ElisaRSR 2 Screen ICA 2 Screen Islet Cell Autoantibody ELISA Kit Instruction Manual

Islet Cell Autoantibody ELISA Kit
Instruction Manual

ElisaRSR 2 Screen ICA 2 Screen Islet Cell Autoantibody ELISA Kit

RSR ElisaRSR TM 2 Screen ICA TM 2 Screen Islet Cell Autoantibody ELISA Kit – Instructions for use

RSR Limited
Parc Ty Glas, Llanishen,
Cardiff CF14 5DU United Kingdom
Tel.: +44 29 2068 9299
Fax: +44 29 2075 7770
Email: [email protected]
Website: www.rsrltd.com

Advena Ltd. Tower Business Centre, 2nd Flr
Tower Street, Swatar, BKR 4013 Malta

INTENDED USE

The RSR 2 Screen Islet Cell autoantibody (2 Screen) ELISA kit is intended for use by professional persons only, for the quantitative determination of both GAD and IA-2 autoantibodies in human serum.
Autoantibodies to pancreatic beta-cell antigens are important serological markers of type 1 diabetes mellitus. The antigens recognized by these antibodies include insulin, glutamic acid decarboxylase (GADes kDa isoform), the islet cell antigen named IA-2 or ICA-512, and zinc transporter 8 (ZnT8). RSR’s 2 Screen ELISA allows simultaneous measurement of GAD and IA-2 autoantibodies in the same sample.

REFERENCES

S. Chen et al
Sensitive non-isotopic assays for autoantibodies to IA2 and to a combination of both IA2 and GAD65. Clinica Chimica Acta 2005 357: 74-83
C. Torn et al
Diabetes Antibody Standardization Program: evaluation of assays for autoantibodies to glutamic acid decarboxylase and islet antigen-2. Diabetologia 2008 51:846-852.

PATENTS

The following patents apply: European patent EP 1 448 993 B1, Chinese patent ZL 02822274.1, Indian patent 226484, Japanese patent 5711449 and US patents US 8,129,132 B2, US 9,435,797 B2 and US 10,481,156 B2.

ASSAY PRINCIPLE

In RSR’s 2 Screen ELISA, GAD and IA-2 autoantibodies (Ab) in patient sera, calibrators, and controls are allowed to interact with GAD65 and IA-2 coated onto ELISA plate wells (1″ incubation). The samples are then discarded, leaving any GAD or IA-2 autoantibodies in the patient sera, calibrators, or controls bound to the GAD65 and IA-2 coated wells. A mixture of GAD65-Biotin and IA-2-Biotin is then added and during a second incubation step (through the ability of GAD and IA-2 autoantibodies to act divalently), a bridge is formed between the GAD65 or IA-2 bound to the wells and GAD65-Biotin or IA-
2-Biotin respectively. The amount of GAD65/IA-2- Biotin bound is determined in a third incubation step by the addition of Streptavidin Peroxidase (SA-POD), which binds specifically to Biotin.
Excess unbound SA-POD has then washed away and the addition of 3,3′,5,5′ tetramethylbenzidine (TMB) results in the formation of a blue color. This reaction is stopped by the addition of a stop solution causing the good contents to turn from blue to yellow. The absorbance of the yellow reaction mixture at 450nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of GAD or IA-2 Ab in the test sample. Reading at 405nm allows quantitation of high absorbances.
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. 100pL is sufficient for one assay (duplicate 50pL determinations). Repeated freeze-thawing or increases in storage temperature must be avoided. Do not use lipemic or haemolysed serum samples. Do not use plasma in the assay. When required, thaw tests sera at room temperature and mix gently to ensure homogeneity. Centrifuge serum prior to assay (preferably for 5 min at 10-15,000 rpm in a microfuge) to remove particulate matter. Please do not omit this centrifugation step if sera are cloudy or contain particulates.

SYMBOLS

Symbol

| Meaning
---|---
| EC Declaration of Conformity
| In Vitro Diagnostic Device
****| Catalog Number

| Lot Number
| Consult Instructions
| Manufactured By
| Expiry Date
| Store
| Positive Control
| Negative Control

MATERIALS REQUIRED AND NOT SUPPLIED

Pipettes capable of dispensing 25µL, 50 µL, and 100µL.
Means of measuring out various volumes to reconstitute or dilute reagents supplied.
Pure water
ELISA Plate reader suitable for 96 well formats and capable of measuring at 450nm and 405nm ELISA Plate shaker, capable of 500 shakes/min (not an orbital shaker).
ELISA Plate cover

