47 10.88 16.79 26.07 - 12.47 17.47 27.47 - 87.2 96.1 94.9

• Linearity

Sample Dilution Observed (ng/ml) Expected (ng/ml) Recovery O/E (%) 1 - 1:2 1:4 1:8 12.56 6.51 2.98 1.74 - 6.28 3.14 1.57 - 103.7 94.9 110.8 2 - 1:2 1:4 1:8 28.46 14.02 7.27 3.88 - 14.23 7.12 3.56 - 98.5 102.2 109.1 Inter-assay (Run-to-Run) (n=3)

Serum samples were spiked with different amounts of human resistin and assayed.

(14)

14. Definition of Resistin Master Calibrator

A recombinant protein is used as the calibrator. The recombinant resistin is a 19,5 kDa dimeric protein consisting of two 92 amino acid polypeptide chains which are disulfidelinked.

Resistin concentration depends on the method used for total protein deter-mination.

(15)

15. Troubleshooting and FAQs

Volunteer Serum (ng/ml) Plasma (ng/ml)

No. Heparin Citrate EDTA

1 14.1 11.2 12.0 13.3 2 7.9 6.6 6.7 6.5 3 11.3 10.2 9.9 12.2 4 9.0 8.3 8.0 9.0 5 5.9 6.2 7.0 8.2 6 9.3 8.9 7.8 9.3 7 6.2 6.3 6.6 7.7 8 5.6 5.9 5.9 5.6 9 5.1 6.0 4.8 5.4 10 6.9 6.5 8.1 7.9 11 9.1 7.7 10.0 9.3 12 7.6 5.2 4.8 5.5 13 7.6 6.2 4.7 6.5 14 7.0 7.1 6.7 7.3 15 6.2 4.4 5.3 6.6 16 5.4 4.9 3.3 5.5

1) Weak signal in all wells Possible explanations:

· Omission of a reagent or a step

· Improper preparation or storage of a reagent

· Assay performed before reagents were allowed to come to room tem perature

2) High signal and background in all wells Possible explanations:

· Improper or inadequate washing

· Overdeveloping; incubation time should be decreased before addition of Stop Solution

3) High coefficient of variation (CV) Possible explanation:

· Improper or inadequate washing

4) Effect of sample matrix (serum/plasma)

Samples from 16 healthy individuals were taken and treated by different meth-ods, results shown below:

(16)

Effect of Sample Matrix

0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Volunteers C o n ce n tr at io n o f H u m an R e si st in (n g /m l) Serum Plasma - Heparin Plasma - Citrate Plasma - EDTA

Sample (n=16) Mean (ng/ml) Plasma / Serum (%)

Serum 7.76

-Plasma - Heparin 6.97 89.8 Plasma - Citrate 6.97 89.7 Plasma - EDTA 7.86 101.2

Mean values of human resistin in serum, heparin plasma, citrate plasma and EDTA plasma:

However, the normal range study with serum samples from healthy donors the following range has been established with the Human Resistin ELISA: 8.1± 4.0 ng/ml (average ± 2 SD, N = 123). It is recommended that each laboratory establishes its own normal range of serum resistin. The refer-ence range should be regarded as guideline only.

(17)

Human Resistin ELISA (page 17 of 24) Plasma (ng/ml) Volunteer No. Number of f/t cycles Serum

(ng/ml) Heparin Citrate EDTA 1x 14.1 11.2 12 13.3 3x 12.8 12.2 11.8 13.1 1 5x 11.6 10.5 10.4 11.5 1x 9.3 8.9 7.8 9.3 3x 9.4 8.3 7.3 9.1 6 5x 9.2 8.7 7.1 8.4 1x 5.1 6 4.8 5.4 3x 5.3 4.9 4.7 4.7 9 5x 3.8 4.5 3.9 4.1 Plasma (ng/ml) Volunteer No . Incubation: Temperature,Period Serum

