Product Manual

Product name

Immunoglobulin IgA Complex Detection Kit (ELISA)

Packaging specifications

96 T/box

Intended Use

It is used for the quantitative determination of the content of IgA complex in human serum or plasma, and is mainly used clinically for the auxiliary diagnosis of IgA nephropathy.

Test principle

This kit uses indirect enzyme-linked immunosorbent assay (ELISA). The HQP001 protein is coated on the plate, a human IgA calibrator or sample to be measured is added, and then the HRP-labeled anti-human IgA antibody (enzyme-labeled antibody) is added, and the unbound components are removed after incubation and sufficient washing, and a sandwich complex of solid-phase antibody-antigen-enzyme-tagged antibody is formed on the solid phase surface of the microplate. With the addition of chromogenic substrates, A and B, the substrate is catalyzed by HRP to produce a blue product, which is finally converted to yellow under the action of the stop solution (2M sulfuric acid), and the absorbance (OD value) is determined on a microplate reader, and the absorbance (OD value) is positively correlated with the concentration of human IgA in the sample to be measured. Fitting the calibrator curve, the concentration of human IgA complexes in the sample can be calculated.

Main components

This kit is a recombinant protein, does not contain human components, the antigen has been inactivated and has no specific potential harm, and has been tested by good laboratory procedures to determine as a suitable biological reagent. The calibrators are traced to the international standard substance ERM-DA470L/PCC.

Note: Components are not interchangeable between different lot size kits.

Materials required but not provided

1. Microplate reader
2. Centrifuge
3. Refrigerator
4. Single-channel pipettes
5. 8-channel pipette
6. Centrifuge tube rack
7. Vortex oscillator
8. Precision pipettes and disposable tips
9. Centrifuge tubes
10. Pipette (2ml, 5ml, 10ml, 25ml)
11. Markers
12. Double distilled or deionized water
13. Dispensing tank
14. Absorbent paper
15. Bottle washing or automatic plate washer
16. 37°C water bath or incubator
17. 500ml graduated cylinder
18. Powder-free disposable latex gloves

Storage conditions and expiration dates

1. Store at 4°C, do not freeze, valid for 6 months.
2. After opening and using, place the plate in a resealable bag with desiccant, seal the bag tightly, and return all reagents to the 4°C freezer.
3. After opening, store according to the recommended conditions, the calibrator, HQP001 protein and secondary antibody-HRP are valid for 4 weeks, and the other ingredients are stable during the expiration period indicated on the label.

Applicable equipment

Microplate reader with 450nm and 630nm wavelength filters.

Sample requirements

1. Serum: After coagulation of whole blood or procoagulant tube samples at room temperature, centrifuge at 1600 rpm for 10 minutes, and take supernatant for detection. Usually, serum samples are stored at 4-8°C for 1 week stable, -20°C for 1 month, -80°C for more than 3 months, it is recommended to store in small samples to avoid repeated freeze-thaw.
2. Plasma: processed within 30 minutes after anticoagulant sampling, centrifuged at 1600 rpm for 10 minutes, and detected by supernatant. Usually, plasma samples are stored at 4-8 °C for 1 week stable, -20°C for 1 month, -80°C for more than 3 months, it is recommended to store in small samples to avoid repeated freeze-thaw.
3. Avoid using hemolytic, hyperlipidemic samples. If the concentration of the test substance in the sample is higher than the maximum value of the standard, please make an appropriate dilution according to the actual situation. It is recommended that serum/plasma be diluted in a volume of 1:1000 to be tested.

