HAV IgM, Page 1

Atlas Link

MICROWELL ELISA

Anti-HAV IgM

An Enzyme-Linked Immunosorbent Assay

for the Detection of Anti-Hepatitis A IgM

Catalog No. 1517

(96 tests)

INTENDED USE

The ATLAS LINK Anti-HAV IgM EIA is a qualitative enzyme immunoassay for the detection of anti-HAV IgM in human serum or plasma.

SUMMARY AND PRINCIPLE OF THE TEST

Viral hepatitis is a systematic disease primarily involving the liver. Most cases of acute viral hepatitis in children and adults are caused by one of the following: Hepatitis A virus (HAV), the etiologic agent of viral hepatitis type A; hepatitis B virus and more recently recognized hepatitis C, D etc. Outbreak of type A hepatitis are common in families and institutions, summer camps and especially among armed forces troops. The most likely mode of transmission under these conditions is be the fecal-oral route through close personal contact.

Anti-HAV appears in the IgM fraction during the acute phase, peaking about 3 weeks after elevation of liver enzymes. During convalescence, anti-HAV is the IgG fraction, where it persists for decades.

The Anti-HAV IgM EIA is a solid-phase, two-step sandwich immunoassay. Microtiter wells are coated with purified inactivated HAV antigens. A serum specimen is added to the antibody coated microtiter wells. Anti-HAV IgM, if present, will bind onto antigen coated microwells. The plate is then washed to remove unbound materials. Horseradish peroxidase conjugated anti human will be added to microwells as tracer and bound to human anti-HAV IgM caught on the microwells. Finally, a solution of substrate is added to the wells and incubated. A blue color will develop in proportion to the amount of anti-HAV IgM present in the specimen. The enzyme-substrate reaction can be stopped and the result is visualized or read by EIA plate reader for absorbence at wavelength of 450 nm.

MATERIAL AND EQUIPMENT REQUIRED BUT NOT INCLUDED IN KIT

1.Microwell holder (12 wells, 48 wells or 96 wells)

2.Humidified box.

3.37C incubator.

4.Microtiter plate washer or wash bottles with a small opening

  1. EIA reader
  2. Test tube for sample dilution.

REAGENTS PROVIDED IN THIS KIT

1.MICROTITER WELLS: 96 in a sealed bag.

2.NEGATIVE CONTROL: One vial of 1.2 ml of anti-HAV IgM negative control.

3.POSITIVE CONTROL: One vial of 1.2 ml containing anti-HAV IgM.

4.BLOCKING SOLUTION: One vial of 12 ml.

5.ENZYME CONJUGATE: One vial of 6 ml.

6.WASH BUFFER CONCENTRATE (100x): One bottle of 7.5 ml for 96 tests. The buffer should be diluted 1:100 with distilled water before use.

7.SUBSTRATE SOLUTION A: One bottle of 6 ml.

  1. SUBSTRATE SOLUTION B: One botttle of 6 ml.
  2. STOP SOLUTION: One bottle of 6 ml.

STORAGE

1.All kit components are stored at 2 to 10c

PRECAUTIONS FOR USERS

1.Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.

2.Wear gloves during the whole procedure and avoid reagents or specimens spilling-out.

3.Wipe up the spills using 5% hypochloride solution.

4.Decontaminate all liquids or solid wastes before disposing.

5.Do not mix components from kits with different lot numbers.

6.This kit is for in vitro diagnostic use only.

SPECIMEN COLLECTION

Fresh human serum are collected for the assay. All specimens should be stored at 2-8C if not assayed immediately, or frozen if samples are not assayed within 3 days. Do not use plasma as sample for testing.

ASSAY PROCEDURE

  1. Allow all reagents to reach room temperature before use (25-30C). Break off number of wells needed and place in the well holder.
  2. Predilute the Wash Solution Concentrate 100 fold with distilled water (7.5 + 742.5 ml of water). Diluted WASH SOLUTION can be stored at room temperature for 1 month or 18 months if refrigerated.
  3. Dilute the serum samples to be tested with diluted Wash buffer 1: 10 in a glass or plastic test tube (100l of sample + 900l of diluted Wash buffer)
  4. Dispense 2 drops (100 ul) of Blocking solution (MS3-BS) into each well to be tested.
  5. Add 10 ul of pre-diluted sample into each respective well with Blocking solution, tap the plate 30 seconds to mix it well.
  6. Dispense 2 drops (100 ul) of Positive control and 2 drops (100 ul) of Negative Control into respective wells without Blocking solution.
  7. Place the microtiter plates into a humidified box and incubate at 37C for 60 minutes.
  8. Wash each well 6 times by filling each well with diluted was buffer, then inverting the plate vigorously to get all waster out and blocking the rim of wells on absorbent paper for a few seconds (Tip: let each wells filled with wash solution and soaked for 1-2 minutes in the last washing.)
  9. Then add 1 drop (50l) of Enzyme Conjugate into each well. Mix it gently by swirling the microtiter plate on a flat bench for 1 minute.
  10. Incubate at 37C for 30 minutes and repeat steps # 8.
  11. Add 1 drop (50l) of Substrate Solution A into each well, followed by adding 1 drop (50l) of Substrate Solution B. Gently mix and incubate at room temperature for 10 minutes.
  12. Add 1 drop (50l) of stop solution to each well to stop the color reaction. Read O.D. at 450 nm with a EIA reader.

INTERPRETATION OF RESULTS

Positive:

  • OD value of sample

= 3.0 or > 3.0

OD value of negative

  • Positive result suggests detection of anti-HAV IgM in serum.

Equivocal:

  • OD value of sample

=2.5-2.9

OD value of negative

  • Any sample with equivocal should be re-tested again in 7-10 days with fresh blood withdrawn.

Negative:

  • OD value of sample

<2.5

OD value of negative

  • Negative result suggests no detectable level of anti HAV IgM in serum using current assay.

LIMITATIONS OF THE ASSAY

1.Anti-HAV IgM EIA is limited to the detection and semi-quantitation of anti-HAV IgM in serum.

2.As in other sensitive immunoassays, there is the possibility that non-repeatable reaction may occur due to inadequate washing. Therefore, aspirate the well or eliminate the entire content of wells completely before adding the washing solution.

  1. As with all diagnostic tests, a definitive clinical diagnosis should not be made only based on the results of a single test. A complete evaluation by physician is needed for a final diagnosis.

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Atlas Link, 12720 Dogwood Hills Lane, Fairfax, VA 22033 USA

Phone: (703) 266-5667, FAX: (703) 266-5664