(ng/ml) Heparin Citrate EDTA - 20 0C 14.1 11.2 12 13.3 4 0C, 1 day 14.5 13.2 13.1 10.7 1 4 0C, 10 days 14.2 10.9 13.2 12.9 - 20 0C 9.3 8.9 7.8 9.3 4 0C, 1 day 10.2 8.4 8.2 9. 9 6 4 0C, 10 days 8.8 8.7 7.4 8.7 - 20 0C 5.1 6 4.8 5.4 4 0C, 1 day 5.1 5.4 4.6 5.7 9 4 0C, 10 days 5.4 5.5 4.1 5.5

5) Effect of freezing/thawing on the concentration of Human Resistin in samples

No decline was observed in concentration of human resistin in serum and plasma samples after repeated (3x) freezing/thawing cycles.

6) Stability of samples at 4°C

Samples should be stored at –20°C. However, no decline was observed in con-centration of human resistin in serum and plasma samples when stored at 4°C for 10 days. To avoid microbial contamination, add NaN3 to a final concentration 0,1% to the samples.

7) Why are calibrator solutions diluted 1:3 prior to use in ELISA and what does it mean for calculation of results?

The dilution buffer suppresses the matrix effect of samples. It is a common prac-tice to use the same dilution for samples and calibrators because of simple calcu-lation of results: concentrations of samples can be read directly off the curve without considering a dilution factor. Thus the same dilution 1:3 is used for cali-brators, controls and serum/plasma samples in Human Resistin ELISA.

Samples exceeding human resistin level of 50 ng/ml should be measured at higher degree of dilution (e.g. 1:9) and dilution factors need to be taken into consideration when calculating human resistin concentrations then (the dilution factor is 3 in this case).

(18)

References to Resistin

References to GenWay’s Human Resistin ELISA

• Meier U, Gressner AM: Endocrine regulation of energy metabolism: review of pathobiochemical and clinical chemical aspects of leptin, ghrelin, adiponectin, and resistin. Clin Chem; 50(9):1511-25 (2004)

• Steppan CM, Lazar MA: The current biology of resistin. J Intern Med;

255(4):439-47 (2004) • Kershaw EE, Flier JS: Adipose tissue as an endocrine organ. J Clin Endocrinol Metab; 89(6):2548-56 (2004)

• Fasshauer M, Paschke R: Regulation of adipocytokines and insulin resistance. Diabetologia; 46(12):1594-603 (2003)

• Steppan CM, Lazar MA: Resistin and obesity-associated insulin resistance. TRENDS in Endocrinology and Metabolism. 13 (1), 18-23, (2002) • Hartman HB, Lazar MA at al.: Mechanisms Regulating Adipocyte Expression of Resistin. The Journal of Biological Chemistry; 277 (22), 19754-19761, (2002)

• Banerjee RR, Lazar MA: Dimerization of resistin and resistin–like moleules is determined by a single cystein. The Journal of Biological Chemistry; 276, 25970- 25973, (2001)

• Steppan C.M. et al.: The Hormone Links Obesity to Diabetes. Nature; 409, 307-312, (2001)

• Way J.M. et al.: Adipose Tissue Resistin Expression Is Severly Suppressed in Obesity and Stimulated by Peroxisome Proliferator-activated Receptor g Agonists. The Journal of Biological Chemistry; 276 (28), 25651-25653, (2001)

• Anderlova K, Kremen J, Dolezalova R, Housova J, Haluzikova D, Kunesova M, Haluzik M: The influence of very-low-calorie-diet on serum leptin, soluble leptin receptor, adiponectin and resistin levels in obese women. Physiol Res; 2005 Aug 5; [Epub ahead of print]

• Pilz S, Horejsi R, Moller R, Almer G, Scharnagl H, Stojakovic T, Dimitrova R, Weihrauch G, Borkenstein M, Maerz W, Schauenstein K, Mangge H: Early athero-sclerosis in obese juveniles is associated with low serum levels of adiponectin. J Clin Endocrinol Metab; 90(8):4792-6 (2005)

• Ohmori R, Momiyama Y, Kato R, Taniguchi H, Ogura M, Ayaori M, Nakamura H, Ohsuzu F: Associations between serum resistin levels and insulin resistance, inflammation, and coronary artery disease. J Am Coll Cardiol; 46(2):379-80 (2005)

