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HUMAN ELISA

Product Data Sheet

Cat. No.: RD194334200R

For Research Use Only

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CONTENTS

1. INTENDED USE 3 2. STORAGE, EXPIRATION 3 3. INTRODUCTION 4 4. TEST PRINCIPLE 5 5. PRECAUTIONS 5 6. TECHNICAL HINTS 6 7. SUPPLIED 6 8. MATERIAL REQUIRED BUT NOT SUPPLIED 7 9. PREPARATION OF 7 10. PREPARATION OF SAMPLES 9 11. PROCEDURE 10 12. CALCULATIONS 12 13. PERFORMANCE CHARACTERISTICS 13 14. DEFINITION OF THE STANDARD 18 15. PRELIMINARY POPULATION AND CLINICAL DATA 18 16. METHOD COMPARISON 20 17. TROUBLESHOOTING AND FAQS 21 18. REFERENCES 22 19. EXPLANATION OF SYMBOLS 23

This is manufactured by: BioVendor – Laboratorní medicína a.s.

Use only the current version of Product Data Sheet enclosed with the kit!

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1. INTENDED USE

The RD194334200R Human Lactoferrin ELISA is a sandwich for the quantitative measurement of human lactoferrin.

Features

• It is intended for research use only • The total assay time is less than 3.5 hours • The kit measures lactoferrin in , plasma (EDTA), bronchoalveolar lavage fluid (BALF), (CSF), , breast , saliva and stool extract • Special Dilution Buffer is needed for measurement of human lactoferrin in stool extract and is not included. For protocol for preparation of stool extracts and other details, please contact us at [email protected] • Assay format is 96 wells • Standard is native based • Components of the kit are provided ready to use, concentrated or lyophilized

2. STORAGE, EXPIRATION

Store the complete kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box).

For stability of opened reagents see Chapter 9.

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3. INTRODUCTION

Lactoferrin is 703- iron-binding that belongs to the family. Lactoferrin capability of binding iron is two times higher than that of transferrin. Two ferric ions can be bound by one lactoferrin molecule. There are three forms of lactoferrin according to its iron saturation: apolactoferrin (iron free), monomeric lactoferrin (one ferric ion), and hololactoferrin (binds two Fe3+ ions). The tertiary structure of hololactoferrin and apolactoferrin is different.[4,5] Lactoferrin was originally isolated from and subsequently was identified in from exocrine glands and in specific granules of . Neutrophils after are main source of lactoferrin in plasma. Lactoferrin has been found in most mucosal secretions such as uterine fluid, saliva, bile, pancreatic juice, small intestine secretions, nasal , and .[4,13] Lactoferrin is also present in urine and fecal samples, though levels in these samples are relatively low. The kidney produces lactoferrin in a highly ordered manner but only a minor fraction of the protein is secreted into urine.[6] The biological properties of lactoferrin are mediated by specific receptors on the surface of target cells and can be found, for example, on mucosal epithelial cells, hepatocytes, , , polymorphonuclear leukocytes, , thrombocytes, fibroblasts, and on some . Lactoferrin possesses various biological functions including its roles in iron , proliferation and differentiation, and antibacterial, antiviral, and antiparasitic activity. Many of these functions do not appear to be connected with its iron binding ability. During most inflammatory reactions lactoferrin concentration increases in all biological fluids, and several authors classify lactoferrin as an acute-phase protein. However, the relationship between its concentration and physiological or pathological effects on body functions is not yet well characterized. [1,2,9,11,12] Lactoferrin has even been reported to inhibit the development of experimental metastases in mice.[3] Lactoferrin has also been identified as a potent anabolic factor affecting osteocytes, where it induces osteoblast proliferation, survival, differentiation, reduces of osteoblasts by 50-70% and inhibits osteoclast formation.[8,15] The influence of lactoferrin on lipid metabolism was discovered through animal studies. Oral administration of bovine lactoferrin reduced plasma cholesterol levels and retarded hepatic lipid accumulation in mice and decreased serum TAG to 72% of the control level in .[7,10] Another study of abdominally obese men and women showed that ingestion of lactoferrin reduced visceral fat.[14] Fecal lactoferrin level has investigated for its use as a non-invasive marker in the distinction of inflammatory bowel disease (IBD) and non-inflammatory condition. [16]

Areas of investigation: Bone and metabolism Energy metabolism and body weight regulation , and Inflammatory bowel disease Lipoprotein metabolism Oncology

