ElisaRSR 3 Screen ICA 3 Screen Islet Cell Autoantibody Elisa Kit Instruction Manual

ElisaRSR 3 Screen ICA 3 Screen Islet Cell Autoantibody Elisa Kit
Instruction Manual

INTENDED USE

The RSR 3 Screen Islet Cell Autoantibody (3 Screen) ELISA kit is intended for use by professional persons only, for the quantitative determination of GAD, IA-2, and ZnT8
autoantibodies (Ab) in human serum. Ab to pancreatic beta cell antigens is important serological markers of type 1 diabetes mellitus (type 1 DM). The antigens recognized by these antibodies include insulin, glutamic acid decarboxylase (GAD65 kDa isoform), the islet cell antigen named IA-2 or ICA-512 and zinc transporter 8 (ZnT8). RSR’s 3 Screen  ELISA allows simultaneous measurement of GAD, IA-2, and ZnT8 Ab in the same sample.

REFERENCES

M. Amoroso et al “3 Screen islet cell autoantibody ELISA: A sensitive and specific ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8.”
Clin. Chim. Acta. 2016 462:60 – 64 A. G. Ziegler et al “3 Screen ELISA for high-throughput detection of beta cell autoantibodies in capillary blood.” Diabetes Technol. Ther.  2016 18:687 – 693

PATENTS

The following patents apply:

European patents EP 1 448 993 B1, EP 1 563 071 B1 and EP 2 118 309 B1, Chinese patents ZL 02822274.1, CN 1738900 B and ZL 200780051859.3, Indian patents 226484  and 279741, Japanese patents 5711449, 4498144 and 5694668 and US patents US 8,129,132 B2, US 9,435,797 B2, US 7,851,164 B2, US 9,023,984 B2 and US 10,481,156 B2.

ASSAY PRINCIPLE

In RSR’s 3 Screen ELISA, GAD, IA-2, and ZnT8 Ab in patients’ sera, reference preparation or calibrators (optional) and controls are allowed to interact with GAD65, IA-2 and  ZnT8 coated onto ELISA plate wells. After a 16 – 20 hour incubation, the samples are discarded leaving any GAD, IA-2, and/or ZnT8 Ab in the patient sera, reference
preparation or calibrators (optional) and controls bound to the GAD65, IA-2, and ZnT8 coated wells.
A mixture of GAD65-Biotin, IA-2-Biotin, and ZnT8- Biotin is then added and during a 2 nd incubation step where, through the ability of GAD, IA-2, and ZnT8 Ab to act divalently, a bridge is formed between the GAD65, IA-2 or ZnT8 immobilized on the plate and GAD65-Biotin, IA-2-Biotin and ZnT8Biotin respectively. Unbound  GAD65/IA-2/ZnT8Biotin is then removed in a wash step and the amount of bound GAD65/IA-2/ZnT8-Biotin is determined (in a 3 rd incubation step) by 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’- tetramethyl-benzidine (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 yellow. The absorbance  of the yellow reaction mixture at 450nm and 405nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of GAD, IA-2, and/or  ZnT8 Ab in the test sample. Reading at 405nm allows quantitation of high absorbances. It is recommended that low absorbance values are measured at 450nm. If it is possible  to read at only one wavelength 405nm 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 o C. 50 µL is sufficient for one assay (RSR recommends duplicate  25 µL determinations). Repeated freeze thawing or increases in storage temperature should be avoided. Do not use lipemic or haemolysed serum samples. Do not use plasma in the assay. When required, bring test sera to room temperature and mix gently to ensure homogeneity. Centrifuge sera 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.

SYMBOLS

MATERIALS REQUIRED AND NOT SUPPLIED

Pipettes capable of dispensing 25µL and 100µL.
Means of measuring out various volumes to reconstitute or dilute reagents.
Pure water.
ELISA Plate reader is 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 kits and all components (A-M) at 2–8ºC.

A| 3 Screen 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- 25°C) for at least 30 minutes before opening.
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Ensure wells are firmly fitted into the frame provided. After opening return any unused wells to the original foil bag with the desiccant provided and seal with
adhesive tape. Place foil bag in the self-seal plastic bag and store at 2-8°C for up to 3 months.
B| Negative Control
0.3 mL Ready for use
Cl| GADAb Positive Control
0.3 mL Ready for use
C2| IA-2 Ab Positive Control 0.3 mL
Ready for use
C3| ZnT8 Ab Positive Control 0.3 mL
Ready for use
D| Reference Preparation
0.3 mL Ready for use
E1-5| Calibrators (optional)
5, 15, 100, 400 and 2000 u/mL (units are arbitrary RSR units)
5 x 0.3 mL
Ready for use
F| Concentrated Wash Solution
125 mL Concentrated
Dilute 10 X with pure water before use. Store at 2-8°C up to kit expiry.
G| 3 Screen-Biotin 3 vials
Lyophilised
---|---
Immediately before use reconstitute each vial with 5.5 mL of reconstitution buffer for 3 Screen-Biotin (H). When more than one vial is used, pool and mix gently before use.
H| Reconstitution Buffer for 3 Screen-Biotin
2 x 15 rnL Coloured red
Ready for use
J| Streptavidin Peroxidase (SA-POD) 0.7 mL
Concentrated
Dilute 1 in 20 with diluent for SA-POD (K). For example, 0.5mL (J) + 9.5mL (K). Store at 2-8°C for up to 28 weeks 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