• Bokarewa M, Nagaev I, Dahlberg L, Smith U, Tarkowski A: Resistin, an adipo-kine with potent proinflammatory properties. J Immunol 174(9):5789-95 (2005)

(19)

• Pagano C, Marin O, Calcagno A, Schiappelli P, Pilon C, Milan G, Bertelli M, Fanin E, And-righetto G, Federspil G, Vettor R: Increased serum resistin in adults with praderwilli syn-drome is related to obesity and not to insulin resistance. J Clin Endocrinol Metab;

90(7):4335-40 (2005)

• Gerber M, Boettner A, Seidel B, Lammert A, Bar J, Schuster E, Thiery J, Kiess W, Kratzsch J: Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance. J Clin Endocrinol Metab; 90(8):4503-9 (2005)

• Carmina E, Orio F, Palomba S, Cascella T, Longo RA, Colao AM, Lombardi G, Lobo RA: Evidence for altered adipocyte function in polycystic ovary syndrome. Eur J Endocrinol; 152(3):389-943 (2005)

• Chen CC, Li TC, Li CI, Liu CS, Wang HJ, Lin CC: Serum resistin level among healthy sub-jects: Relationship to anthropometric and metabolic parameters. Metabolism; 54(4):471-5 (2005)

• Reilly MP, Lehrke M, Wolfe ML, Rohatgi A, Lazar MA, Rader DJ: Resistin is an inflammatory marker of atherosclerosis in humans. Circulation; 111(7):932-9 (2005)

• Chen D, Dong M, Fang Q, He J, Wang Z, Yang X: Alterations of serum resistin in normal pregnancy and pre-eclampsia. Clin Sci; 108(1):81-4. (2005)

• Housova J, Wilczek H, Haluzik MM, Kremen J, Krizova J, Haluzik M.: Adipocytederived hormones in heroin addicts: the influence of methadone maintenance treatment. Physiol Res; 54(1):73-8 (2005)

• Svensson J, Herlitz H, Lundberg PA, Johannsson G: Adiponectin, Leptin, and Erythrocyte Sodium/Lithium Countertransport Activity, but not Resistin, are Related to Glucose Metabo-lism in Growth Hormone Deficient Adults. J Clin Endocrinol Metab; [Epub ahead of print] (2005)

• Lehrke M, Reilly MP, Millington SC, Iqbal N, Rader DJ, Lazar MA: An Inflammatory Cascade Leading to Hyperresistinemia in Humans. PLoS Med; 1(2):e45 (2004) • Schaffler A, Buchler C, Muller-Ladner U, Herfarth H, Ehling A, Paul G, Scholmerich J, Zietz B: Identification of variables influencing resistin serum levels in patients with type 1 and type 2 diabetes melli-tus. Horm Metab Res; 36(10):702-7 (2004)

• Shetty GK, Economides PA, Horton ES, Mantzoros CS, Veves A: Circulating adiponectin and resistin levels in relation to metabolic factors, inflammatory markers, and vascular reactivity in diabetic patients and subjects at risk for diabetes. Diabetes Care; 27(10):2450-7 (2004) • Wolfe BE, Jimerson DC, Orlova C, Mantzoros CS: Effect of dieting on plasma leptin, soluble leptin receptor, adiponectin and resistin levels in healthy volunteers. Clin Endocrinol (Oxf); 61(3):332-8 (2004)

• Azuma K, Katsukawa F, Oguchi S, Murata M, Yamazaki H, Shimada A, Saruta T: Correlation between serum resistin level and adiposity in obese individuals. Obes Res; 11(8):997-1001 (2003)

• Azuma K, Oguchi S, Matsubara Y, Mamizuka T, Murata M, Kikuchi H, Watanabe K, Katsu-kawa F, Yamazaki H, Shimada A, Saruta T: Novel resistin promoter

(20)

polymorphisms: association with serum resistin level in Japanese obese individuals. Horm Metab Res; 36(8):564-70 (2004)

