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50 (dilution factor) × 10 (100 μl converted to 1 ml conversion factor)
Concentration (mU/100μl)
ASA anti-sperm antibody ELISA kit instructions
ASA anti-sperm antibody ELISA kit
(Germany IBL imported original, article number: RE52001)
Germany IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
An enzyme-free kit for in vitro quantitative detection of anti-sperm surface antigen autoantibodies in human serum.
1 , description
In Western countries, the proportion of infertile couples accounts for 10-15% of the total population (Haas et al, 1980). At present, people cannot accurately know the incidence of infertility caused by immunological factors. Because of the many different detection methods, there is considerable controversy about the role of anti-sperm antibodies in infertility. Among the traditional detection methods for detecting anti-sperm antibodies, sperm agglutination detection and sperm fixation detection have been widely used. These methods and other agglutination tests are time consuming and the results are not coordinated with their operation. There are many advantages to detecting antisperm antibodies by ELISA compared to conventional assays. IBL's anti-sperm antibody ELISA kit combines these advantages with high sensitivity and high specificity. This kit is simple to operate and can detect a wide variety of serums.
Note: Additional information for this lab can be obtained from the IBL website (http://) or directly from Hamburg IBL.
2 , the principle of experiment
IBL's Sperm Antibody Enzyme Kit provides the experimental materials needed for quantitative detection of anti-sperm surface antigen antibodies in human serum and semen.
This test kit utilizes a non-competitive principle. The sperm surface antigen coated in the micropores of the coated plate was extracted from the pool using the method Alexander mentioned in 1984. The serum of the suspected immunologically infertile patient (diluted) was added to the coated wells for incubation, after which the plates were washed, and then peroxidase-labeled anti-human immunoglobulins (IgA, IgG and IgM) were added and incubated. After the incubation, the plate was washed, TMB substrate solution was added, and the displayed color intensity was measured by a microplate reader. The intensity of the color shown in the microwells is directly proportional to the concentration of bound immunoglobulin. The experimental results can be read from the standard curve and the unit of results is units/ml.
3 , precautions and preventive measures:
1) Reagents are for in vitro diagnostics and professional use only.
2) See the Material Safety Data Sheet for information on hazardous materials in the kit.
3) All reagents in the kit, including the detected human serum/plasma, are negative for HIV/II, HbsAg and HCV, but such viruses (HIV/II, HbsAg and HCV) or other infections may appear in the reagents. The possibility of sexual pathogens cannot be completely ruled out. Therefore, all reagents should be treated as potentially biohazardous during use and handling.
4) Please do not use the mouth to blow the reagents, do not touch the reagents and samples to the skin and mucous membranes.
5) Samples and kits for reagent handling, do not smoke, eat and use cosmetics.
6) Wear rubber gloves when handling samples and reagents. Microbially contaminated reagents and samples can lead to erroneous results.
7) Dispose of samples and reagents in accordance with the appropriate national biosafety management method.
8) Do not use expired reagents
9) Carry out the experimental operation according to the volume (loading volume and pretreatment step) specified in the instruction manual. The ideal experimental results are obtained with a calibrated pipette.
10) According to the National Biosafety Management Regulations, the chemicals in the experiment and the reagents used or prepared should be treated as hazardous waste.
11) The material safety data of the product can be uploaded from the IBL homepage and can be directly consulted by IBL. The Safety Data Sheet complies with the following standards: EU-Guideline 91/155 EWG, ANSI-Standard, ISO-Sstandard 11014, OSHA (US).
4 , kit composition
4.1 kit components
The amounts indicated on the reagent bottles are theoretical volumes and all reagent bottles are filled with reagents. For solutions with ingredients below 250μl, please note that all solutions are at the bottom of the reagent bottle.
4.1.1 Coated plates, 12 x 8 wells, coated with sperm surface antigen.
4.1.2 Standard, 3, lyophilized powder, dissolve each lyophilized powder in 1 ml of washing solution. The reconstituted standard (Standard S5) contains 100 μl of anti-sperm antibody in 100 μl.
4.1.3 Enzyme conjugate, 601 times concentrated, 1 branch, 100 μl, containing peroxidase-labeled anti-human IgG, IgA, IgM, diluted with a dilution ratio of 1:601 before use.
