DRG EIA-5955 21 Hydroxylase Autoantibody ELISA Instructions

DRG EIA-5955 21 Hydroxylase Autoantibody ELISA

Product Information

• The 21-Hydroxylase Autoantibody ELISA (EIA-5955) is a diagnostic kit manufactured by DRG International, Inc.
• It is used for the detection and quantification of 21-Hydroxylase autoantibodies in patient samples.
• The kit includes various components such as coated wells, controls, reagents, and buffers.

Components

and
sealed in a foil bag.
Negative Control| 0.7 mL Ready for use
Positive Controls I & II| 2 x 0.7 mL Ready for use
Reference Preparation| 0.7 mL Ready for use
Calibrators (optional)| 0.3, 1.0, 10, 100 U/mL (arbitrary DRG units) 4 x 0.7 mL Ready
for use
Reaction Enhancer| 6 mL, colored red Ready for use
Reconstitution Buffer for 21-OH-Biotin| 2 x 10 mL Ready for use
Streptavidin Peroxidase (SA-POD)| 0.7 mL Concentrated
Diluent for SA-POD| 15 mL Ready for use
Peroxidase Substrate (TMB)| 15 mL Ready for use
Stop Solution| 12 mL Ready for use
Concentrated Wash Solution| 125 mL Concentrated

Product Usage Instructions

1. Ensure the wells are firmly fitted in the frame provided. After opening, return any unused wells to the original foil bag with the desiccant provided and seal with adhesive tape. Place the foil bag in the self-seal plastic bag provided.
2. Reconstitute the Reaction Enhancer with room temperature reconstitution buffer immediately before use (within 30 minutes), using 5.5 mL per vial. If using multiple vials, pool them and mix gently.
3. Prepare the samples and controls according to the assay requirements.
4. Aspirate and wash/aspirate the wells three times with diluted wash solution using an ELISA plate washing machine.
5. Add the prepared samples, controls, and calibrators (if applicable) to the appropriate wells.
6. Cover the frame and shake the wells for 1 hour at room temperature on an ELISA plate shaker (500 shakes per minute). Repeat wash step 4.
7. Add Streptavidin Peroxidase (SA-POD) to each well and incubate for the specified time.
8. Repeat wash step 4.
9. Add Peroxidase Substrate (TMB) to each well and incubate for the specified time.
10. Add Stop Solution to stop the reaction.
11. Measure the absorbance of each well at 450 nm using a microplate reader.
12. Calculate the index values using the absorbance data at 450 nm or 405 nm, as described in the manual.
13. Interpret the results based on the calculated index values and the assay cut-off.

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

INTENDED USE

• The 21-Hydroxylase Autoantibody (21-OH Ab) ELISA kit is intended for use by professional persons only, for the quantitative determination of 21-OH Ab in human serum.
• Autoimmune destruction of the adrenal cortex is the most common cause of Addison’s disease and autoantibodies to the adrenal-specific enzyme steroid 21 hydroxylase are important markers of adrenal autoimmunity.
• This can be the case if the disease presents as Addison’s disease or as part of the autoimmune polyglandular syndromes (APS) type I or type II.

REFERENCES / LITERATURE

• J. Furmaniak and B. Rees Smith
• Editorial: Adrenal and Gonadal Autoimmune Diseases.
• J. Clin. Endocrinol. Metab. 1995 80: 1502 – 1505
• S. Chen et al
• Autoantibodies to Steroidogenic Enzymes in Autoimmune Polyglandular Syndrome, Addison’s Disease, and Premature Ovarian Failure.
• J. Clin. Endocrinol. Metab. 1996 81: 1871-1876
• H. Tanakaetal
• Steroid 21-Hydroxylase Autoantibodies: Measurements with a New Immunoprecipitation Assay.
• J. Clin. Endocrinol. Metab. 1997 82: 1440-1446
• G. Cocoetal
• Estimated Risk for Developing Autoimmune Addison’s Disease in Patients with Adrenal Cortex Autoantibodies.
• J. Clin. Endocrinol. Metab. 2006 91: 1637-1645
• E. S. Husebye et al
• Consensus Statement on the Diagnosis, Treatment, and Follow-up of Patients with Primary Adrenal Insufficiency.
• J. Intern. Med. 2014 275: 104-115

