PRINCIPLE

A. HBeAg Assay

The HBeAg assay is based on Sandwich Principle (Antibody‧Antigen‧Antibody), the reaction processes are as follows:

• 1. Plate (Anti-HBe) + Specimen (containing HBeAg) →Anti-HBe‧HBeAg
• 2. Anti-HBe‧ HBeAg + Anti-HBe‧HRPO → Anti-HBe‧HBeAg‧Anti-HBe‧HRPO sandwich complex
• 3. Anti-HBe‧HBeAg‧Anti-HBe‧HRPO sandwich complex + TMB Solution (colorless) → light blue to blue color
• 4. Light blue to blue color + 2N H2SO4 → light yellow to yellow color absorbing at 450nm

A. Anti-HBe Assay

The Anti-HBe assay is based on Neutralization Principle. Anti-HBe in specimen is inhibited by incubation with serum containing HBeAg, but not inhibited by incubation with specimen lacking anti-HBe, the specification of the antibody is confirmed. The reaction process are summarized as follows:

1. Plate (Anti-HBe) + Specimen (containing Anti-HBe) + Neutralizing Solution (HBeAg) → Plate（Anti-HBe）and HBeAg‧Anti-HBe

*If the specimen does not contain Anti-HBe, HBeAg in Neutralizing Solution will react with Anti-HBe coated on the plate and continue the following reaction.

2. Plate（Anti-HBe‧HBeAg）+ Anti-HBe‧HRPO → Plate（Anti-HBe‧HBeAg‧Anti-HBe‧HRPO）sandwich complex

3. Plate（Anti-HBe‧HBeAg‧Anti-HBe‧HRPO）sandwich complex+ TMB Solution (colorless) → light blue to blue color

4. Light blue to blue color + 2N H2SO4 → light yellow to yellow color absorbing at 450nm

REAGENTS PROVIDED IN KIT

Item No. 1-8 should be refrigerated at 2 to 8℃ and the others stored at room temperature (20-30℃)

ITEM 96 TESTS

1. Anti-HBe Plate: One microtiter plate coated with antibody to HBeAg（Anti-HBe）. 1 Plate

2. Anti-HBe‧Peroxidase Solution: Containing anti-HBe • Peroxidase (horseradish) conjugate dissolved in protein stabilizers. Preservatives: 0.003% Gentamycin and 0.01% Thimerosal. 1 bottle
10 ml

3. HBeAg Positive Control: Containing HBeAg positive serum diluted in buffer with protein stabilizers. Preservatives: 0.003% Gentamycin and 0.01% Thimerosal. 1 bottle 1 ml

4. HB Negative Control: Containing normal human serum, which is free of HBeAg, Anti-HBe and HBsAg. Preservatives: 0.003% Gentamycin and 0.01% Thimerosal. 1 bottle
1.5 ml

5. Anti-HBe Positive Control: Containing Anti-HBe positive serum dissolved in buffer with protein stabilizers. Preservatives: 0.003% Gentamycin and 0.01% Thimerosal. 1 bottle 1 ml

6. Neutralizing Solution for Anti-HBe: Containing HBeAg positive serum diluted in buffer with protein stabilizers. Preservatives: 0.003% Gentamycin and 0.01% Thimerosal. 1 bottle
5 ml

7. TMB Substrate Solution A: 0.6 mg/ml of 3,3',5,5'-tetramethylbenzidine in an organic base. 1 bottle, 10 ml

8.TMB Substrate Solution B: Citrate Acid Buffer containing 0.03% H2O2. 1 bottle, 10 ml

9. Washing Solution D (20X) Concentrate: Phosphate buffer with Tween-20 1 bottle, 50 ml

10. 2N Sulfuric Acid 1 bottle, 10 ml
ACCESSORIES: (provided as needed)

11. Black Cover.

12. Adhesive Slips.

13. Absorbent Pads.  

OTHER MATERIALS AND DEVICES NEEDED 

1. 100μl and 50μ1 micropipettes and tips.

2. Water bath or incubator with temperature control at 37±1℃.

3. Plate washing equipment.

4. ELISA Reader: Precision ELISA Reader capable for 450nm wavelength reading is necessary.

PRECAUTIONS

1.  This kit is for medical technicians or physicians used only.

2.  This reagent kit is for in vitro diagnosis only.

3.  Bring all kit reagents and samples to room temperature (20-30℃) and mix gently before use.

4.  Do not use kit beyond its expira­tion date.

5.  Do not interchange reagents between different lots.

6. Reagents must be protected from microbial contamination.

7.  HBeAg Positive Control and Neutralizing Solution for Anti-HBe are made of HBeAg positive serum, and are considered the etiological agent, therefore it must be treated as infectious material.