4.1.4 TMB substrate solution, 1 bottle, 10ml, ready to use, containing TMB and stabilizer.
4.1.5 TMB Stop Solution, 1 bottle, 10 ml, ready to use, containing 1 M H 2 SO 4 , Note: Avoid skin contact.
4.1.6 Washing solution, 20 times concentrated, 1 bottle, 50ml, diluted with 1:200 in double distilled water, the prepared washing solution can be stably stored for 4 weeks in the environment of 2-8 °C.
4.1.7 Diluent, 3 bottles, lyophilized, each bottle is diluted with 20ml washing solution, the prepared diluent can be stably stored for 2 days in 2-8 °C environment, if frozen in -20 °C environment Can be stored for a longer period of time.
4.1.8 Quality control serum, 3, lyophilized, each diluted with 1 ml of washing solution. The quality control serum should be temporarily configured for each experiment and can only be used once. The acceptable range of quality control serum can be found in the quality control sheet.
4.1.9 sealing paper, 5 pieces
4.10 equipment required for the experiment but the kit does not provide
1) Calibration of precision pipettes and sampling tips: 0-20μl, 10-100μl, 100-1000μl
2) Multichannel pipette, 50-200μl
3) Washing machine and washing bottle
4) 37 ° C incubator
5) Microplate reader with 450nm filter
4.2 Storage and stability of the kit
The kit should be shipped at 2-8 °C. Once the coated slats are removed, they should be resealed. When the slats are disassembled and stored at 2-8 ° C, their immunological activity can be stabilized for 6 weeks.
4.3 Preparation of reagents
The required number of slats and all reagents were equilibrated to room temperature before the start of the experiment. The kit can be tested in 3 separates and the volume described below can be used to detect 4 coated strips (32 detection wells). If more slats are used in the experiment, the volume should be changed accordingly.
4.3.1 Washing solution, 600 ml of double distilled water was added to 30 ml of concentrated washing solution.
4.3.2 Diluent, add 20 ml of ready-to-use wash solution to each lyophilized dilution.
4.3.3 Standard, quality control serum
a) S5 (500 mU/100 μl), each standard was diluted with 1 ml of a ready-to-use washing solution. The reconstituted standard (Standard S5) contains 100 μl of anti-sperm antibody in 100 μl.
b) Other concentrations of samples can be obtained by serial dilution with dilutions, as follows:
S5 500mU/100μl
S4 250mU/100μl = 500μl S5 + 500μl dilution
S3 125mU/100μl = 500μl S4 + 500μl dilution
S3 62.5mU/100μl = 500μl S3 + 500μl dilution
S2 31.3mU/100μl = 500μl S2 + 500μl dilution
S0 0 mU/100μl = 500μl dilution (zero standard)
c) Quality control serum, 1 ml of ready-to-use washing solution was added to each lyophilized quality control serum.
4.3.4 Enzyme conjugate, add 9 ml of the dilution solution to 15 μl of the concentrated enzyme conjugate, and mix well.
4.4 Prepare reagent storage and stability
4.4.1 The configured washing solution can be stably stored for 4 weeks in an environment of 2-8 °C. The phosphate precipitates in the solution when it is refrigerated and dissolves at room temperature.
4.4.2 Sample diluent, the prepared diluent can be stored stably for 2 days in the environment of 2-8 °C, and can be stored for a longer time if stored in the environment of -20 °C.
4.4.3 Standards and quality control products should be temporarily configured for each experiment and can only be used once.
4.4.4 Enzyme conjugate, the configured enzyme conjugate can be stably stored at room temperature for 20 min.
4.5 Kit processing
According to the national official treatment kit, please refer to the material safety data sheet of the product for specific information.
4.6 Treatment of broken kit
If the kit is damaged, please contact IBL or submit your complaint, but no more than one week after you get the kit. Seriously damaged ingredients cannot be used in the experiment and saved until the final solution is found. After that, dispose of it according to official regulations.
5 , sample
5.1 collection
Serum: venous blood collection according to general principles. Do not use obvious hemolysis, jaundice, and lipemia samples.
Semen: The semen sample should be centrifuged at 1000 rpm for 15 minutes, after which the supernatant is used for the experiment (semen).