ASSAY PRINCIPLE

• In this 21-OH Ab ELISA kit, 21-OH Ab in patients’ sera, reference preparation or calibrators (optional), and controls are allowed to interact with 21-OH coated onto ELISA plate wells. After a 16 – 20 hour incubation, the samples are discarded leaving 21-OH Ab bound to the 21-OH coated on the wells. 21-OH-Biotin is added in a 2nd incubation step where, through the ability of 21-OH Ab to act divalently, a bridge is formed between the 21-OH immobilized on the plate and 21-OH-Biotin.
• The amount of 21-OH-Biotin bound is then determined in a 3rd incubation step involving the addition of streptavidin peroxidase (SA-POD), which binds specifically to biotin. Excess, unbound SA-POD has then washed away and the addition of the peroxidase substrate 3,3’,5,5’-tetramethlybenzidine (TMB) results in the formation of a blue color. This reaction is stopped by the addition of a stop solution, causing the well contents to turn yellow.
• The absorbance of the yellow reaction mixture at 450 nm and 405 nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of 21-OH Ab in the test sample.
• Reading at 405 nm allows quantitation of high absorbances. It is recommended that low absorbance values are measured at 450 nm. If it is possible to read at only one wavelength 405 nm may be used.

STORAGE AND PREPARATION OF TEST SERUM SAMPLES

• Sera to be analyzed should be assayed soon after separation or stored, preferably in aliquots, at or below -20 °C. 100 µL is sufficient for one assay (duplicate 50 µL determinations).
• Repeated freeze-thawing or increases in storage temperature should be avoided. Do not use lipaemic or haemolysed serum samples. Do not use plasma in the assay.
• When required, bring the test sera to room temperature and mix gently to ensure homogeneity.
• Centrifuge serum prior to assay (preferably for 5 min at about 10,000 rpm i.e. about 10,000 g in a microfuge) to remove particulate matter. Please do not omit this centrifugation step if sera are cloudy or contain particulates.

MATERIALS REQUIRED AND NOT SUPPLIED

• Pipettes capable of dispensing 50 µL and 100 µL.
• Means of measuring various volumes to reconstitute or dilute reagents supplied.
• Pure water.
• ELISA Plate reader suitable for 96 well formats and capable of measuring at 450 nm and 405 nm.
• ELISA Plate shaker, capable of 500 shakes/min (not an orbital shaker).
• ELISA Plate cover.
• ELISA Plate washing machine.

PREPARATION OF REAGENTS SUPPLIED

Store unopened kit and all kit components (A – N) at 2 °C – 8 °C.

A| 21-OH Coated Wells

12 breakapart strips of 8 wells (96 in total) in a frame and sealed in a foil bag. Allow foil bag to stand at room temperature (20 °C – 25 °C) for 30 minutes before opening.

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Ensure wells are firmly fitted in the frame provided. After opening return any unused wells to the original foil bag with desiccant provided and seal with adhesive tape. Place foil bag in the self-seal plastic bag provided and store at 2 °C – 8 °C for up to 6 months.
B| Negative Control

0.7 mL

Ready for use

C1 – C2| Positive Controls I & II

2 x 0.7 mL

Ready for use

D| Reference Preparation

0.7 mL

Ready for use

E1 – E4| Calibrators (optional)

0.3, 1.0, 10, 100 U/mL (arbitrary DRG units)

4 x 0.7 mL

Ready for use

F| Reaction Enhancer

6 mL, coloured red Ready for use

G| 21- OH–Biotin

3 vials Lyophilised

Reconstitute with room temperature reconstitution buffer (H) immediately before use (within 30 minutes),

5.5 mL per vial. When more than one vial is to be used, pool the vials and mix gently.