PREPARATION OF REAGENTS SUPPLIED

Store unopened kit and components at 2 – 8°C

A| GAD 65 and IA-2 Coated Wells
12 break-apart strips of 8 wells (96 in total) in a frame and sealed in a foil bag. Allow standing at room temperature (20 – 25ºC) for at least 30 minutes before opening.
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Ensure strip wells are firmly fitted into the frame provided. After opening return any unused wells to the original foil packet with the desiccant provided and seal with adhesive tape. Place foil bag in the self-seal plastic bag and store at 2-8oC for up to 8 months.
B| Reaction Enhancer 4 mL colored red Ready for use
C1- 6| Calibrators
4, 10, 20, 70, 145 and 450 u/mL (units are NIBSC 97/550) 6 x 0.7 mL Ready for use
D1| GAD Ab Positive Control
0.7 mL Ready for use
D2| IA-2 Ab Positive Control
0.7 mL Ready for use
D3| Negative Control
0.7 mL Ready for use
E| GAD 65/IA-2-Biotin **(GAD 65 Biotin plus IA-2 Biotin) 3 vials lyophilised
Reconstitute each vial with the amount of reconstitution buffer for GAD65/IA-2-Biotin (F) shown on the vial label.  When more than one vial is used,  pool the reconstituted vials and mix gently before use. Use on the day of reconstitution.
F| Reconstitution Buffer for GAD 65/IA-2-Biotin 2 x 15 mL colored blue Ready for use
G| Streptavidin Peroxidase (SA-POD) 1 x 0.7 mL
Concentrated
Dilute 1 in 20 with diluent for SA-POD (H). For example, 0.5mL (G) + 9.5mL (H). Store at 2 – 8oC for up to 18 weeks after dilution.
H| Diluent for SA-POD 15 mL Ready for use
I| Peroxidase Substrate (TMB) 15 mL
Ready for use
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J| Concentrated Wash Solution 125 mL
Concentrated
Dilute 10 X with pure water before use. Store at 2 – 8oC up to kit expiry.
K| Stop Solution** 12 mL
Ready for use

ASSAY PROCEDURE

Allow all reagents to stand at room temperature (20 – 25ºC) for at least 30 minutes before use. A repeating Eppendorf-type pipette is recommended for steps 2, 6, 9, 11, and 12.

Day 1| 1.| Pipette ** of patient sera, calibrators (C1-6), and controls (D1, D2, and D3) into respective wells in duplicate, leaving one well empty for blank (see step 13).
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2.| Pipette ** of reaction enhancer (B) into each well (except blank).
3.| Cover the frame and shake the wells for 5 seconds on an ELISA plate shaker (500 shakes per min).
4.| Incubate the plate at 2 – 8oC (without shaking) overnight (16-20 hours)
Day 2| 5.| After this overnight incubation, aspirate the samples and wash the plate 3 times with wash solution (J) using a plate washer. (If a plate washer is not available, discard the samples by briskly inverting the frame of strip wells over a suitable receptacle, wash the wells 3 times manually and after the final wash invert the frame of wells and tap gently on a clean dry absorbent surface to remove excess wash solution).
6.| Pipette ** of reconstituted GAD65/IA-2-Biotin (E) into each well (except blank). Avoid splashing the material out of the wells during addition.
7.| Cover the plate, and incubate at 18 – 22 oC for 1 hour on an ELISA plate shaker (500 shakes per min).
8.| Repeat wash step 5.
9.| Pipette ** of diluted SA-POD (G) into each well (except blank) and incubate at room temperature for 20 minutes, on an ELISA plate shaker (500 shakes per min).
Day 2 continued| 10.| After the incubation, wash the wells three times with diluted wash solution (J) as in step 5 (in the case of washing manually,  use an additional final wash step with pure water to remove any foam).
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11.| Pipette 100 m L of TMB (I) into each well (including blank) and incubate in the dark at room temperature for 20 minutes without shaking.
12.| Pipette 100 m L stop solution (K) into each well (including blank) and shake the plate for approximately 5 seconds on a plate shaker (500 shakes per min). Ensure substrate incubations are the same for each well.
13.| Within 10 minutes read the absorbance of each well at 405nm and then 450nm using an ELISA plate reader, blanked against a well-containing 100 m L of TMB substrate (I) and 100 m L Stop solution (K) only.

RESULT ANALYSIS

A calibration curve can be established by plottingcali a better concentration on the x-axis (log scale)against the absorbance of the calibrators on the Y-axis (linear scale). The GAD  and/or IA-2 Ab concentrations in patient sera can then be read off the calibration curve [Plotted at RSR as a spline lug/lin curve (smoothing factor = 0)]. Other data reduction methods can be used. The negative control (D3) has a concentration of 0 u/mL, but can be assigned a value of 0.4 u/mL to facilitate computer processing of data. Absorbance readings at 405nm can be converted to 450nm absorbance values by multiplying by the appropriate factor (approximately 3.5, depending on the equipment being used). Values less than 25 u/mL should be read off a 450 nm curve.

