RiaRSR N-VGCC Ab N-Type Voltage-Gated Calcium Channel Autoantibody RIA Kit Instruction Manual

RSR
RiaRSRTmN- VGCC Ab
N-Type Voltage-Gated Calcium
Channel (N-VGCC) Autoantibody RIA
Kit – Instructions for use

FOR RESEARCH USE ONLY

RSR Limited

Parc Ty Glas, Llanishen, Cardiff CF14 5DU United Kingdom
Tel.: +44 29 2068 9299
Fax: +44 29 2075 7770
Email: info@rsrltd.com
Website: www.rsrltd.com

INTENDED USE

The RSR N-type voltage-gated calcium channel (N-VGCC) autoantibody RIA kit is intended for use by professional persons only, for the quantitative determination of N-type VGCC autoantibodies (N-VGCC Ab) in human serum. Serum autoantibodies reactive with N-VGCC were reported initially in dysfunction of the neuromuscular junction specifically associated with Lambert-Eaton Myasthenic Syndrome (LEMS). Subsequently, N-VGCC Abs were detected in disorders of the central nervous system, including cerebellar degeneration, and in paraneoplastic autoimmunity, particularly associated with Small Cell Lung Cancer (SCLC). Functional N-VGCCs consist of a pore-forming al-subunit together with ancillary p, y, and a2/8 subunits and are expressed widely in the central and peripheral nervous systems. N-VGCC Abs primarily bind to the al -subunit. The kit is easy to use and provides a specific and sensitive assay for N-type VGCC Ab and is designed to complement the RiaRSRTMVGCC Ab RIA kit for the detection of autoantibodies against P/Q-type VGCCs.

REFERENCES

M. Motomura et al An improved diagnostic assay for Lambert-Eaton myasthenic syndrome.
J. Neurol. Neurosurg. Psychiatry (1995) 58:85-87
V. A. Lennon et al Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes. N. Engl. J. Med. (1995) 332:1467-1474.

ASSAY PRINCIPLE

In RSR’s N-VGCC Ab RIA, N-VGCC Ab in patient sera and controls are allowed to interact with detergent-solubilized N-type VGCCs extracted from rabbit brain tissue and complexed with 1251-labeled co-conotoxin GVIA. After incubation at 2 — 8°C overnight, the resulting antigen-antibody complexes are immunoprecipitated by the addition of anti-human IgG. After the second incubation of 11/2 hours, assay buffer is added and the samples centrifuged. Unbound 1251-labelled co-conotoxin GVIA is removed from the tubes by aspiration of the supernatant. The level of radioactivity remaining in the tube is proportional to the antibody level in the test sample.

STORAGE AND PREPARATION OF SERUM SAMPLES

Sera to be analyzed should be assayed soon after separation or stored, preferably in aliquots, at 2 -8°C for up to 2 weeks, or at -20°C or below for longer periods. 15 μL is sufficient for one assay. Repeated freeze-thawing or increases in storage temperature must be avoided. Do not use lipemic or haemolysed serum samples. Citrate, EDTA, and heparin plasma may be used in the assay. When required, thaw tests sera at room temperature and mix gently to ensure homogeneity. Dilute 1:10 using assay buffer (e.g. 15 μL serum plus 135 μL assay buffer). Centrifuge diluted serum prior to assay (preferably for 5 min at about 10,000 rpm i.e. about 10,000g in a microfuge) to remove any particulate matter.

SYMBOLS

MATERIALS REQUIRED AND NOT SUPPLIED

4.5 mL Conical plastic tubes Pipettes capable of dispensing 50 pL, 0.75 mL, and 1 mL
Pure water
Vortex mixer
Refrigerated centrifuge capable of 1500g
Gamma counter

PREPARATION OF REAGENTS SUPPLIED FOR 25 TUBE KIT

Store unopened kits and all components at 2 – 8°C.

A| 1251-labeled N-VGCC  1 5kBq/vial (at manufacture)
2 vials
Lyophilised
---|---
Reconstitute each vial by addition of
0.75 mL pure water and vortex gently to dissolve. Use immediately.
B| Negative Control
0.25 mL
Ready for use
C1-2| Positive Controls I & II
2 x 0.25 mL
Ready for use. See vial label for the concentration range
D| Anti-Human IgG
2 mL
Ready for use
E| Assay Buffer
60 mL
Ready for use and keep at 2 – 8°C except when in use.

ASSAY PROCEDURE

Allow all reagents, except assay buffer, to stand at room temperature (20 – 25°C) for at least 30 minutes before use. An Eppendorf-type repeating pipette is recommended for steps 2, 4, 6, and 9.