5.2 storage
Serum, semen: When the collected sample is stored at 2-8 ° C, it must be detected within 24 hours. If you want to store the serum for a longer period of time, you can freeze the serum at -20 °C to avoid repeated freezing and thawing.
5.3 pretreatment
Serum, semen: Dilute the patient sample with a dilution of 1:51 (for example: 10 μl sample + 500 μl dilution) and mix well.
6 , experimental steps
General Note: All reagents and samples should be equilibrated to room temperature before the start of the experiment. Foam generation should be avoided when all reagents are mixed. To avoid cross-reactivity, new sampling tips should be used for each reagent, standard, and sample. Before the start of the experiment, we recommend that you prepare all the reagents, remove the cover, place the required number of slats on the rack, and so on. These preparations ensure that the time interval between each loading step is the same and uninterrupted. Secure the required number of slats to the pallet to prepare for loading.
6.1 Experimental steps
6.1.1 Add 100 μl of standard, control and sample to the corresponding wells
6.1.2 Incubate for 60 minutes at 37 ° C after sealing.
6.1.3 Wash the plate 3 times with 300 μl of washing solution per hole (recommended to use a washing machine), and pat dry on absorbent paper. Note: Whether the washing step is correct or not will significantly affect the sensitivity and precision of the experiment.
6.1.4 Add 100 μl of enzyme conjugate to each well.
6.1.5 After the plate was closed, incubate at 37 ° C for 60 minutes.
6.1.6 Wash the plate 3 times with 300 μl of washing solution per well
6.1.7 Add 100 μl of TMB substrate solution to each well.
6.1.8 Incubate for 10-15 minutes at room temperature (18 ° C - 24 ° C).
6.1.9 Add 50 μl of Stop Solution to each well to stop the substrate reaction and gently shake the plate to mix the solution evenly.
6.1.10 The OD value was read at 450 nm within 30 min after the addition of the stop solution. (Reference wavelength: 600-650nm)
6.2 Calculation of results
Calculation of standards, controls and patient sample results:
On a double logarithmic scale paper, a standard curve is applied to the standard concentration by the OD value of the standard. The total antisperm antibody concentration (mIU/100 μl) of the unknown sample (diluted) was calculated using a standard curve. Finally, multiply the result by 0.5 to obtain the concentration (U/ml) of the undiluted sample.
U/ml=Mu/100μl ×
= mU/100μl×0.5
1000 (mU becomes the conversion factor of U)
Automatic calculation:
Using a three-regression, four-parameter or double-logarithmic equation to make a standard curve generally achieves better results. As for the accuracy of the results, please refer to item 7 (experimental characteristics). If the antibody concentration in the sample is higher than the highest standard, it should be diluted with the corresponding dilution and retested. Finally, multiply the result by the corresponding dilution factor.
The following is a typical example of the standard curve for an anti-sperm antibody ELISA kit:
sample
OD value
S0
0.057
0.00
S1
S2
S3
0.264
31.3
0.484
62.5
0.886
125
S4
1.353
250
S5
2.170
500
6.3 automatic detection
IBL's laboratory to prove that the anti-sperm antibody ELISA kit can be used with a fully automated enzyme-free analyzer. Instructions for operation can be obtained directly from IBL or downloaded from the IBL homepage. For details, please check the "Automatically detect" item.
7 , experimental characteristics
7.1 Expected value
It is recommended that each laboratory determine the normal range of values ​​for its laboratory. In the normal range of studies, all participants in the study were clearly healthy individuals, and the normal range of individuals should be 95%.
Normal range (serum, semen): <150 mU/100 μl, corresponding in the undiluted sample range: <75 U/ml.
7.2 specificity
The purified sperm-specific antigen is coated in the microwell of the coated plate. It has not been found to cross-react with other antibodies.
7.3 sensitivity
The lowest detectable concentration of this kit is 0.14 mU/100 μl (equal to the average of the zero standard OD values ​​minus two standard deviations), corresponding to 0.07 U/ml in the undiluted sample.