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4. TEST PRINCIPLE

In the BioVendor Human Lactoferrin ELISA, standards and samples are incubated in microplate wells pre-coated with monoclonal anti-human lactoferrin . After 60 minutes incubation at 37°C and washing, biotin labelled monoclonal anti-human lactoferrin antibody is added and incubated at 37°C for 60 minutes with captured lactoferrin. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation at 37°C and the last washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of lactoferrin. A standard curve is constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

5. PRECAUTIONS

• For professional use only • Notice: Wear gloves, face mask (or another mouth covering) and laboratory coat when handling ELISA components and during ELISA assay. Saliva, nasal secretion, and tears contain lactoferrin and contamination in any ELISA step could cause false positive results • Do not drink, eat or smoke in the areas where immunodiagnostic materials are being handled • This kit may contain components of human or animal origin. These materials should be handled as potentially infectious • Avoid contact with the acidic Stop Solution and Substrate Solution, which contains hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents. Stop and/or Substrate Solutions may cause skin/eyes irritation. In case of contact with the Stop Solution and the Substrate Solution wash skin/eyes thoroughly with water and seek medical attention, when necessary • The materials must not be pipetted by mouth

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6. TECHNICAL HINTS

• Reagents with different lot numbers should not be mixed • Use thoroughly clean glassware • Use deionized (distilled) water, stored in clean containers • Avoid any contamination among samples and reagents. For this purpose, disposable tips should be used for each sample and reagent • Substrate Solution should remain colourless until added to the plate. Keep Substrate Solution protected from light • Stop Solution should remain colourless until added to the plate. The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution. Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution • Dispose of consumable materials and unused contents in accordance with applicable national regulatory requirements

7. REAGENT SUPPLIED

Kit Components State Quantity Antibody Coated Microtiter Strips ready to use 96 wells Biotin Labelled Antibody Conc. (100x) concentrated 0.13 ml Streptavidin-HRP Conjugate ready to use 13 ml Master Standard lyophilized 2 vials Biotin-Ab Diluent ready to use 13 ml Dilution Buffer ready to use 50 ml Wash Solution Conc. (10x) concentrated 100 ml Substrate Solution ready to use 13 ml Stop Solution ready to use 13 ml Product Data Sheet + Certificate of Analysis - 1 pc

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8. MATERIAL REQUIRED BUT NOT SUPPLIED

• Deionized (distilled) water • Test tubes for diluting samples • Glassware (graduated cylinder and bottle) for Wash Solution (Dilution Buffer) • Precision pipettes to deliver 5-1000 l with disposable tips • Multichannel pipette to deliver 100 l with disposable tips • Absorbent material (e.g. paper towels) for blotting the microtitrate plate after washing • Vortex mixer • Incubator 37°C • Microplate washer (optional). [Manual washing is possible but not preferable.] • Microplate reader with 450  10 nm filter, preferably with reference wavelength 630 nm (alternatively another one from the interval 550-650 nm) • Software package facilitating data generation and analysis (optional)

9. PREPARATION OF REAGENTS

All reagents need to be brought to room temperature prior to use

Always prepare only the appropriate quantity of reagents for your test

Do not use components after the expiration date marked on their label

• Assay reagents supplied ready to use:

Antibody Coated Microtiter Strips Stability and storage: Return the unused strips to the provided aluminium zip-sealed bag with desicant and seal carefully. Remaining Microtiter Strips are stable 3 months stored at 2-8°C and protected from the moisture.

Streptavidin-HRP Conjugate Biotin-Ab Diluent Dilution Buffer Substrate Solution Stop Solution Stability and storage: Opened reagents are stable 3 months when stored at 2-8°C.

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• Assay reagents supplied concentrated or lyophilized:

Human Lactoferrin Master Standard Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution of standard!!! Reconstitute the lyophilized Master Standard with Dilution Buffer just prior to the assay. For measurement of human lactoferrin in stool extract reconstitute the lyophilized Master Standard with special Dilution Buffer. Let it dissolve at least 15 minutes with occasional gentle shaking (not to foam). The resulting concentration of the human lactoferrin in the stock solution is 80 ng/ml.