ASSAY PROCEDURE

Allow all reagents and test sera to stand at room temperature (20-25ºC) for at least 30 minutes before use except for 3 Screen-Biotin and reconstitution buffer for 3 Screen-Biotin. A repeating Eppendorf-type pipette is recommended for steps 4, 7, 10, and 11.

Day 1| 1.| Pipette 25 IL of negative control (B), positive controls (C1-3), reference preparation (D) or (if used) calibrators (E1-5), and patients’ sera into respective wells (A), (in duplicate is
recommended), leaving one well empty for blank (see step 12).
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2.| Cover the frame and shake for approximately 5 seconds on an ELISA plate shaker (500 shakes per min) and incubate at 2-8°C (without shaking) overnight (16-20 hours)
Day 2| 3.
.| Use an ELISA plate washer to aspirate and wash the plate 3 times with diluted wash solution (F). If a plate washer is not available, discard the good contents by briskly inverting the frame of wells over a suitable receptacle, wash the wells 3 times manually and finally tap the inverted wells gently on a clean dry absorbent surface.
4.| Pipette 100 gl. of cold reconstituted 3 Screen-Biotin (G) into each well (except blank). Avoid splashing the material out of the wells during addition.
5.| Cover the frame, and incubate at 2-8°C for 1 hour (without shaking).
6.| Repeat wash step 3.
7.| Pipette 100 gL of diluted SA-POD (J) into each well (except blank).
8| Cover the frame and incubate at room temperature (20-25o C) for 20 minutes on an ELISA plate shaker (500  shakes per min).
3| Repeat wash step 3. If manual washing is being carried out using one additional wash step with pure water (to remove any foam) before finally tapping the inverted wells dry.
10| Pipette 100 mL of TMB (L) into each well (including blank) and incubate in the dark at room temperature  (20-25oC) for 20 minutes without shaking.
11| Pipette 100 mL stop solution (M) into each well (including blank) cover the frame and shake for approximately 5 seconds on a plate shaker (500 shakes per min).  Ensure substrate incubations
are the same for each well.
12| Within 10 minutes, read the absorbance of each well at 405nm and then 450 nm using an ELISA plate reader,  blanked against a well containing 100 mL of TMB (L) and 100 mL stop solution (M) only.

RESULT ANALYSIS

Calculation of results without calibrators

Index Calculation
The index values are calculated as follows:

The index value can also be calculated using absorbance data at 405nm 97% of 1200 healthy adult male blood donor sera gave index values of less than 30 suggesting that index values of 30 or more could be considered positive in this group (see page 4).

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

| A450
nm| Index
value| A405
nm| Index
value
---|---|---|---|---
Reference
Preparation (D)| 0.702| 100| 0.222| 100
Negative
Control (B)| 0.030| 4.3| 0.009| 4.1
Positive
Control (C1)| 1.300| 185| 0.412| 186
Positive
Control (C2)| 0.387| 55| 0.123| 55
Positive
Control (C3)| 0.181| 26| 0.057| 26

ASSAY INDEX VALUE CUT OFF

Calculation of results with calibrators 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 GAD, IA-2, and/or ZnT8 Ab concentrations in patients’ sera can then be read off the calibration curve [plotted at RSR as a spline log/lin curve (smoothing factor = 0)]. Other data reduction methods can be used. The negative control (B) has a  concentration of 0 u/mL, but can be assigned a value of 0.5 u/mL to facilitate computer processing of data.

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

| A450
nm| Conc.
u/mL| A405
nm| Conc.
u/mL
---|---|---|---|---
Negative
Control (B)| 0.030| 0| 0.009| 0
El| 0.094| 5| 0.030| 5
E2| 0.167| 15| 0.052| 15
E3| 0.702| 100| 0.222| 100
E4| 2.233| 400| 0.711| 400
E5| 6.637*| 2000| 2.| 2000
Positive
Control (C1)| 1.300| 213| 0.412| 206
Positive
Control (C2)| 0.387| 48| 0.123| 51
Positive
Control (C3)| 0.181| 17| 0.057| 17

*derived from 405nm value
For absorbance readings at 450nm above 3.0, the absorbance reading at 405nm can be converted to 450nm absorbance values by multiplying by the appropriate factor (3.4 in the case of equipment used at RSR).