H| Reconstitution Buffer for 21-OH-Biotin

2 x 10 mL Ready for use

J| Streptavidin Peroxidase (SA- POD)

0.7 mL Concentrated

Dilute 1 in 20 with diluent for SA-POD (K). For example, 0.5 mL (J) + 9.5 mL (K). Store for up to 16 weeks at 2 °C – 8 °C after dilution.
K| Diluent for SA-POD

15 mL Ready for use

L| Peroxidase Substrate (TMB)

15 mL Ready for use

M| Stop Solution 12 mL Ready for use
N| Concentrated Wash Solution 125 mL Concentrated
Dilute 1 in 10 with pure water before use. Store at 2 °C – 8 °C up to kit expiry date.

ASSAY PROCEDURE

• On day 1 allow all the reagents required for steps 1-3 to stand at room temperature (20 °C – 25 °C) for at least 30 minutes prior to use.
• On day 2 allow all the reagents required for steps 4-13 (except for the coated wells) to stand at room temperature (20 °C – 25 °C) for at least 30 minutes prior to use.
• The coated wells from day 1 must remain at 2 °C – 8 °C until ready to proceed with step 4 below.
• Do not reconstitute 21-OH-Biotin until step 5 below.
• A repeating Eppendorf-type pipette is recommended for steps 2, 5, 8, 11, and 12.

Day 1| 1.| Pipette 50 µL (in duplicate) of patient sera, negative control (B), positive controls (C1 – C2), reference preparation (D), and (if used) calibrators (E1 – E4) into respective wells (A).

Leave one well empty for blank (see step 13).

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2.| Pipette 50 µL reaction enhancer (F) into each well (except blank).
3.| Cover the frame and shake the wells on an ELISA plate shaker (500 shakes per minute) for 1 minute.

Incubate overnight (16 – 20 hours) at 2 °C – 8 °C without shaking.

Day 2| 4.| Aspirate and wash/aspirate the wells three times with diluted wash solution (N) by use of an ELISA plate washing machine.
5.| Reconstitute 21-OH-Biotin (G) using reconstitution buffer (H) which has reached room temperature. Pipette 100 µL into each well (except blank).
6.| Cover the frame and shake the wells for 1 hour at room temperature on an ELISA plate shaker (500 shakes per minute).
7.| Repeat wash step 4.
8.| Pipette 100 µL of diluted SA-POD (J) into each well (except blank).
9.| Cover the frame and shake the wells for 20 minutes at room temperature on an ELISA plate shaker (500 shakes per minute).
10.| Repeat wash step 4.
 | 11.| Pipette 100 µL of TMB (L) into each well (including blank) and incubate for 20 minutes in the dark at room temperature without shaking.
12.| Pipette 50 µL of stop solution (M) into each well (including blank), cover the frame and shake for approximately 5 seconds on an ELISA plate shaker. Ensure substrate incubations are the same for each well.
13.| Within 20 minutes, read the absorbance of each well at 450 nm and 405 nm using an ELISA plate reader, blanked against the well containing 100 µL of TMB (L) and 50 µL stop solution (M) only.

RESULT ANALYSIS

Calculation of results without calibrators

• Index Calculation
• The index values are calculated as follows:
• Index = test sample absorbance at 450nm x 100
  • reference preparation absorbance at 450nm
• The index value can also be calculated using absorbance data at 405 nm.
• TYPICAL RESULTS (Example only; not to be used for calculation of actual results)

ASSAY CUT OFF

Calculation of results with calibrators (optional)

• A calibration curve can be established by plotting calibrator concentration on the x-axis (log scale) against the absorbance of the calibrators on the y-axis (linear scale).
• The 21-OH Ab concentrations in patients’ sera can then be read off the calibration curve [plotted at DRG as a spline log/lin curve (smoothing factor = 0)]. Other data reduction systems can be used.
• The negative control (B) can be assigned a value of 0.03 U/mL to assist in the computer processing of assay results.