Prepare set of standards using Dilution Buffer as follows: Volume of Standard Dilution Buffer Concentration Stock - 80 ng/ml 250 l of stock 250 l 40 ng/ml 250 l of 40 ng/ml 250 l 20 ng/ml 250 l of 20 ng/ml 250 l 10 ng/ml 250 l of 10 ng/ml 250 l 5 ng/ml 250 l of 5 ng/ml 250 l 2.5 ng/ml

Prepared Standards are ready to use, do not dilute them. Stability and storage: Do not store the reconstituted Master Standard and/or diluted standard solutions.

Biotin Labelled Antibody Conc. (100x) Prepare the working Biotin Labelled Antibody solution by adding 1 part Biotin Labelled Antibody Concentrate (100x) to 99 parts Biotin-Ab Diluent. Example: 10 l of Biotin Labelled Antibody Concentrate (100x) + 990 l of Biotin-Ab Diluent for 1 strip (8 wells). Stability and storage: Do not store the diluted Biotin Labelled Antibody solution.

Wash Solution Conc. (10x) Dilute Wash Solution Concentrate (10x) ten-fold in distilled water to prepare a 1x working solution. Example: 100 ml of Wash Solution Concentrate (10x) + 900 ml of distilled water for use of all 96-wells. Stability and storage: The diluted Wash Solution is stable 1 month when stored at 2-8°C. Opened Wash Solution Concentrate (10x) is stable 3 months when stored at 2-8°C.

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10. PREPARATION OF SAMPLES

The kit measures lactoferrin in serum, plasma (EDTA), bronchoalveolar lavage fluid (BALF), cerebrospinal fluid (CSF), urine, breast milk, saliva and stool extract.

Samples should be assayed immediately after collection or should be stored at -20°C. Thoroughly mix thawed samples just prior to the assay and avoid repeated freeze/thaw cycles, which may cause erroneous results. Avoid using hemolyzed or lipemic samples.

Collection of blood samples must be performed carefully because human lactoferrin can be released from neutrophils into surrounding fluid during the incorrect process of blood coagulation leading to elevated serum or plasma levels and false positive results.

An appropriate dilution should be assessed by the researcher in advance to batch measurement.

Serum and plasma samples: Dilute samples 25x with Dilution Buffer just prior to the assay, e.g. 5 l of sample + 120 l of Dilution Buffer for singlets, or preferably 10 l of sample + 240 l of Dilution Buffer for duplicates. Mix well (not to foam). Vortex is recommended.

Recommended starting dilution for BALF is 20x.

Recommended starting dilution for CSF and urine is 3x.

Recommended starting dilution for breast milk is 20 000x.

Recommended starting dilution for saliva is 1 000x.

Stool extract: For protocol for preparation of stool extracts and other details, please contact us at [email protected]. Recommended starting dilution for stool extract is 3x. Special Dilution Buffer is needed for measurement of human lactoferrin in stool extract and is not included.

Stability and storage: Samples should be stored at -20°, or preferably at -70°C for long-term storage. Avoid repeated freeze/ thaw cycles. Do not store the diluted samples. Note: It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results.

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11. ASSAY PROCEDURE

1. Pipet 100 l of diluted standards, Dilution Buffer (=Blank) and samples, preferably in duplicates, into the appropriate wells. See Figure 1 for example of work sheet. 2. Incubate the plate at 37°C for 1 hour without shaking. 3. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel. 4. Add 100 l of Biotin Labelled Antibody into each well. 5. Incubate the plate at 37°C for 1 hour without shaking. 6. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel. 7. Add 100 l of Streptavidin-HRP Conjugate into each well. 8. Incubate the plate at 37°C for 30 minutes without shaking. 9. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel. 10. Add 100 l of Substrate Solution into each well. Avoid exposing the microtiter plate to direct sunlight. Covering the plate with e.g. aluminium foil is recommended. 11. Incubate the plate for 10 minutes at room temperature. The incubation time may be extended [up to 20 minutes] if the reaction temperature is below than 20°C. Do not shake the plate during the incubation. 12. Stop the colour development by adding 100 l of Stop Solution. 13. Determine the absorbance of each well using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550 - 650 nm). Subtract readings at 630 nm (550 - 650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 12.

Note 1: If some samples and standard/s have absorbances above the upper limit of your microplate reader, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine lactoferrin concentration of off- scale standards and samples. The readings at 405 nm should not replace the readings for samples that were “in range” at 450 nm.