Test method

1. Pretreatment
   1. Remove the kit from the 4°C freezer and equilibrate to room temperature, noting that the coating buffer and 20x sample dilutions may crystallize and need to be completely dissolved before use.
   2. Coating: HQP001 protein lyophilized powder was reconstituted in 11mL coating buffer. 1ml can be taken from 11ml of coating buffer into HQP001 protein lyophilized powder tube for reconstitution, gently and repeatedly pipette to fully dissolve the lyophilized protein, and then transferred to the coating buffer to mix.
   3. Add the protein coating solution to each well of the plate according to 100μL/well, and gently shake it after adding to evenly lay the bottom of the plate. Seal with sealing film, room temperature, 3h incubation.
   4. Calculate the amount of blocking solution according to 200μL/well, and dilute the blocking solution mother solution into 1x working solution. According to 200μ/well, wash 14 times to calculate the amount of washing solution, that is, Nx200x14 (N is the number of sample wells). Dilute the wash solution mother solution into 1x working solution. (Note that there is no crystallization before dilution of the mother liquor.)
   5. Take out the sealed plate incubated at room temperature for 3h, carefully tear off the seal, quickly shake off the solution in the wells vertically, and pat dry on clean absorbent paper. Add the washing solution according to 200μL//well, let stand for 1min, quickly shake off the solution in the wells vertically, and vigorously pat dry on clean absorbent paper. After that, repeat the wash 2 times following this washing procedure.
   6. Blocking: Add the blocking solution according to 200μL/well, and place the sealing film in a 4°C refrigerator for 1h.
   7. Add the washing solution according to 200μL//well, let stand for 1min, quickly shake off the solution in the wells vertically, and vigorously pat dry on clean absorbent paper. After that, repeat the wash 2 times following this washing procedure.
   8. Standard diluting: Dilute sample dilution into 1x working solution, add 1mL of 1x sample dilution to the standard quality tube, and mixed with a pipette tip, at this time the concentration of the standard is 500ng/mL, and then diluted into STD1-STD6 at a concentration of 50ng/mL~300ng/mL.

      	Standard concentration	IgA complex in the standard
      STD1	300ng/ml	30U
      STD2	250ng/ml	25U
      STD3	200ng/ml	20U
      STD4	150ng/ml	15U
      STD5	100ng/ml	10U
      STD6	50ng/ml	5U

   9. The sample to be tested is diluted 1000-fold with 1x sample diluent.
2. Test steps
   1. The standard, sample to be tested, and negative control were added to the well of the plate at 100μl/well. Seal, incubate at room temperature for 0.5h.

      	1	2	3	4	5	6	7	8	9	10	11	12
      A	STD1		Sample 1		Sample 9		Sample 17		Sample 25		Sample 33
      B	STD2		Sample 2		Sample 10		Sample 18		Sample 26		Sample 34
      C	STD3		Sample 3		Sample 11		Sample 19		Sample 27		Sample 35
      D	STD4		Sample 4		Sample 12		Sample 20		Sample 28		Sample 36
      E	STD5		Sample 5		Sample 13		Sample 21		Sample 29		Sample 37
      F	STD6		Sample 6		Sample 14		Sample 22		Sample 30		Sample 38
      G	Sample dilution		Sample 7		Sample 15		Sample 23		Sample 31		Sample 39
      H	ddH2O		Sample 8		Sample 16		Sample 24		Sample 32		Sample 40

   2. After standing for 0.5h, quickly shake off the solution in the wells, and vigorously pat dry on absorbent paper, and pay attention to avoid mutual contamination of the liquids between the wells during operation.
   3. Add the washing solution according to 200μl/well, let stand for 1min, quickly shake off the solution in the wells vertically, and vigorously pat dry on clean absorbent paper. After that, repeat the wash 2 times according to this washing procedure.
   4. Add secondary antibody HRP: Dilute HRPase-labeled anti-human IgA antibody with 1x sample diluent at a ratio of 1:2000, and add the diluted HRPase-labeled anti-human IgA antibody to the well of the enzyme plate at 100μl/well, seal, and incubate at room temperature for 0.5h.
      Note: When preparing HRPase-labeled anti-human IgA antibody, it is recommended to prepare at 1.2xNx100 μl to avoid insufficient preparation volume. (N is the number of sample wells)
   5. After incubation for 0.5h, quickly shake off the enzyme plate liquid, pat dry vigorously on absorbent paper, add washing solution according to 200μl/well, stand for 1min, quickly shake off the solution in the wells vertically, and vigorously pat dry on clean absorbent paper. After that, repeat the wash 2 times according to this washing procedure.
   6. Color rendering: Prepare chromogenic solution, A solution and B solution 1:1 mixed. The chromogenic solution should be prepared and used freshly. Be protected from light. The chromogenic solution and stop solution are harmful to the human body, and the operation should be careful not to touch the skin.
      Note: The stop solution is a strong acid corrosive liquid, avoid contact with skin and metal, and operate according to laboratory safety requirements when discarded.
   7. Add the chromogenic solution according to 100μL/well, and develop the color at room temperature protected from light for 10min. Pay attention to the degree of color change, such as if the color is too blue should terminate the reaction early according to the situation, if the color is too light can extend the reaction time.
   8. Pull the plug: Add 50μL of stop solution to each well to stop the reaction, gently shake and measure the results on a microplate reader.
   9. Set the microplate reader parameters: dual wavelength, first wavelength 450 nm, second wavelength 630 nm.
   10. Test results, data analysis.