1.| Pipette 50 μL (in duplicate) of negative control (B), positive controls (C1-2), and diluted patient sera (diluted 1:10 in assay buffer), into labeled assay tubes (the controls are supplied ready diluted).
---|---
2.| Pipette 50 μL of freshly reconstituted 1251-
labeled N-VGCC (A) into each tube and into two additional empty tubes for total counts.
3.| Mix each tube gently on a vortex mixer; cover the tubes with a suitable cover and incubate at 2 – 8°C for 16 – 20 hours.
4.| Pipette 50 μL of anti-human lgG (D) into each tube (excluding the two total count tubes).
5.| Mix each tube gently on a vortex mixer; cover the tubes with a suitable cover and incubate at 2 – 8°C for
1 ½hours.
6.| Pipette 1 mL of cold (2 – 8°C) assay buffer (E) into each tube (excluding the two total count tubes) and mix gently on a vortex mixer.
7.| Centrifuge each tube at 1500g for 20 minutes at 4°C.
8.| Aspirate or decant the supernatant.
9.| Pipette 1 mL of cold (2 – 8°C) assay buffer (E) into each tube (excluding the two total count tubes) and resuspend the pellet gently using a vortex mixer.
10.| Repeat steps 7 and 8.
11.| Count each tube (including total count tubes) for 1 minute using a gamma counter.

RESULT ANALYSIS

The radioactivity in the pellet represents the amount of 125I-labeled w-conotoxin GVIA bound by the N-VGCC Ab. This is often expressed as picomoles of labeled toxin bound per liter of test serum, and the relationship between this parameter and pellet radioactivity can be calculated from the knowledge of (values for K and A are on the QC record sheet):

1. the specific activity (K Ci/mmol) of the 125I-labeled toxin at the time it was labeled;
2. the decay of 1251 in the labeled toxin—N-VGCC complex in the period between labeling and the day of the assay, (decay factor A);
3. the volume of neat serum used in the assay (C μL) (C = 5 for 50 μL of the 1:10 diluted sample);
4. the counter efficiency of the gamma counter used (B);

The formula is as follows:-
pmol/L = 1000 x (cpm test sample — cpm negative control) x A/(C x K x B x 2.22)
TYPICAL RESULTS (example only; not for use in calculation of actual results)

| cpm| pmol/L
---|---|---
Negative Control| 1461| 0
Positive Control I| 10064| 652
Positive Control II| 3915| 186

ASSAY CUT OFF

This cut-off has been validated at RSR. However, each laboratory should establish its own normal and pathological reference ranges for N-VGCC 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 59 individual healthy blood donors were assayed in the N-VGCC Ab RIA. 58 (98.3%) were identified as being negative for N-VGCC Ab.
Clinical Sensitivity
Serum samples from 20 patients positive for P/Q-type VGCC Ab in the VGCC Ab RIA were assayed in the N-VGCC Ab RIA. 6 (30%) were positive for N-VGCC Ab.

Clinical Accuracy
None of 30 patients with autoimmune diseases other than those with suspected LEMS and related neurological disorders were positive for N-VGCC Ab. This study indicated no interference from autoantibodies to GAD or the TSH receptor in the RSR N-VGCC Ab RIA.

SAFETY CONSIDERATIONS

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 reconstituted reagents. Refer to Safety Data Sheet for more detailed safety information. The kit contains radioactive material. Users should make themselves aware of, and observe, any national and local legislation and codes of practice governing the use, storage, transportation, and disposal of radioactive materials. Avoid all actions likely to lead to ingestion. Avoid contact with skin and clothing. Wear protective clothing and, where appropriate, personal dosimeters. Radioactive materials should only be used by authorized personnel and in designated areas. Wash hands thoroughly after handling. Monitor hands and clothing before leaving the designated area. Materials of human origin used in the preparation of the kit have 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 before leaving the laboratory. Sterilize all potentially contaminated waste, including test specimens, before disposal. Materials of animal origin used in the preparation of the kit have 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, or contact with skin, eyes, or 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 assay buffer, to stand at room temperature (20-25°C) for at least 30 minutes before use

Pipette:| 50 μL negative control ((B) ready to use, do not dilute), positive controls ((C1-2) ready to use, do not dilute) and diluted patient sera (diluted 1:10 in assay buffer)
Pipette:| 50 μL 125I-labeled N-VGCC (A) (freshly reconstituted) into all tubes plus two additional empty tubes for total counts
Tubes:| Mix gently on a vortex mixer and cover
Incubate:| 16 – 20 hours at 2 — 8°C
Pipette:| 50 μL anti-human IgG (D) into all tubes (excluding the two total count tubes)
Tubes:| Mix gently on a vortex mixer and cover
Incubate:| 1½ hours at 2 — 8°C
Pipette:| 1 mL cold assay buffer (E) (excluding the two total count tubes)
Tubes:| Mix gently on a vortex mixer
Tubes:| Centrifuge at 1500g for 20 minutes at 4°C
Tubes:| Aspirate or decant supernatants
Pipette:| 1 mL cold assay buffer (E) (excluding the two total count tubes)
Tubes:| Mix on a vortex mixer to resuspend the pellet
Tubes:| Centrifuge at 1500g for 20 minutes at 4°C
Tubes:| Aspirate or decant supernatants
Count tubes for 1 minute using a gamma counter

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