DRG International EIA-4555 Testosterone Elisa Instructions

DRG International EIA-4555 Testosterone Elisa

Product Information

Specifications

• Product Name: S100B (Human) ELISA EIA-4555
• Manufacturer: DRG International, Inc.
• Intended Use: Sandwich enzyme immunoassay for quantitative measurement of human S100B
• Areas of Investigation: Neural tissue damage, Acute myocardial infarction, Oncology

Product Usage Instructions

Precautions

• For professional use only.
• Wear gloves and laboratory coats when handling immunodiagnostic materials.
• Avoid eating, drinking, or smoking in areas where materials are handled.
• Dispose of consumable materials properly.

Technical Hints

• Avoid mixing reagents with different lot numbers.
• Use clean glassware and deionized water stored in clean containers.
• Avoid contamination among samples and reagents by using disposable tips for each.
• Protect Substrate Solution from light and ensure proper mixing of Stop Solution.

Reagent Preparation

• All reagents should be brought to room temperature before use.
• Centrifuge liquid-containing vials before opening.
• Prepare only the required quantity of reagents for the test.
• Do not use components past their expiration date.

Assay Reagents

• Ready to Use: Assay reagents supplied ready to use.
• Concentrated or Lyophilized: Reconstitute the lyophilized Master Standard as per the Certificate of Analysis to prepare standard stock solution just before the assay. Dissolve for 25-30 minutes with gentle shaking.

FAQ

Frequently Asked Questions

• Q: Can components with different lot numbers be mixed?
  • A: No, reagents with different lot numbers should not be mixed to maintain accuracy.
• Q: Can I reuse disposable tips for multiple samples?
  • A: No, disposable tips should be used for each sample to prevent contamination.

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The Human S100B ELISA is a sandwich enzyme immunoassay for the quantitative measurement of human S100B.

Features

• It is intended for research use only.
• The total assay time is less than 4.5 hours.
• The kit measures S100B protein in serum and cerebrospinal fluid samples.
• Assay format is 96 wells.
• Quality Controls are human serum based. Animal serum is used for Master Standard and Dilution Buffer preparations.
• Standard is native protein based.
• Components of the kit are provided ready to use, concentrated or lyophilized.

STORAGE, EXPIRATION

• Store the complete kit at 2 °C – 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.

INTRODUCTION

S-100B is a member of highly homologous Ca2+ binding proteins family that possess two EF-hand motifs. The family of S100 proteins consists of 19 members. Most S100 proteins exist as dimers (frequently homodimers) within cells. Exclusively expressed in vertebrates, S100 is implicated in various intracellular and extracellular regulatory activities. Studies indicate that S100 proteins are involved in the inhibition of protein phosphorylation, inhibition of cytoskeletal constituent assembly, regulation of Ca2+ homeostasis, stimulation of enzyme activities, and interaction with transcription factors. S100B is abundant in the nervous system where it is predominantly expressed in astrocytes, oligodendrocytes and Schwann cells.

When secreted by astrocytes, S100B has neurotrophic effects during development and nerve regeneration at physiologic (nanomolar) concentrations. However, high (micromolar) concentrations of S100B have shown to be neurotoxic, participating in the physiology of neurodegenerative disorders. The clinical values have been demonstrated in stroke, cerebral complications associated with cardiac arrest and in patients with severe as well as minor head injuries. Patients with progressive melanoma disease also show elevated serum concentrations of S100B.

Areas of investigation:

• Neural tissue damage
• Acute myocardial infarction
• Oncology

TEST PRINCIPLE

In the Human S100B ELISA, standards, quality controls and samples are incubated in microplate wells pre-coated with monoclonal anti-human S100B antibody. After 120 minutes incubation and washing, biotin-labelled monoclonal anti-human S100B antibody is added to the wells and incubated for 60 minutes with captured S100B. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation 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 S100B. A standard curve is constructed by plotting absorbance values against concentrations of Standards, and concentrations of unknown samples are determined using this standard curve.

PRECAUTIONS

• For professional use only.
• Wear gloves and laboratory coats when handling immunodiagnostic materials.
• Do not drink, eat or smoke in the areas where immunodiagnostic materials are being handled.
• This kit contains components of human origin. These materials were found non-reactive for HBsAg, HCV antibody and for HIV 1/2 antigen and antibody. However, these materials should be handled as potentially infectious, as no test can guarantee the complete absence of infectious agents.
• This kit contains components of 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.

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.