Samples with high GAD, IA-2, and/or ZnT8 Ab concentrations can be diluted in the kit negative control (B). 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.

ASSAY CONCENTRATION CUT OFF

This cut-off and the cut-off based on index value has been validated at RSR. However, each laboratory should establish its own normal and pathological reference ranges for 3 Screen. Also, it is recommended that each laboratory include its own panel of control samples in the assay.

CLINICAL EVALUATION

Clinical Specificity and Sensitivity
In an analysis of 1200 healthy adult male blood donors sera, 1166 (97%) gave index values of less than 30. An index of 30 was equivalent to 20 u/mL. Out of the 34 sera giving index values of 30 or greater, 33 (97%) were positive in individual assays for GADAb and/or IA-2 Ab and/or ZnT8 Ab.
Analysis of sera from 147 patients with type 1 DM (mostly with longstanding disease) indicated that 126 (86%) gave 3 Screen index values of 30 or more. There was good agreement between 3 Screen results and individual assays for GADAb and/or IA-2 Ab and/or ZnT8 Ab (concordance 94%).

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 1.3 u/mL, and the index value was 8.3.

Intra Assay Precision

Sample| Mean
u/mL
(n=25)| CV
(%)| Mean
index
(n=25)| CV
(%)
---|---|---|---|---
1| 23| 7.9| 32| 4
2| 25| 4.5| 33| 2.5
3| 38| 5.7| 42| 4.4
4| 145| 4.6| 140| 4.1
5| 405| 4.4| 336| 3.4

Inter Assay Precision

Sample| Mean
u/mL
(n=20)| CV
(%)| Mean
index
(n=20)| CV
(%)
---|---|---|---|---
A| 71| 5.8| 72| 3.2
B| 95| 5.1| 93| 3
C| 121| 4.7| 114| 3.1
D| 192| 4.1| 167| 3.6
E| 260| 4.8| 212| 3.5
F| 489| 3.3| 334| 2.5
G| 1158| 3.3| 553| 3.1

Clinical Accuracy
Out of 108 sera with Graves’ disease, 6 (5.6%) were 3 Screen positive (index ≥ 30). 5 of the 6 were also positive for GADAb and/or IA-2 Ab and/or ZnT8 Ab in individual Ab assays.
In the case of Addison’s disease, 3 out of 10 (30%) of patients were 3 Screen positive (index ≥
30) as were 3 out of 29 (10%) coeliac disease sera and 1 out of 20 (5%) sera from patients with rheumatoid arthritis. All 3 Screen positive sera in these 3 patient groups were also positive for GADAb and/or IA-2 Ab and/or ZnT8 Ab in individual Ab assays.

Interference
No interference was observed when samples were spiked with the following materials: hemoglobin at 500 mg/dL, bilirubin at 20 mg/dL, Intralipid up to 3000 mg/dL or  Biotin at 14 µg/mL.

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 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 (except 3 Screen-Biotin and reconstitution buffer for 3 Screen-Biotin) and test sera to reach room temperature (20-25°C) before use

Day 1| Pipette:| 25 4 negative and positive controls (B and C1-3), reference preparation (D) or calibrators (if used E1-5) and test sera into ELISA plate (A) (except blank)
Mix:| Shake for 5 seconds at 500 shakes/min
Incubate:| Overnight (16-20) hours at 2-8°C (without shaking)
Day 2| Aspirate/Decant:| ELISA plate (A)
Wash:| ELISA plate (A) three times (dry on absorbent material for manual wash (F))
Pipette:| 100 AL cold 3 Screen-Biotin (G) (reconstituted with (H)) into each well (A) (except blank)
Incubate:| 1 hour at 2-8°C (without shaking)
Aspirate/Decant:| ELISA plate (A)
Wash:| ELISA plate (A) three times (dry on absorbent material for manual wash (F))
Pipette:| 100 JAL SA-POD (J) (diluted 1:20) into each well (except blank)
Incubate:| 20 minutes at room temperature with shaking at 500 shakes/min
Aspirate/Decant:| ELISA plate (A)
Wash:| ELISA plate (A) three times, (additional rinse with pure water and dry on absorbent material for manual wash (F))
Pipette:| 100 AL TMB (L) into each well (A) (including blank)
Incubate:| 20 minutes at room temperature in the dark (without shaking)
Pipette:| 100 ILL stop solution (M) into each well (including blank (A)) and shake for 5 seconds
Read absorbance at 405nm and 450nm within 10 minutes of addition of stop solution

ElisaRSR™ 3 Screen ICA™

3 Screen Islet Cell AutoantibodyELISA 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.

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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