• Harsch IA, Koebnick C, Wallaschofski H, Schahin SP, Hahn EG, Ficker JH, Lohmann T, Konturek PC: Resistin levels in patients with obstructive sleep apnoea syndrome - the link to subclinical inflammation? Med Sci Monit; 10(9):CR510-5 (2004)

• Vendrell J, Broch M, Vilarrasa N, Molina A, Gomez JM, Gutierrez C, Simon I, Soler J, Richart C: Resistin, adiponectin, ghrelin, leptin, and proinflammatory cytokines: relation-ships in obesity. Obes Res; 12(6):962-71 (2004)

• Heilbronn LK, Rood J, Janderova L, Albu JB, Kelley DE, Ravussin E, Smith SR: Relation-ship between serum resistin concentrations and insulin resistance in nonobese, obese, and obese diabetic subjects. J Clin Endocrinol Metab; 89(4):1844- 8 (2004)

• Fujinami A, Obayashi H, Ohta K, Ichimura T, Nishimura M, Matsui H, Kawahara Y, Yamazaki M, Ogata M, Hasegawa G, Nakamura N, Yoshikawa T, Nakano K, Ohta M:

Enzyme-linked immunosorbent assay for circulating human resistin: resistin concentrations in normal subjects and patients with type 2 diabetes. Clin Chim Acta; 339(1-2):57-63 (2004)

• Youn BS, Yu KY, Park HJ, Lee NS, Min SS, Youn MY, Cho YM, Park YJ, Kim SY, Lee HK, Park KS: Plasma resistin concentrations measured by enzyme-linked immunosorbent assay using a newly developed monoclonal antibody are elevated in individuals with type 2 dia-betes mellitus. J Clin Endocrinol Metab; 89(1):150-6 (2004)

• Bajaj M, Suraamornkul S, Hardies LJ, Pratipanawatr T and DeFronzo RA: Plasma resistin concentration, hepatic fat content, and hepatic and peripheral insulin resistance in

pioglitazone-treated type II diabetic patients. Int J Obes Relat Metab Disord; 28(6):783-9 (2004)

• De Courten BV, Degawa-Yamauchi M, Considine RV and Tataranni PA: High Serum Resis-tin Is Associated With an Increase in Adiposity But Not a Worsening of Insulin Resistance in Pima Indians. Diabetes; 53(5):1279-1284 (2004)

• Hana V, Silha JV, Justova V, Lacinova Z, Stepan JJ and Murphy LJ: The effects of GH replacement in adult GH-deficient patients: changes in body composition without concomi-tant changes in the adipokines and insulin resistance. Clin Endocrinol (Oxf); 60(4):442-50 (2004)

• Krsek M, Silha JV, Jezkova J, Hana V, Marek J, Weiss V, Stepan J. and Murphy LJ.: Adipo-kine levels in Cushing’s syndrome; elevated resistin levels in female patients with

Cushing’s syndrome. Clin Endocrinol (Oxf); 60(3):350-7 (2004)

• Degawa-Yamauchi M, Bovenkerk JE, Juliar BE, Watson W, Kerr K, Jones R, Zhu Q, Consi-dine RV: Serum resistin (FIZZ3) protein is increased in obese humans. J Clin Endocrinol Metab; 88(11):5452-5 (2003)

• Stejskal D, Adamovska S, Bartek J, Jurakova R, Proskova J: Resistin - concentrations in persons with type 2 diabetes mellitus and in individuals with acute inflammatory disease. Biomed Papers; 147(1):63-9 (2003)

(21)

For more references on this product please visit www.genwaybio.com • Stejskal D, Ruzicka V, Adamovska S, Jurakova R, Proskova J, Jedelsky L, Bartek J: Adiponectin concentrations as a criterion of metabolic control in persons with type 2 diabetes mellitus? Biomed Papers; 147(2):167-72 (2003)

• Schaffler A, Ehling A, Neumann E, Herfarth H, Tarner I, Scholmerich J, Muller-Ladner U, Gay S: Adipocytokines in synovial fluid. JAMA; 290(13):1709-10 (2003)

