SS-A/Ro-Ab ELISA Kit

Sample

serum or plasma (EDTA, citrate, heparin).

Species Reactivity

Human

Detection Method

dELISA

Intended Use

SS-A / Ro 60 IgG ELISA is an enzyme-linked immunosorbent assay (ELISA) intended for the quantitative or qualitative determination of IgG class antibodies directed against SS-A / Ro 60 in human serum or plasma samples.
This product is intended for research use only.

Contents of Kit

1. MTP, 1 Microtiter Plate, coated with the SS-A/Ro antigen described above and hermetically packed in a foil laminate pouch together with a desiccant bag. The plate consists of 12 strips, each of which can be broken into 8 individual wells.
2. ENZCONJ, IgG Enzyme Conjugate, 14 mL, ready-to-use, red coloured. Buffered solution containing stabilising protein, methylisothiazolone and bromonitrodioxane.
3. CAL, A-F Calibrator A-F, 2 mL each, 0, 0.70, 2.0, 7.0, 20 and 70 U SS-A/Ro IgG / mL, ready-touse, gradually blue coloured. Contain TBS, BSA, Tween and Na-azide.
4. CONTROL + & CONTROL -, Positive Control and Negative Control, 2.0 mL each, ready-to-use, green and red coloured, respectively. Contain TBS, BSA, Tween and Na-azide.
5. SAMPLEDIL, Sample Diluent, 100 mL, ready-to-use, orange coloured. Contains Tris-buffered saline (TBS), bovine serum albumin (BSA), Tween and Na-azide.
6. TMB SUBS, TMB Substrate Solution, 14 mL, ready-to-use, colourless. Contains a buffered solution of TMB and H₂O₂. Contained in a vial impermeable to light.
7. WASHBUF CONC, Wash Buffer, 100 mL, 10×-concentrate, blue coloured. Contains TBS, Tween and bromonitrodioxane.
8. STOP TMB, Stop Solution (0.2 M H₂SO₄), 14 mL, colourless, ready-to-use. Caution: sulfuric acid is corrosive.
9. Instructions for Use
10. Lot-specific certificate of analysis

Storage

Store kit at 2 - 8°C, do not freeze. It is stable up to the expiry date stated on the label of the box. Do not use kit beyond its expiry date.

Performance Characteristics

Standardisation:
The test is standardised with a purified serum preparation containing IgG antibodies specifically directed at the SS-A / Ro 60 kDa antigen. This preparation is calibrated against a set of gradually positive sera, solely reserved for this purpose. The degree of sample reactivity is measured in arbitrary units (U/mL) since no international standard is available.
Homogeneity of the solid phase:
Measurement of the solid phase homogeneity is a regular QC part of each production lot. This is determined by 288-fold measurement of a positive but non-saturating sample on 3 selected plates. Acceptance criterion: mOD-coefficient of variation (cv) over the plates < 8%. The figure below shows a representative excerpt of such an analysis.

Precision

For the assessment of the test precision, the variability of results under the following conditions was determined: a. within 1 assay and between 3 assays, b. between 3 operators and c. between 2 kit lots.
a. Intra- and inter-assay variability (n = 24 and 72, respectively)

b. Operator to operator variability (n = 12)

c. Variability between 2 kit lots (n = 6)

Detection Range

The detection limit is defined as that concentration of analyte that corresponds to the mean absorbance of Sample Diluent plus 3-fold standard deviation (s). It was determined as < 0,2 U SS-A / Ro IgG per mL sample (n = 24). Recommended measuring range: 0,5 - 50 U / mL.

General Description

Sjögren's syndrome (SS) is a chronic inflammatory, autoimmune disorder of the exocrine glands. Especially the lacrimal and salivary glands are affected: lymphocytic infiltration causes swelling and progressive loss of function, often accompanied by development of the sicca syndrome. Systemic lupus erythematosus (SLE) is another autoimmune-mediated, chronic inflammatory illness, with variable clinical presentation, ranging from localised skin lesions to a destructive systemic disorder without cutaneous changes.
Autoantibodies directed against the subcellular ribonucleoprotein (RNP) particles SS-A / Ro (soluble substance A / patient Robair-antigen) and SS-B / La (soluble substance B / patient Lane-antigen) are regarded as specific serological marker for SS (prevalence about 65 and up to 90 %, respectively); as well as for SLE (prevalence 5 - 50 %), in particular and with higher prevalence for some of its variants, e.g.subacute cutaneous LE, neonatal LE, ANA-negative SLE.
The SS-A / Ro antigen consists of two distinct proteins, 52 and 60 kDa, complexed with one small, uridinerich, specific RNA chain, either hY1, hY2, hY3 or hY5. The physiological role of this complex is only in part resolved, as is the question of its primary location, nucleus or cytoplasma. The majority of SS-A /Ro-positive sera contains antibodies directed at both Ro60 and Ro52; nevertheless, the Ro60 protein is regarded as the most significant autoantigen. Ro60 positive and simultaneously Ro52 negative sera mostly indicate SLE.
The present ELISA is intended for the quantitative or qualitative determination of IgG antibodies directed against Ro-60 (isolated from bovine thymus) in human serum or plasma. Ro 52 is excluded from the immobilised antigen preparation since some Jo-1 positive sera (primarily indicative for myositis) also cause positive signals on this substrate. The test is fast (incubation time 30 / 30 / 30 minutes) and flexible (divisible solid phase, ready-to-use reagents). Six calibrators allow quantitative measurements; a negative and a positive control check the assay performance.

Standard Curve