TYPICAL RESULTS (Example only; not for calculation of actual results)

• For absorbance readings at 450 nm above 3.0, the absorbance reading at 405nm can be converted to 450 nm absorbance values by multiplying by the appropriate factor (3.4 in the case of equipment used at DRG).
• Samples with 21-OH Ab concentrations above 100 U/mL can be diluted (e.g. 10 x and/or 100 x) in 21-OH Ab negative serum. Some sera will not dilute in a linear way.

ASSAY CUT OFF

• This cut off and the cut off based on index value (see above) has been validated at DRG. However, each laboratory should establish its own normal and pathological reference ranges for 21-OH Ab levels.
• Also, it is recommended that each laboratory include its own panel of control samples in the assay.

CLINICAL EVALUATION

Clinical Specificity

• Sera from 928 healthy blood donors were tested in the 21-OH Ab ELISA kit. 922 (99.4%) sera were identified as being negative for 21-OH Ab.
• The remaining 6 (0.6%) of healthy blood donor sera (0.59, 0.93, 1.2, 2.4, >100 and >100 U/mL) were all found to contain IgM antibodies to 21-OH.

Clinical Sensitivity

• Sera from 100 patients diagnosed with autoimmune Addison’s disease were tested in the 21-OH Ab ELISA kit. 86 (86%) were identified as being positive for 21-OH Ab.

Lower Detection Limit

• The negative control was assayed 20 times and the mean and standard deviation were calculated.
• The lower detection limit at +2 standard deviations was 0.13 U/mL, the index value was 12.

Intra Assay Precision

Sample| Mean U/mL (n=25)| CV (%)| Mean index (n=25)| CV (%)
---|---|---|---|---
1| 0.30| 2.7| 39| 2.2
2| 0.89| 6.1| 92| 4.8
3| 2.0| 6.3| 154| 3.5
4| 5.4| 18.1| 249| 7.3
5| 55| 9.9| 512| 2.3

Inter Assay Precision

Sample| Mean U/mL (n=20)| CV (%)| Mean index (n=20)| CV (%)
---|---|---|---|---
A| 0.39| 4.1| 43| 4.9
B| 1.0| 7.4| 102| 4.6
C| 2.7| 17.9| 164| 8.9
D| 10.7| 11.5| 284| 6.2
E| 58.7| 14.0| 500| 8.4

Clinical Accuracy

• Analysis of 185 sera from patients with autoimmune diseases other than Addison’s disease indicated no interference from autoantibodies to thyroglobulin, thyroid peroxidase, TSH receptor, glutamic acid decarboxylase, zinc transporter 8, aquaporin-4, voltage-gated potassium channel, double-stranded DNA, acetylcholine receptor or from rheumatoid factor. A serum sample from a further patient with Type 1 DM (GADAb positive) gave an index value of 409 and a concentration of 44 U/mL.
• This sample was assayed in a 21-OH Ab RIA kit (RIA-4244) and was positive with a 21-OH Ab concentration of 100 U/mL. A serum sample from a further patient with Type 1 DM (ZnT8 Ab positive) gave an index value of 60 and a concentration of 0.53 U/mL. This sample was assayed in the 21 OH Ab RIA and was negative. A further sample that was AChRAb positive gave an index value of 68 and a concentration of 0.61 U/mL.

Interference

No interference was observed when samples were spiked with the following materials; haemoglobin at 500 mg/dL, bilirubin at 20 mg/dL, or Intralipid up to 3000 mg/dL.