REAGENT SUPPLIED

MATERIAL REQUIRED BUT NOT SUPPLIED

• Deionized (distilled) water
• Test tubes for diluting samples
• Glassware (graduated cylinder and bottle) for Wash Solution
• Precision pipettes to deliver 10-1000 μL with disposable tips
• Multichannel pipette to deliver 100 μL with disposable tips
• Absorbent material (e.g. paper towels) for blotting the microtitrate plate after washing
• Vortex mixer
• Orbital microplate shaker capable of approximately 300 rpm
• 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)

PREPARATION OF REAGENTS

• All reagents need to be brought to room temperature prior to use.
• Centrifuge liquid containing microtube vials before opening.
• 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 desiccant and seal carefully. Remaining Microtiter Strips are stable 3 months stored at 2 °C – 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 °C – 8 °C.

Assay reagents supplied concentrated or lyophilized

Human S100B Master Standard

Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution of standard!!!
Reconstitute the lyophilized Master Standard according to the Certificate of Analysis to prepare standard stock solution just prior to the assay. Let it dissolve for 25-30 minutes with occasional gentle shaking (not to foam). The resulting concentration of the human S100B in the stock solution is 1600 pg/ml.

Prepare set of standards using Dilution Buffer as follows:

• Mix thoroughly but gently (not to foam) after each dilution step. 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.

Quality Controls High, Low

Refer to the Certificate of Analysis for current volume of distilled water needed for reconstitution and for current Quality Control concentration!!!

Reconstitute each Quality Control (High and Low) with distilled water just prior to the assay. Let it dissolve for 25-30 minutes with occasional gentle shaking (not to foam).
Dilute Quality Controls prior to the assay 4x with Dilution Buffer, e.g. 40 μL of Quality Control + 120 μL of Dilution Buffer for singlets, or preferably 60 μL of Quality Control + 180 μL of Dilution Buffer for duplicates.

Stability and storage:

• The reconstituted Quality Controls must be used immediately or aliquoted and frozen at -20 °C for 3 months. Avoid repeated freeze/thaw cycles.
• Do not store the diluted Quality Controls.

Note: Concentration of analyte in Quality Controls need not be anyhow associated with normal and/or pathological concentrations in serum or another body fluid. Quality Controls serve just for control that the kit works in accordance with Instruction for Use and Certificate of Analysis and that ELISA test was carried out properly.

Biotin Labelled Antibody Conc. (100x)

• Prepare the working Biotin Labelled Antibody solution by adding 1 part of Biotin Labelled Antibody Concentrate (100x) to 99 parts of Biotin-Ab Diluent.
• Example (for 1 strip, i.e. 8 wells): 10 μL of Biotin Labelled Antibody Conc. (100x) + 990 μL Biotin-Ab Diluent.

Stability and storage:

• Opened Biotin Labelled Antibody Conc. (100x) is stable 3 months when stored at 2 °C – 8 °C.
• Do not store diluted Biotin Labelled Antibody working 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 Conc. (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 °C – 8 °C.
• Opened Wash Solution Concentrate (10x) is stable 3 months when stored at 2 °C – 8 °C.

PREPARATION OF SAMPLES

The kit measures S100B in serum and cerebrospinal fluid (CSF) samples. EDTA plasma and citrate plasma are not suitable kinds of samples because of unfavourable interactions between sample and Dilution Buffer components. No similar interaction has been observed with heparin plasma samples therefore it can be assumed that heparin plasma samples may be used in this ELISA. However heparin plasma samples could not be validated with this ELISA because suitable samples paired with serum samples from the same individuals were not available.

Samples should be assayed immediately after collection or should be stored at -20 °C. Mix thawed samples thoroughly just prior to the assay and avoid repeated freeze/thaw cycles, which may cause erroneous results. Avoid using hemolyzed or lipemic samples.
An appropriate sample dilution should be assessed by the researcher in advance to batch measurement.

Recommended starting dilution for serum and CSF samples is 4x. Dilute samples 4x with Dilution Buffer just prior to the assay, e.g. 40 μL of sample + 120 μL of Dilution Buffer for singlets, or preferably 60 μL of sample + 180 μL of Dilution Buffer for duplicates. Mix thoroughly but gently (not to foam). Vortex is recommended.

Stability and storage:

• Samples should be stored at -20 °C, or preferably at -70 °C for long-term storage. Avoid repeated freeze/ thaw cycles.
• Do not store the diluted samples.
• See Chapter 13 for stability of cerebrospinal fluid sample when stored at 2 °C – 8 °C and effect of freezing/thawing on the concentration of S100B protein.
• Note: It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results.
• Ask for more detailed information at drg@drg-diagnostics.de if assaying heparin plasma samples or other kinds of samples.