Inspection quality control

The scope of quality control of the control substance is printed on the label.

Each experiment should include both low-value controls and high-value controls.

Each laboratory should establish its own quality control scope.

Recommendation: If the quality control results do not meet expectations, the laboratory should not issue a test report.

Result calculation

After the detection is completed, the standard concentration is used as the ordinate, the corresponding absorbance (OD value) is used as the abscissa, and the standard curve equation is created by using the four-parameter Logistic curve fitting (4-pl) using computer software, and the concentration value of the sample is calculated by the absorbance (OD value) of the sample. If the sample is diluted, the concentration value measured by the above method is multiplied by the dilution factor to determine the final concentration of the sample.

Interpretation of test results

This reagent is used to study clinical samples of people of different genders and ages, calculated by percentile distribution method, with 5th as the lower limit of the reference range, and the reference values of this project is:

Normal base range: <15.1907U

The base range is on the high side: 15.1907-18.2839U

Unusually high: >=18.2839U

Limitations of the test method

1. The test results are for clinical reference only and cannot be used alone as the basis for diagnosis or exclusion of cases, and the clinical management of patients should be considered comprehensively based on their symptoms/signs, medical history, other laboratory tests and treatment response.
2. Use within the validity period indicated on the kit, expired products must not be used.
3. Do not mix with kits or components from other manufacturers.

Product performance indicators

• Sensitivity: 3.725U
• Detect linear range: 3.725-25U
• Specificity: The content of IgA complex in human samples is measured, and there was no obvious reaction with other analogues.
• Repeatability: The coefficient of variation is less than 10% both intraplate and interplate.

Note: U represents the concentration of IgA complex in 1 ng standard

Notes

Warnings and precautions: IVD For in vitro diagnostic use

• There is currently no known test method to completely ensure that human material is not infectious, so all human material should be considered potentially infectious. For substances that contain or may contain the source of infection, they should be implemented in accordance with biosafety level 2 or other corresponding biosafety specifications. Ingredients of animal origin should be treated as potentially infectious.
• If the detection reagent stored in the refrigerator is used, it is recommended that it should be taken out of the refrigerator before testing, placed at room temperature and then opened for use, otherwise the test results will be affected.
• General precautions should be taken when handling all laboratory reagents.
• All waste disposal should be carried out in accordance with local regulations.
• Wear gloves when handling samples or reagents.
• Clean and disinfect spilled samples or reagents with appropriate disinfectants.

References

1. [ZHANG2021]ZhangX,et.al.Poly-lgA Complexes and Disease Severity in lgA Nephropathy. CJASN.16:16521664,2021.doihttps://doiorg/10.2215/CjN.01300121
2. [LV2019]LvJC,et.al.A molecular probe for serological diagnosis of 1gA nephropathy. Patent application number：201910857929.9

Basic information

• Name of the registrant/manufacturing company:
  Shenzhen Luwei Biotechnology Co., Ltd
• Address:
  10th Floor, Block B, Kelu Building, Baoshen Road, North District, Nanshan Science and Technology Park, Shenzhen, Guangdong Province, China
• Contact:
  0755-22670570
• Name of after-sales service unit:
  Shenzhen Luwei Biotechnology Co., Ltd
• Production address:
  Room 1305, 13th Floor, Building 5, No. 17 Bulan Road, Nanwan Street, Longgang District, Shenzhen, Guangdong Province, China