8.  Do not smoke or eat in areas where specimens or reagents are handled.

9.  Do not pipette by mouth.

10. Wear gloves when handling reagents or specimens, and wash hands thoroughly afterwards.

11. Infectious specimens and nonacid containing spills should be wiped up thoroughly with 5% sodium hypochlorite.

12. All waste materials should be properly disinfected before disposal. Both liquid and solid waste should be autoclaved for at least 1 hour at 121℃. Solid waste can also be incinerated. Non-acidic liquid waste can be treated with sodium hypochlorite diluted to a final concentration of 1%. Liquid waste containing acid must be neutralized before similar treatment and should stand for 30 minutes to obtain effective disinfection.

13. TMB substrate solution A contains dimethyl sulfoxide, an irritant to skin and mucous membranes. Avoid contact of TMB substrate solution and sulfuric acid with skin and mucous membrane.

SPECIMEN COLLECTION

AND STORAGE

1. Either serum or plasma can be used with this diagnostic kit. Whole blood specimens should be separated as soon as possible in order to avoid hemolysis. Also, clots must be removed.

2. Specimens must be stored at 2-8℃ and avoided heat-inactivation to minimize deterioration. For long-term storage, they should be frozen below -20℃. Storage in self-defrosting freezer is not recommended.

3. Avoid multiple freeze-thaw procedures.

4. Frozen specimens must be thawed thor­oughly and mixed before test.

5. Do not use heat-inactivated specimen.

REAGENT STORAGE

1. The kit must be stored at 2-8℃. Do not freeze.

2. Strips of the plate should be used within one month after open the original aluminum foil bag. The unused strips should be kept in the aluminum foil bag and tapped the opening tightly.

3. Return the reagents to 2-8℃ im­mediately after use.

4. Washing Solution D (20X) Concentrate should be stored at room temperature to avoid crystallization. If the crystal has been precipitated before use, warm up the solution in a 37℃ water bath till crystal dissolved.

PLATE WASHING

PROCEDURE

NOTE: Dilute Washing Solution D (20X) Concentrate with distilled or deionized water to 1:20 dilution. Do not use tap water.

A. AUTOMATIC OR SEMI-AUTOMATIC PLATE WASHER

Any commercial automatic microplate washer or other liquid aspirating/dispensing devices can be used for washing purpose. The user should test the devices to determine the proper volume of water and wash cycles to insure proper washing.

We suggested 6 wash cycles with at least 350μl per well per wash and soaking for 10 seconds is necessary.

B.       MANUAL PLATE WASH

Cover the reaction plate with an Absorbent Pad. Invert the plate and allow the liquid absorb onto the pad, then return the plate back to upright position.  Fill each well with 350μl of washing buffer.  Aspirate the water after soaking 10 seconds.  Repeat this procedure 6 times.

Blot dry by inverting the plate and tapping firmly onto absorbent paper.  All residual washing buffer should be blotted dry.

WARNING:

Improper washing can cause false results.

TEST PROCEDURE

A. HBeAg ASSAY

1. Bring all reagents and specimens to room temperature (20-30℃) before beginning the assay. Swirl the reagents gently before use.  Adjust a incubator to 37±1℃.

2. Reserve two wells for Blanks. Add 100μ1 of Control (3 Negative Control and 2 HBeAg Positive Control) and 100μ1 of each Specimen into wells, respectively.

NOTE:

Use an individual tip for each sample to avoid cross-contamination.

3. Gently tap the plate.

4. Remove the protective backing of the adhesive slip and press it on the plate, so that it is tightly sealed.

5. Incubate the plate in a 37±1℃incubator or water bath for 1 hour.

6. At the end of the incubation period, remove and discard the adhesive slip and wash the plate by following the "PLATE WASHING PROCEDURE."

7.  Add 100μ1 of Anti-HBe • Peroxidase Solution into each reaction well except 2 blanks.

NOTE: Do not touch the edge of well to avoid false results.

8. Repeat steps 3 and 4.

9. Incubate the plate in a 37±1℃ incubator or water bath for 1 hour.

10. Repeat step 6 to wash the plate.

11. Choice one of the following two meth­ods for color development:

NOTE:

TMB Substrate Solution A should be colorless to light blue, otherwise, should be discarded.  The mixture of TMB Substrate Solution A and B should be used within 30 minutes after mix. The mixture should be avoided from intense light.

A. Mix equal volume of TMB Substrate Solution A and B in a clean container immediately prior to use. Add 100μl of the mixture solution to each well including 2 blank wells.

B. Add 50μl of TMB Substrate Solution A first, then add 50μl of TMB Substrate Solution B into each well including 2 blanks. Mix well gently.