ASSAY PROCEDURE

1. Pipet 100 μL of diluted Standards, Quality Controls, 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 room temperature (ca. 25 °C) for 120 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.
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 μL of Biotin Labelled Antibody solution into each well.
5. Incubate the plate at room temperature (ca. 25 °C) for 60 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.
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 μL of Streptavidin-HRP Conjugate into each well.
8. Incubate the plate at room temperature (ca. 25 °C) for 30 minutes, shaking at ca. 300 rpm on an orbital microplate shaker.
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 μL 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 15 – 30 minutes at room temperature. Longer incubation time (up to 30 minutes) is recommended if the colour development is slow. Do not shake the plate during the incubation.
12. Stop the colour development by adding 100 μL 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 S100B 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 5-times: Aspirate wells and pipet 0.35 mL Wash Solution into each well. Aspirate wells and repeat 4-times. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

 | strip 1+2| strip 3+4| strip 5+6| strip 7+8| strip 9+10| strip 11+12
---|---|---|---|---|---|---
A| Standard 320| Blank| Sample 8| Sample 16| Sample 24| Sample 32
B| Standard 160| Sample 1| Sample 9| Sample 17| Sample 25| Sample 33
C| Standard 80| Sample 2| Sample 10| Sample 18| Sample 26| Sample 34
D| Standard 40| Sample 3| Sample 11| Sample 19| Sample 27| Sample 35
E| Standard 20| Sample 4| Sample 12| Sample 20| Sample 28| Sample 36
F| Standard 10| Sample 5| Sample 13| Sample 21| Sample 29| Sample 37
G| QC High| Sample 6| Sample 14| Sample 22| Sample 30| Sample 38
H| QC Low| Sample 7| Sample 15| Sample 23| Sample 31| Sample 39

Figure 1: Example of a work sheet.

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 S100B pg/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 S100B in samples and in Quality Controls calculated from the standard curve must be multiplied by their respective dilution factor, because samples and controls have been diluted prior to the assay, e.g. 75.5 pg/mL (from standard curve) x 4 (dilution factor) = 302 pg/mL.

PERFORMANCE CHARACTERISTICS

Typical analytical data of Human S100B ELISA are presented in this chapter.

Sensitivity

• The measurement range of the assay is 10 – 320 pg/mL (final concentration in a well after dilution).

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.

Specificity

The antibodies used in this ELISA are specific for human S100B with no detectable cross reactivities to human S100A1, S100A4, S100A5, S100A8, S100A9, S100A10, S100A11, S100A14, S100A15, S100A16, S100G.
High S100B concentration was detected in diluted (1000x – 2000x) mouse brain homogenate samples using this ELISA.
Sera of several mammalian species were measured in the assay.
Positive reactivity has been found in these animal sera: bovine, cat, goat, hamster, rhesus monkey, rabbit and rat. For details please contact us at drg @drg-diagnostics.de.

S100B protein amino acid sequence is highly conserved (e.g. there is only 1 amino acid difference between mouse and human S100B protein sequences), therefore cross reactivity with some animal S100B proteins may be expected. On the other hand, very low or zero S100B levels found in animal serum samples can reflect in some cases only absence of S100B in those particular serum samples from animals with the uninjured brain.
Presented results are multiplied by respective dilution factor

Precision

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

Inter-assay (Run-to-Run) (n=4)

Spiking Recovery

Serum and cerebrospinal fluid (CSF) samples were spiked with different amounts of human S100B antigen and assayed.

Sample| O bserved (pg/mL)| E xpected (pg/mL)| Recovery O/E

(%)

---|---|---|---
Serum sample| 568.6

863.8

1168.9

1760.8

| – 888.6

1208.6

1848.6

| – 97.2

96.7

95.3

CSF sample| 268.2

424.4

478.1

706.4

| – 368.2

468.2

668.2

| – 115.3

102.1

105.7

Linearity

Serum and CSF samples were serially diluted with Dilution Buffer and assayed.

Sample| Dilution| Observed (pg/mL)| Expected (pg/mL)| Recovery O/E (%)
---|---|---|---|---
Serum sample| –

2x 4x 8x

| 420.5

212.3

111.2

58.6

| – 210.3

105.1

52.6

| – 101.0

105.7

111.4

CSF sample| –

2x 4x 8x

| 827.3

481.3

237.1

124.7

| – 413.7

206.8

103.4

| – 116.4

114.6

120.6

Stability of samples stored at 2 °C – 8 °C

Samples should be stored at -20 °C. However, no decline in concentration of S100B was observed in cerebrospinal fluid samples after 14 days when stored at 2 °C – 8 °C. To avoid microbial contamination, samples were treated with -aminocaproic acid and sodium azide, resulting in the final concentration of 0.03% and 0.1%, respectively.

1

| -20 °C| 148.3
2 °C – 8 °C, 1 day| 142.9
2 °C – 8 °C, 7 days| 141.7
2 °C – 8 °C, 14 days| 149.2

2

| -20 °C| 190.3
2 °C – 8 °C, 1 day| 202.5
2 °C – 8 °C, 7 days| 196.4
2 °C – 8 °C, 14 days| 188.2

3

| -20 °C| 183.1
2 °C – 8 °C, 1 day| 183.1
2 °C – 8 °C, 7 days| 168.3
2 °C – 8 °C, 14 days| 172.8

Effect of Freezing/Thawing

Concentration of human S100B in cerebrospinal fluid samples declined more than 30% after repeated (3x) freeze/thaw cycles. It is strongly recommended to avoid repeated freezing/thawing of the samples.