Samples with high GADAb and IA-2Ab concentrations can be diluted in the kit negative control (D3). For example, 15 µL of the sample plus 135 µL of negative control to give a  10x dilution. Other dilutions (e.g. 100x) can be prepared from a 10x dilution or otherwise as appropriate. Some sera will not dilute in a linear way.
TYPICAL RESULTS (Example only; not to be used for calculation of actual results)

Index Calculation

If results are to be expressed as an index, only the 4 u/mL calibrators need to be included in the assay (all controls should still be included). The index values are calculated as  follows:

Healthy blood donor sera give index values of less than 1 suggesting that index values of 1 or more can be considered positive for GADAb and/or IA-2 Ab.

ASSAY CUT OFF

| u/mL
---|---
Negative| < 4 u/mL
Positive| ≥ 4.0 u/mL

This cut-off has been validated at RSR. However, each laboratory should establish its own normal and pathological reference ranges for GAD and/or IA-2 Ab levels. Also, it is recommended that each laboratory include its own panel of control samples in the assay.

CLINICAL EVALUATION

Clinical Specificity and sensitivity Sera from 70 healthy blood donors were all negative in the 2 Screen ELISA, although occasional healthy blood donors may have detectable  GAD autoantibodies. Autoantibodies to GAD and/or IA2 were detected in 84% (n=216) of samples from patients with type 1 diabetes of various disease durations. In the  DASP 2005 the study, the RSR 2 Screen ELISA showed 98% (n=100) specificity and 96% (n=50) sensitivity.
Lower Detection Limit
The kit negative control was assayed 20 times and the mean and standard deviation were calculated. The lower detection limit at +2 standard deviations was 0.43 u/mL.

Intra Assay Precision

Inter Assay Precision

Clinical Accuracy

Analysis of sera from patients with autoimmune diseases other than type 1 DM indicated no interference from autoantibodies to the TSH receptor, thyroglobulin, thyroid peroxidase, dsDNA the acetylcholine receptor, or rheumatoid factor.

Interference

No interference was observed when samples were spiked with the following materials; hemoglobin up to 5mg/mL, bilirubin up to 20 mg/dL, or intralipid up to 3000 mg/dL.

SAFETY CONSIDERATIONS

Streptavidin Peroxidase (SA-POD)
Signal word: Warning
Hazard statement(s)
H317: May cause an allergic skin reaction
Precautionary statement(s)
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
Peroxidase Substrate (TMB)
Signal word: Danger
Hazard statement(s)
H360: May damage fertility or the unborn child

Precautionary statement(s)

P280: Wear protective gloves/protective clothing/eye protection/face protection
P308 + P313: IF exposed or concerned: Get medical advice/attention
This kit is intended for in vitro use by professional persons only. Follow the instructions carefully.
Observe expiry dates stated on the labels and the specified shelf life for coated wells, reconstituted reagents, and diluted reagents. Refer to Safety Data Sheet for more detailed safety information. Material of human origin used in the preparation of the kit has been tested and found nonreactive for HIV1 and 2 and HCV antibodies and HBsAg but should, nonetheless, be handled as potentially infectious. Wash hands thoroughly if contamination has occurred 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 the preservative. 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

Allow all reagents and samples to reach room temperature (20 – 25ºC) before use

Pipette:| 50µ L Calibrators, Controls, Patient Sera (except blanks)
Pipette:| 25µ L Reaction Enhancer (except blanks)
Mix:| Shake for 5 seconds at 500 shakes/min
Incubate| Overnight (16-20) hours at 2 – 8oC (without shaking)
Aspirate/Decant:| Plate
Wash:| Plate three times (dry on absorbent material for manual wash)
Pipette:| 100µ L GAD/IA-2 Biotin (reconstituted) into each well (except blanks)
Incubate:| 1 hour at 18 – 22 oC with shaking at 500 shakes/min
Aspirate/Decant:| Plate
Wash:| Plate three times (dry on absorbent material for manual wash)
Pipette:| 100µ L SAPOD (diluted 1:20) into each well (except blanks)
Incubate:| 20 minutes at room temperature with shaking at 500 shakes/min
Aspirate/Decant:| Plate
Wash:| Plate three times, (additional rinse with pure water and dry on absorbent material for manual wash)
Pipette:| 100µ L TMB into each well (including blanks)
Incubate:| 20 minutes at room temperature in the dark (without shaking)
Pipette:| 100µ L stop solution into each well (including blanks) and shake for 5 seconds
Read absorbance at 405nm and 450nm within 10 minutes of stop solution addition.

References

• RSR Limited In Vitro Diagnostics Services- Manufacturer of Medical Diagnostic Devices, Reagents & Kits for the Test and Diagnosis of Autoimmune Diseases
• RSR Limited In Vitro Diagnostics Services- Manufacturer of Medical Diagnostic Devices, Reagents & Kits for the Test and Diagnosis of Autoimmune Diseases

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