12. Cover the plate with Black Cover and incubate at room temperature for 30 minutes.

13. Stop the reaction by adding 100μl 2N H2SO4 to each well including 2 blanks.

14. Determine absorbance of Controls and test specimens within 15 minutes with a spectrophotome­ter at 450nm or 450/650nm (450nm reading wavelength with 650nm reference wavelength). Use the lighter one of the blank wells to blank spectrophotometer. Use the lighter color of two blank wells to blank spectrophotometer.

NOTE:

The color of the blank should be colorless to pale yellow, otherwise, the test must be repeated.

B. Anti-HBe ASSAY

1. Add 50μ1 of Control (3 Negative Control and 2 Anti-HBe Positive Control) and 50μ1 of each Specimen into wells, respectively. Reserve 2 wells for blanks.

2. Add 50μ1 of Neutralizing Solution for Anti-HBe into each well except 2 blank wells.

NOTE:

Use an individual tip for each sample to avoid cross-contamination.

3. Gently tap the plate.

4. Remove the protective backing of the adhesive slip and press it on the plate, so that it is tightly sealed.

5. Incubate the plate in a 37±1℃incubator or water bath for 1 hour.

6. At the end of the incubation period, remove and discard the adhesive slip and wash the plate by following the "PLATE WASHING PROCEDURE."

7.  Add 100μ1 of Anti-HBe• Peroxidase Solution into each reaction well except 2 blanks.

NOTE: Do not touch the edge of well to avoid false results.

8. Repeat steps 3 and 4.

9. Incubate the plate in a 37±1℃ incubator or water bath for 1 hour.

10. Repeat step 6 to wash the plate.

11. Develop the color by following HBeAg ASSAY steps 11 - 14.

CALCULATIONS AND DETERMINATIONS

A. HBeAg Assay

1. Calculation of NCx (Negative Control Mean Absorbance)

Example:

Sample No.     Absorbance

           1                        0.025

           2                        0.028

           3                        0.022

NCx  = (0.025+0.028+0.022)/3

= 0.025

NCx must be ≦0.1, otherwise, the test is invalid.

2. Calculation of  HBeAg PCx (Positive Control Mean Absorbance)

Example:   

Sample No.       Absorbance

           1                  1.246

           2                      1.202

PCx = (1.246 + 1.202) /2 = 1.224

PCx must be ≧0.4, otherwise, the test is invalid.

3. Calculation of P- N Value

P- N = PCx - NCx

Example:

P - N = 1.224 - 0.025 = 1.224

P - N Value must be ≧ 0.3, otherwise, the test is invalid.

4. Calculation of the Cutoff Value

Cutoff Value = NCx + 0.06

Example:

Cutoff Value = 0.025 +0.06 = 0.085

5. Calculation of the Retest Range

Retest Range = Cutoff Value±10%

Example:

Retest Range

= (0.085 - 0.009) to (0.085 + 0.009)

= 0.076 to 0.094

B. Anti-HBe ASSAY

1. Calculation of NCx (Negative Control Mean Absorbance)

Example:   

Sample No.       Absorbance

           1                        0.888

           2                        0.915

           3                        0.909

NCx =(0.888+0.915+0.909)/3= 0.904

NCx must be ≧0.4, otherwise, the test is invalid.

2. Calculation of Anti-HBe PCx (Positive Control Mean Absorbance)

Example:

   Sample No.      Absorbance

          1                          0.044

          2                          0.056

PCx = (0.044+0.056)/2 = 0.050

PCx must ≦ 0.1, otherwise, the test is invalid.

3. Calculation of N - P Value

N - P = NCx - PCx

Example:

 N - P = 0.904 - 0.050 = 0.854

N - P Value must be ≧ 0.3, otherwise, the test is invalid.

4. Calculation of the Cutoff Value

Cutoff Value = ( NCx + PCx)/2

Example:

Cutoff Value = (0.904 + 0.050) / 2

= 0.477

5. Calculation of the Retest Range

Retest Range = Cutoff Value+10%

Example: 

Retest Range

= (0.477 - 0.048) to (0.477 + 0.048) = 0.429 to 0.525

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RESULT NTERPRETATION 
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A. HBeAg ASSAY

1. Specimens with absorbance values LESS than the Cutoff Value are  NEGATIVE for HBeAg.

2. Specimens with absorbance values GREATER than or EQUAL to the Cutoff Value are POSITIVE for HBeAg.

3. If the data is within the Retest Range, the test must be repeated and interpreted as above

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B. Anti-HBe ASSAY

1. Specimens with absorbance values GREATER than the Cutoff Value are NEGATIVE for Anti-HBe.

2. Specimens with absorbance values LESS than or EQUAL to the Cutoff Value are POSITIVE for Anti-HBe.

3. If the data is within the Retest Range, the test must be repeated and interpreted as above.