1

| 1x| 221.3
3x| 132.1
5x| 93.1

2

| 1x| 218.8
3x| 191.5
5x| 174.3

3

| 1x| 304.8
3x| 203.7
5x| 191.0

Comparison of serum and CSF samples

Serum samples and cerebrospinal fluids were taken from 8 patients with head injury or brain disorder and measured in the assay, results shown below:

Reference range

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 reference ranges for human S100B levels with the assay.

DEFINITION OF THE STANDARD

A S100B (ββ homodimer) protein purified from human brain tissue is used as the standard in this assay. S100B is a 21 kDa protein.

METHOD COMPARISON

The Human S100B ELISA has not been compared to the other commercial immunoassays.

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
• Incubation temperature over 30 °C

High coefficient of variation (CV)
Possible explanations:

• Improper or inadequate washing
• Improper mixing Standards, Quality Controls or samples

REFERENCES / LITERATURE

1. Zimmer DB et al.: The S100 Protein Family: History, Function, and Expression. Brain Research Bulletin. 37(4):417-429 (1995)
2. Kärnell R et al: S100B Protein, 5-S-Cysteinyldopa and 6-Hydroxy-5-Methoxyindole-2-Carboxylic Acid as Biochemical Markers for Survival Prognosis in Patients with Malignant Melanoma. Melanoma Research 7(5):393-399 (1997)
3. Jönsson H et al: S100β After Coronary Artery Surgery: Release Pattern, Source of Contamination, and Relation to Neuropsychological Outcome. Ann Thorac Surg. 68(6): 2202-2208 (1999)
4. Hauschild A et al: S100B Protein Detection in Serum Is a Significant Prognostic Factor in Metastatic Melanoma. Oncology 56(4):338-344 (1999)
5. Donato R: S100: a Multigenic Family of Calcium-Modulated Proteins of the EF-Hand Type with Intracellular and Extracellular Functional Roles. Int J Biochem Cell Biol. 33(7) 637-68 (2001)
6. Reynolds MA et al: Early Biomarkers of Stroke. Clinical Chemistry 49(10):1733-1739 (2003)
7. Johnsson P et al: Increased S100B in Blood After Cardiac Surgery Is a Powerful Predictor of Late Mortality. Ann Thorac Surg 75(1):162-168 (2003)
8. Beems T et al: Serum- and CSF-concentrations of Brain Specific Proteins in Hydrocephalus. Acta Neurochir 145(1):37-43 (2003)
9. Gazzolo D et al: S100B protein in urine of preterm newborns with ominous outcome. Pediatr Res. 58(6):1170-4 (2005)
10. Fernandez-Fernandez MR et al: Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor. Proc Natl Acad Sci USA. 102(13):4735-40 (2005)
11. Delgado P et al: Plasma S100B level after acute spontaneous intracerebral hemorrhage. Stroke. 37(11):2837-9 (2006)
12. Ralay Ranaivo H et al.: Glia as a therapeutic target: selective suppression of human amyloid-beta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration. J Neurosci. 26(2):662-70 (2006)
13. Sanchez-Juan P et al: CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease. Neurology. 67(4):637-43 (2006)
14. Steiner J et al: Increased cerebrospinal fluid and serum levels of S100B in first-onset schizophrenia are not related to a degenerative release of glial fibrillar acidic protein, myelin basic protein and neurone-specific enolase from glia or neurones. J Neurol Neurosurg Psychiatry. 77(11):1284-7(2006)
15. Steiner J et al: Evidence for a wide extra-astrocytic distribution of S100B in human brain. BMC Neurosci. 8:2 (2007)
16. Berger RP et al.: Serum biomarker concentrations and outcome after pediatric traumatic brain injury. J Neurotrauma. 24(12):1793-801 (2007)
17. Foerch C et al.: Elevated serum S100B levels indicate a higher risk of hemorrhagic transformation after thrombolytic therapy in acute stroke. Stroke. 38(9):2491-5 (2007)

For more references on this product send a request drg@drg- diagnostics.de

ASSAY PROCEDURE SUMMARY

SYMBOLS USED

CONTACTS

DRG Instruments GmbH, Germany

• Frauenbergstraße. 18, D-35039 Marburg
• Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50
• Website: www.drg-diagnostics.de
• E-mail: drg@drg-diagnostics.de

DRG International, Inc., USA

• 841 Mountain Ave., Springfield, NJ 07081
• Phone: 973-564-7555, Fax: 973-564-7556
• Website: www.drg-international.com
• E-mail: corp@drg-international.com

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks

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