Jo-1 Ab ELISA Kit

Sample

Serum or plasma (EDTA, citrate or heparin)

Species Reactivity

Human

Detection Method

Indirect-ELISA

Intended Use

Jo-1 IgG ELISA is an enzyme-linked immunosorbent assay (ELISA) intended for the quantitative or qualitative determination of IgG class antibodies directed against Jo-1 (Histidyl-tRNA synthetase) in human serum or plasma samples. For research use only.

Contents of Kit

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 Jo-1 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.
Analytical specificity
The test allows the specific determination of human IgG antibodies directed against Jo-1. It has been validated (among other parameters) by means of the commercially available human reference sera of the "Center of Disease Control" (CDC, Atlanta, USA). The following results are typical:

Interference with anticoagulants (EDTA, Citrat, Heparin) in samples has been tested and no interference effects have been observed.
Detection limit (analytical sensitivity)
The detection limit is defined as that concentration of analyte that corresponds to the mean absorbance of sample buffer plus 3-fold standard deviation (s). It was determined as < 0.2 U Jo-1 IgG per mL ssample (n = 24). Recommended measuring range: 0.5 - 60 U/mL
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.


Linearity
In order to assess the dose-response relationship of the test, positive sera were measured in serial 2-fold dilution. Acceptance criterion: linear regression of 4 successive dilutions must yield a correlation factor > 0.98. A typical result is depicted below.

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)

Frequency distribution of Jo-1 IgG
This was analysed in a sera collective of blood donors, equally distributed by sex and age, and a collective of sera found positive for Jo-1 autoantibodies according to a CE-compliant reference ELISA or that were clinically defined. The following distribution of the analyte was observed:

ROC-analysis of these data was used to determine the cut-off as 4.0 U/mL (10). The data presented here suggest a diagnostic specificity and sensitivity of the ELISA of about 99 and 100 %, respectively. These values apply for the measured sera only; other collectives may yield different results. In view of the low number of positive sera, particular caution is required when interpreting test sensitivity.

General Description

Polymyositis (PM) and dermatomyositis (DM) are inflammatory, autoimmune mediated disorders of the connective tissue with unknown etiology. They primarily damage skin and/or muscles, but may also affect other organs as e. g. the lung. Without treatment (standard therapy is immune suppression), they tend to develop into a life-threatening state. Since immunosuppressive agents are known to cause considerable side effects, early diagnosis of PM/DM is essential, in order to keep their dosage as low as possible.
A striking feature of PM/DM is the occurrence of antibodies to aminoacyl-tRNA synthetases; functionally related but immunologically distinct enzymes. About 30 % of all myositis patients carry antibodies against histidyl-tRNA synthetase (EC 6.1.1.21), a 100 kDa dimeric antigen, located in the cytoplasma and known as Jo-1 (derived from a patient's name). Jo-1 Ab positive sera represent about 75 % of all sera with anti-synthetase autoantibodies. With few exceptions, these antibodies tend to be mutually exclusive.
The term Jo-1 (or anti-synthetase) syndrome comprises the symptoms of interstitial pulmonary disease with fibrosing alveolitis. For these symptoms, Jo-1 antibodies possess a high predictive value (80 %). In most cases, this condition is clinically significant and may fatally end in adult respiratory distress syndrome (ARDS).
The present enzyme-linked immunosorbent assay (ELISA) is intended for the quantitative or qualitative determination of IgG antibodies directed against Jo-1 in human serum or plasma. The antigen used is a highly purified preparation of human histidyl-tRNA synthetase, expressed by baculovirus infected insect cells. 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.