7.4 precision
7.4.1 Differences within the board
In the experiment, five different quality control serums were used for double detection to determine intraplate differences. The results of the intraplate variation experiments are as follows:
sample
Quantity
Average value (mU/100μl)
Standard deviation (mU/100μl)
CV (%)
1
15
47.0
2.0
4.3
2
15
64.0
7.6
11.9
3
15
98.0
2.6
2.7
4
15
59.0
4.5
7.6
5
15
151.0
11.8
7.8
7.4.2 Differences between boards
In the experiment, two different quality control sera were used for double detection to determine the difference between the plates. The experimental results of the difference between the plates are as follows:
sample
Quantity
Average value (mU/100μl)
Standard deviation (mU/100μl)
CV (%)
1
15
42.0
3.3
7.9
2
15
48.0
7.4
15.4
3
15
77.0
7.7
10.1
4
15
74.0
7.2
9.7
5
15
130.0
17.6
13.6
7.5 accuracy
7.5.1 Linearity
The serum sample is diluted with the diluent to obtain eight serum samples having different sperm antibody concentrations, and the concentration of the sperm antibody in the released sample is detected by an ELISA kit. All three samples were diluted and the results were as follows:
sample
P1 (mU/100μl)
Reproducibility (%)
P2 (mU/100μl)
Reproducibility (%)
P3 (mU/100μl)
Reproducibility (%)
1:1
205.1
100
241.5
100
259.4
100
1:2
100
98
132.8
110
138.6
107
1:4
50.8
99
64.0
106
68.0
105
1:8
32.7
128
32.0
106
35.4
109
sample
P4 (mU/100l)
Reproducibility (%)
P5 (mU/100μl)
Reproducibility (%)
P6 (mU/100μl)
Reproducibility (%)
1:1
272.8
100
242.2
100
366.5
100
1:2
141.4
104
116.0
96
163.3
89
1:4
61.9
91
57.7
95
64.9
71
1:8
31.4
92
33.2
110
33.8
74
sample
P7 (mU/100μl)
Reproducibility (%)
P8 (mU/100μl)
Reproducibility (%)
1:1
376.9
100
331.0
100
1:2
171.9
91
139.4
84
1:4
83.0
88
82.1
99
1:8
49.0
104
42.9
104
7.5.2 Reproducibility
Four serum samples were concentrated with samples of known concentration to increase the concentration of sperm antibodies (all results are in mU/100 μl).
sample
Endogenous antibody concentration (mU/100μl)
The concentration of the added sample (mU/100μl)
Expected value (mU/100μl)
Measured value (mU/100μl)
Reproducibility (%)
1
52.0
346.0
398.0
307.0
77
52.0
169.0
221.0
175.0
79
52.0
68.0
120.0
116.0
97
52.0
35.0
87.0
80.0
92
52.0
16.0
68.0
69.0
101
2
76.0
346.0
422.0
329.0
78
76.0
169.0
245.0
236.0
96
76.0
68.0
144.0
140.0
97
76.0
35.0
111.0
108.0
97
76.0
16.0
92.0
91.0
99
3
79.0
326.0
405.0
354.0
87
79.0
163.0
242.0
198.0
82
79.0
72.5
151.5
136.0
90
79.0
34.5
113.5
112.0
99
79.0
17.5
96.5
89.0
92
4
120.0
319.0
439.0
356.0
81
120.0
159.5
279.5
231.0
83
120.0
69.5
189.5
180.0
95
120.0
34.0
154.0
149.0
97
120.0
18.0
138.0
130.0
94
7.5.3 Method comparison
IBL anti-sperm antibody ELISA kit is compared with ELIAS anti-sperm antibody ELISA kit
The IBL anti-sperm antibody ELISA kit simultaneously detects 38 patient samples with commercially available anti-sperm antibody ELISA kits. The linear regression results are as follows:
IBL ELISA = 4.0698 × ELIAS ELISA + 1.0441; correlation coefficient = 0.669; quantity = 38
7.5.4 Quality Control
According to national regulations, we recommend that you use quality control products. The use of controls can ensure the daily effectiveness of the results, both normal and pathological quality control must be used. The quality control products of the quality control laboratory and the corresponding results are described on the quality control sheet of the IBL of the kit. The data and scope on the QC sheet usually correspond to the corresponding batch and can be used directly for comparison of results. In order to ensure the accuracy of the results, it is recommended to use national or international quality assessment procedures. The quality control values ​​were analyzed using appropriate statistical methods. If the test result is not within the acceptable range for which the QC material has been determined, the patient's result is considered invalid. In this case, please check the following technical areas: sample and timekeeping equipment; microplate reader; reagent expiration date; storage and incubation conditions; pumping and washing methods. If you have checked the above equipment and found no problems, please contact the IBL agent directly.