Note 2: Manual washing: Aspirate wells and pipet 0.35 ml Wash Solution into each well. Aspirate wells and repeat four-times. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

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strip 1+2 strip 3+4 strip 5+6 strip 7+8 strip 9+10 strip 11+12 A Standard 80 Sample 2 Sample 10 Sample 18 Sample 26 Sample 34 B Standard 40 Sample 3 Sample 11 Sample 19 Sample 27 Sample 35 C Standard 20 Sample 4 Sample 12 Sample 20 Sample 28 Sample 36 D Standard 10 Sample 5 Sample 13 Sample 21 Sample 29 Sample 37 E Standard 5 Sample 6 Sample 14 Sample 22 Sample 30 Sample 38 F Standard 2.5 Sample 7 Sample 15 Sample 23 Sample 31 Sample 39 G Blank Sample 8 Sample 16 Sample 24 Sample 32 Sample 40 H Sample 1 Sample 9 Sample 17 Sample 25 Sample 33 Sample 41

Figure 1: Example of a work sheet.

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12. CALCULATIONS

Most microplate readers perform automatic calculations of analyte concentration. The Standard curve is constructed by plotting the mean absorbance (Y) of Standards against the known concentration (X) of Standards in logarithmic scale, using the four-parameter algorithm. Results are reported as concentration of lactoferrin (ng/ml) in samples.

Alternatively, the logit log function can be used to linearize the standard curve (i.e. logit of the mean absorbance (Y) is plotted against log of the known concentration (X) of standards).

The measured concentration of samples calculated from the standard curve must be multiplied by their respective dilution factor, because samples have been diluted prior to the assay, e.g. 10 ng/ml (from standard curve) x 25 (dilution factor) = 250 ng/ml

Human Lactoferrin ELISA Standard Curve

3.5

3.0

2.5

2.0

1.5

1.0 Absorbance (A450 Absorbance nm - A630 nm) 0.5

0.0 1 10 100

Concentration of lactoferrin (ng/ml)

Figure 2: Typical Standard Curve for Human Lactoferrin ELISA.

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13. PERFORMANCE CHARACTERISTICS

Typical analytical data of BioVendor Human Lactoferrin ELISA are presented in this chapter

• Sensitivity Limit of Detection (LOD) (defined as concentration of analyte giving absorbance higher than mean absorbance of blank* plus three standard deviations of the absorbance of blank: Ablank + 3xSDblank) is calculated from the real lactoferrin values in wells and is 1.1 ng/ml. *Dilution Buffer is pipetted into blank wells.

• Limit of assay Samples with absorbances exceeding the absorbance of the highest standard should be measured again with higher dilution. The final concentration of samples calculated from the standard curve must be multiplied by the respective dilution factor.

Presented results are multiplied by respective dilution factor

• Precision Intra-assay (Within-Run) (n=8)

Sample Mean SD CV (ng/ml) (ng/ml) (%) Serum 1 359.0 11.6 3.2 Serum 2 645.9 21.9 3.4

Inter-assay (Run-to-Run) (n=6)

Sample Mean SD CV (ng/ml) (ng/ml) (%) Serum 1 345.3 21.5 6.2 Serum 2 731.7 33.2 4.5

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• Spiking Recovery Samples were spiked with different amounts of human lactoferrin and assayed.

Sample Observed Expected Recovery O/E (ng/ml) (ng/ml) (%) 324.0 - - 416.6 449.0 92.8 Serum 1 526.0 574.0 91.6 786.9 824.0 95.5 352.3 - - 559.7 552.3 101.3 Serum 2 739.4 752.3 98.3 1153.9 1152.3 100.1 673.2 - - EDTA 771.7 798.2 96.7 plasma 1002.5 923.2 108.6 1222.4 1173.2 104.2 158.1 - - 289.6 258.1 112.2 BALF 380.5 358.1 106.2 533.7 558.1 95.6 18.9 - - 37.8 33.9 111.5 CSF 48.9 48.9 100.0 75.1 78.8 95.3 39.6 - - 61.0 54.6 111.7 Urine 67.4 69.6 96.8 104.3 99.6 104.7

Sample Observed Expected Recovery O/E (g/g) (g/g) (%) 1.6 - - 2.2 2.4 91.7 Stool 2.8 3.1 90.3 4.0 4.6 87.0

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• Linearity Samples were serially diluted with Dilution Buffer and assayed.