SAFETY CONSIDERATIONS

Streptavidin Peroxidase (SA-POD) and Reaction Enhancer

• Signal word: Warning
• Hazard statement(s)
• H317: May cause an allergic skin reaction
• Precautionary statement(s)
• P261: Avoid breathing mist, vapors
• P272: Contaminated work clothing should not be allowed out of the workplace
• P280: Wear protective gloves/protective clothing/ eye protection/face protection
• P302 + P352: IF ON SKIN: Wash with plenty of soap and water
• P333 + P313: If skin irritation or rash occurs: Get medical advice/attention
• P362 + P364: Take off contaminated clothing and wash it before reuse
• P501: Dispose of contents/container to hazardous or special waste collection point, in accordance with local, regional, national and/or international regulation

Peroxidase Substrate (TMB)

• Signal word: Danger
• Hazard statement(s)
• H360D: May damage the unborn child

Precautionary statement(s)

• P202: Do not handle until all safety precautions have been read and understood
• P280: Wear protective gloves/protective clothing/ eye protection/face protection
• P308 + P313: IF exposed or concerned: Get medical advice/attention
• P501: Dispose of contents/container to hazardous or special waste collection point, in accordance with local, regional, national and/or international regulation
• This kit is intended for use by professional persons only. Follow the instructions carefully. Observe the expiry dates stated on the labels and the specified shelf life for coated wells, diluted and reconstituted reagents.
• Refer to the Safety Data Sheet for more detailed safety information. Avoid all actions likely to lead to ingestion. Avoid contact with skin and clothing.
• Wear protective clothing. Material of human origin used in the preparation of the kit has been tested and found non-reactive for HIV1 and 2 and HCV antibodies and HBsAg but should, nonetheless, be handled as potentially infectious. Wash hands thoroughly if contamination has occurred and before leaving the laboratory. Sterilize all potentially contaminated waste, including test specimens before disposal. Material of animal origin used in the preparation of the kit has been obtained from animals certified as healthy but these materials should be handled as potentially infectious. Some components contain small quantities of sodium azide as a preservative. As with all kit components, avoid ingestion, inhalation, injection and contact with skin, eyes and clothing. Avoid the formation of heavy metal azides in the drainage system by flushing any kit component away with copious amounts of water.

ASSAY PLAN

Day 1| Allow all reagents and samples to reach room temperature (20 °C – 25 °C) before use
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Pipette:| 50 µL negative and positive controls (B and C1- C2), reference preparation (D) or calibrators (if used E1 – E4) and patient sera (except blank)
Pipette:| 50 µL reaction enhancer (F) (except blank)
Mix:| Shake on an ELISA plate shaker at 500 shakes/min for 1 minute
Incubate:| Overnight (16 – 20 hours) at 2 °C – 8 °C without shaking
Day 2| Aspirate/Wash:| ELISA plate (A) three times
Pipette:| 100 µL 21-OH-Biotin (G) reconstituted with room temperature reconstitution buffer (H) into each well (except blank)
Incubate:| 1 hour at room temperature on an ELISA plate shaker at 500 shakes/min
Aspirate/Wash:| ELISA plate (A) three times
Pipette:| 100 µL SA-POD (J) (diluted 1:20) into each well (except blank)
Incubate:| 20 minutes at room temperature on an ELISA plate shaker at 500 shakes/min
Aspirate/Wash:| ELISA plate (A) three times
Pipette:| 100 µL TMB (L) into each well (including blank)
Incubate:| 20 minutes at room temperature in the dark (without shaking)
Pipette:| 50 µL stop solution (M) into each well (including blank) and shake for 5 seconds
Read absorbance at 450 nm and 405 nm within 20 minutes of adding stop solution

SYMBOLS USED

• 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

Documents / Resources

| DRG EIA-5955 21 Hydroxylase Autoantibody ELISA [pdf] Instructions
EIA-5955 21 Hydroxylase Autoantibody ELISA, EIA-5955, 21 Hydroxylase Autoantibody ELISA, Hydroxylase Autoantibody ELISA, Autoantibody ELISA
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References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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