• Pfutzner A, Langenfeld M, Kunt T, Lobig M, Forst T: Evaluation of human resistin assays with serum from patients with type 2 diabetes and different degrees of insulin resistance. Clin Lab; 49(11-12):571-6 (2003)

• Lee JH, Chan JL, Yiannakouris N, Kontogianni M, Estrada E, Seip R, Orlova C and Mant-zoros CS: Circulating resistin levels are not associated with obesity or insulin resistance in humans and are not regulated by fasting or leptin administration: crosssectional and interventional studies in normal, insulin-resistant, and diabetic subjects. J Clin Endocrinol Metab; 88(10):4848-56 (2003)

• Silha JV, Krsek M, Skrha JV, Sucharda P, Nyomba BL, Murphy LJ: Plasma resistin, adipo-nectin and leptin levels in lean and obese subjects: correlations with insulin resistance. Eur J Endocrinol; 149(4):331-5 (2003)

• Monzillo LU, Hamdy O, Horton ES, Ledbury S, Mullooly C, Jarema C, Porter S, Ovalle K, Moussa A and Mantzoros CS: Effect of lifestyle modification on adipokine levels in obese subjects with insulin resistance. Obes Res; 11(9):1048-54 (2003)

• Kielstein JT, Becker B, Graf S, Brabant G, Haller H, Fliser D.: Increased resistin blood levels are not associated with insulin resistance in patients with renal disease. Am J Kidney Dis; 42(1):62-6 (2003)

• Silha JV, Krsek M, Hana V, Marek J, Jezkova J, Weiss V, Murphy LJ.: Perturbations in adiponectin, leptin and resistin levels in acromegaly: lack of correlation with insulin resis-tance. Clin Endocrinol (Oxf); 58(6):736-42 (2003)

• Yannakoulia M, Yiannakouris N, Bluher S, Matalas AL, Klimis-Zacas D, Mantzoros CS.: Body fat mass and macronutrient intake in relation to circulating soluble leptin receptor, free leptin index, adiponectin, and resistin concentrations in healthy humans. J Clin Endo-crinol Metab; 88(4):1730-6 (2003)

• Yura S, Sagawa N, Itoh H, Kakui K, Nuamah MA, Korita D, Takemura M, Fujii S.: Resis-tin is expressed in the human placenta. J Clin Endocrinol Metab; 88(3):1394-7 (2003) • Robert A. Hegele, Maria E. Kraw, Matthew R. Ban, Brooke A. Miskie, Murray W. Huff, and Henian Cao: Elevated Serum C-Reactive Protein and Free Fatty Acids Among Nondia-betic Carriers of Missense Mutations in the Gene Encoding Lamin A/C (LMNA) With Partial Lipodystrophy. Arterioscler Thromb Vasc Biol; 23(1):111-116-8 (2003)

• Fain JN, Cheema PS, Bahouth SW, Lloyd Hiler M: Resistin release by human adipose tissue explants in primary culture. Biochem Biophys Res Commun; 300(3):674-8 (2003) Stejskal D, Proskova J, Adamovska S, Jurakova R, Bartek J.: Preliminary experience with resistin assessment in common population. Biomed. Papers; 146(2):47-9 (2002).

(22)

Incubate at RT for 1 hours/300 rpm

Incubate at RT for 1 hour/300rpm

Incubate at RT 1 hour/300 rpm

Incubate at RT for 10 min.

Assay Procedure Summary

Antibody Coated Plate Prepare Calibrators and

dilute them 1:3

Dilute QCs and Samples 1:3

Add Calibrators, QCs and Samples 100 ul Prepare Wash Solution

Wash 3x Add Biotin-Labelled Antibody100 ul Wash 3x Add Streptavidin-HRP Conjugate 100 ul Wash 3x Add Substrate 100 ul

GenWay Biotech, Inc. Human Resistin ELISA. Protein and Antibody Solutions. GenWay. Cat. No.: Human Resistin ELISA (page 1 of 24)

Cat. No.: 40-055-200007