Sample Dilution Observed Expected Recovery (ng/ml) (ng/ml) O/E (%) - 748.1 - - 2x 357.8 374.0 95.6 Serum 1 4x 173.5 187.0 92.8 8x 92.3 93.5 98.7 - 1129.3 - - 2x 520.6 564.7 92.2 Serum 2 4x 261.7 282.3 92.7 8x 135.0 141.2 95.6 - 948.7 - - EDTA 2x 465.3 474.4 98.1 plasma 4x 228.2 237.2 96.2 8x 116.5 118.6 98.3 - 527.3 - - 2x 246.0 263.7 93.3 BALF 4x 113.8 131.8 86.3 8x 56.1 65.9 85.1 - 37.8 - - 2x 19.3 18.9 102.2 CSF 4x 9.1 9.5 96.0 8x 4.7 4.7 100.3 - 169.1 - - 2x 80.7 84.6 95.5 Urine 4x 40.0 42.3 94.6 8x 22.2 21.1 105.0

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Sample Dilution Observed Expected Recovery (g/ml) (g/ml) O/E (%) - 536.9 - - Breast 2x 251.5 268.5 93.7 milk 4x 134.5 134.2 100.2 8x 65.3 67.1 97.3 - 26.8 - - 2x 12.6 13.4 94.0 Saliva 4x 6.7 6.7 100.0 8x 3.3 3.4 97.1

Sample Dilution Observed Expected Recovery (g/g) (g/g) O/E (%) - 18.4 - - 2x 9.0 9.2 97.8 Stool 4x 4.2 4.6 91.3 8x 2.2 2.3 95.7

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• Effect of sample matrix Heparin, citrate and EDTA plasmas were compared to respective serum samples from the same 10 individuals. However, we observed low correlation among serum and plasma (citrate and heparin) lactoferrin values. Results are shown below:

Volunteer Serum Plasma (ng/ml) No. (ng/ml) EDTA Citrate Heparin 1 310.9 270.5 168.9 509.9 2 309.5 307.5 104.8 271.0 3 1033.6 505.6 216.8 187.6 4 218.0 126.4 73.3 438.9 5 230.2 219.7 164.2 128.9 6 361.7 216.6 184.2 188.6 7 503.4 316.9 92.0 224.5 8 210.2 129.5 159.4 92.6 9 197.4 135.7 130.3 119.8 10 201.5 140.0 146.3 123.0 Mean (ng/ml) 357.6 236.8 144.0 228.5 Mean Plasma/Serum - 66.2% 40.3% 63.9% (%) Coefficient of - 0.84 0.23 0.00 determination R2

Figure 3: Lacoferrin levels measured using Human Lactoferrin ELISA from 10 individuals using serum, heparin, citrate and EDTA plasma, respectively.

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14. DEFINITION OF THE STANDARD

A native protein isolated from human breast milk is used as the standard in this assay.

15. PRELIMINARY POPULATION AND CLINICAL DATA

The following results were obtained when serum samples from 166 unselected donors (93 men + 73 women) 21-65 years old were assayed with the BioVendor Human Lactoferrin ELISA in our laboratory.

• Age and sex dependent distribution of Lactoferrin

Age n Mean Median SD Min Max Sex (years) Lactoferrin (ng/ml) 21-29 19 303.6 245.6 170.0 129.0 679.3 30-39 29 376.8 319.2 233.8 172.7 1368.2 Men 40-49 31 342.5 314.4 169.0 94.7 824.1 50-65 14 377.5 368.8 138.4 134.5 606.6 22-29 13 385.2 356.2 156.3 172.0 677.5 30-39 28 370.5 338.7 169.4 137.4 886.4 Women 40-49 23 372.9 333.9 194.5 157.6 974.7 50-61 9 239.7 244.0 96.7 105.1 431.5

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Figure 4: Lactoferrin concentration plotted against donor age and sex.

• Reference range The data quoted in these instructions should be used for guidance only. It is recommended that each laboratory include its own panel of control samples in the assay. Each laboratory should establish its own normal and pathological references ranges for lactoferrin levels with the assay.

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16. METHOD COMPARISON

The BioVendor Human Lactoferrin ELISA was compared to other commercial immunoassay by measuring 34 serum samples. The following correlation graph was obtained:

Figure 5: Method Comparison

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17. TROUBLESHOOTING AND FAQS

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 temperature • Improper wavelength when reading absorbance

High signal and background in all wells Possible explanations: • Improper or inadequate washing • Overdeveloping; incubation time with Substrate Solution should be decreased before addition of Stop Solution

High coefficient of variation (CV) Possible explanation: • Improper or inadequate washing • Improper mixing Standards and samples

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18. REFERENCES

References to lactoferrin:

1. Yamauchi K, Tomita M, Giehl TJ, Ellison RT: Antibacterial activity of lactoferrin and pepsinderived lactoferrin fragment. Infection and 1993, 61:719-728 2. Baynes RD, Bezwoda WR: Lactoferrin and the inflammatory response. Advance in Exp Med and Biol 1994, 357:133-141 3. Bezault JA, Bhimani R, Wiprovnick BJ, Furmanski P: Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice. Res 1994, 54:2310-2312 4. Levay PF, Viljoen M: Lactoferrin: A general review. Haematologica 1995, 80:252-267 5. Steijns JM, van Hooijdonk ACM: Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. British J of Nutrition 2000, 84:S11-S17 6. Abrink M, Larsson E, Gobl A, Hellman L: Expression of lactoferrin in the kidney: implications for innate immunity and iron metabolism. Kidney Int 2000, 57:2004-20010 7. Takeuchi T, Shimizu H, Ando K, Harada E: Bovine lactoferrin reduces triacylglycerol and NEFA accompanied by decrease hepatic cholesterol and triacylglycerol contents in rodents. British J of Nutrition 2004, 91:533-538 8. Naot D, Grey A, Reid IR, Cornish J: Lactoferrin – A novel bone growth factor. Clin Med and Research 2005, 2:93-101 9. Valenti P, Antonini G: Lactoferrin: an important host defense against microbial and viral attack. Cel and Mol Life 2005, 62:2576-2587 10. Tamano S, Sekine K, Takase M, Yamauchi K, Iigo M, Tsuda H: Lack of chronic oral of chemopreventive bovine lactoferrin in F344/DuCrj rats. Asian Pacific J Cancer Prev 2008, 9:313-316 11. Jenssen H, Hancock REW: properties of Lactoferrin. Biochimie 2009, 91:19-29 12. Actor JK, Hwang S-A, Kruzel ML: Lactoferrin as a Natural Immune Modulator. Curr Pharm Des 2009, 15(17):1956-1973 13. González-Chávez SA, Arévalo-Gallegos S, Rascón-Cruz Q: Lactoferrin: structure, function and applications. I J Antimic Ag 2009, 33:301.e1-301.e8 14. Ono T, Murakoshi M, Suzuki N, Iida N, Ohdera M, Iigo M, Yoshida T, Sugiyama K, Nishino H: Potent anti-obesity effect of enteric-coated lactoferrin: decrease in visceral fat accumulation in Japanese men and women with abdominal obesity after 8-week administration of enteric-coated lactoferrin rablets. British J of Nutrition 2010, 1-8 15. Naot D, Palmano K, Cornosh J: Lactoferrin – A potential anabolic intervention in osteoporosis. Osteoporosis, publisher In Tech 2012, 803-821 16. Zhou X, Xu W, Tang X, Luo L, Tu J, Zhang Ch, Xu X, Wu Q, Pan W: Fecal lactoferrin in discriminating inflammatora bowel disease from irritable bowel syndrome: a diagnostic meta-analysis. BMC Gastroenterology 2014. 14:121

For more references on this product see our WebPages at www.biovendor.com

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19. EXPLANATION OF SYMBOLS

Catalogue number

Content

Lot number

Attention, see instructions for use

Potential biological hazard

Expiry date

Storage conditions

Name and registered office of the manufacturer

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Assay Procedure Summary

Antibody Coated Microtiter Plate

Dilute samples Reconstitute Master Standard, prepare set of standards

Add Standards and samples 100 l

Prepare Wash Solution Incubate at 37°C for 1 hour

Wash 5x

Prepare Biotin Labeled Antibody solution

Add Biotin Labelled Antibody solution

100 l

Incubate at 37°C for 1 hour

Wash 5x

Add Streptavidin HRP Conjugate 100 l

Incubate at 37°C for 30 min

Wash 5x

Add Substrate Solution 100 l

Incubate at RT for 10 min

Add Stop Solution 100 l

Read absorbance and calculate results

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12

11

10

9

8

7

6

5

4

3

2

1

F

E

A B C D H G

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NOTES

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NOTES

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