8 , the limitations of the experiment
8.1 Interfering substances
Improper sample handling or changing experimental procedures may affect the results of the experiment. See “Sample Collection†for the effects of improper sample handling on the experiment. Sodium azide and thimerosal concentrations above 0.1% will affect this experiment. Therefore, the above components may cause erroneous experimental results when the concentration in the quality control serum or sample is higher than the concentration.
8.2 High dose hook effect
No high dose hook effect was found in the antisperm antibody ELISA kit.
9 , legal aspects
9.1 Reliability of results
The experiment must be carried out in strict accordance with the manufacturer's instructions. In addition, the experimenter must strictly abide by laboratory optimization management rules or other applicable national standards or laws. This is highly dependent on the use of quality control reagents. To ensure the accuracy and accuracy of the experiment, each experiment must contain a sufficient number of controls. If all the controls are within a certain range, all other test parameters are also within the given test range, and the test results are valid. Please contact IBL if you have any questions.
9.2 Complaint
As for the complaint form (preferably using the maternity's complaint form) to be handed in in writing, and the complaint must contain all the information and experimental results of the kit. The format of the complaint can be obtained from IBL.
9.3 treatment
Even if all the experimental results are consistent with the requirements described in item 9.1, the treatment method cannot be judged based on the experimental results. Any experimental results are only part of the patient's clinical symptoms. Only when the experimental results are consistent with all the clinical symptoms of the patient can we determine the treatment based on the experimental results. The results of the experiment itself have never been the only factor in determining the treatment.
9.4 Responsibility
Any modification, exchange or mixing of different kit components of the kit will have a negative impact on the expected results and the validity of all experimental results. This modification and exchange of the kit cannot require the supplier to replace it. If the customer complains about the results of the wrong experiment, it is considered invalid. Regardless of the form of accommodation, the responsibility of the manufacturer does not exceed the price of the kit itself. The manufacturer is not responsible for the damage caused by the kit during transportation.
10 , references
1) Alexander NJ, Sampson JH and Flugham JL. Pregnancy rates in patients treated for anti-sperm-antibodies with prednisone. Int. J. Fertil., 28 : 63 (1983).
2) Alexander NJ and Bearwood D. An immunosorption assay for antibodies to spermatozoa: Comparison with agglutination and immobilization tests. Fert. Steril., 41 : 270 (1984).
3) Bronson R, Cooper G, Rosenfeld D. Sperm antibodies: their role in infertility. Fertil. Steril., 42 (2) : 171-183 (1984).
4) Haas GG, Cines DB and Schreiber AD. Immunological infertility identification of patients with anti-sperm-antibody. N. Eng. J. Med., 303 : 722 (1980).
5) Haas GG, Kubota K, Quebbeman JF, Jijon A, Menge AC, Beer AE. Circμlating antisperm antibodies in recurrently aborting women. Fertil. Steril., 45 (2) : 209-215 (1986).
6) Hjort T, Meinertz H. Anti-Sperm Antibodies in Women. in: Nilsson O, Mattsson R (Editors). Immunocontraception. Ares-Serono Symposia Publications, Rome, 151-159 (1995).
7) Isojima ShLi, Ashitaka TSh and Y. Immunologic analysis of spermimmobilizing factor found in sera of women with unexplained sterility. Am. J. Obstet. Gynecol., 101 : 677 (1968).
8) Kibrick S, Belding DL and Merril B. Methods for detection of antibodies against mammalian spermatozoa. Fertil. Steril., 3 : 403 (1952).
9) Marshburn PB, Kutteh WH. The role of antisperm antibodies in infertility. Fertil. Steril., 61 (5): 799-811 (1994).
10) Meinertz H, Hjort T. Anti-Sperm Antibodies in the Male. in: Nilsson O, Mattsson R (Editors). Immunocontraception. Ares-Serono Symposia Publications, Rome, 83-89 (1995).
11) Menge AC, Medley NE, Mangioni CM, Dietrich JW. The incidence and influence of anti-sperm-antibodies in infertile human couples on sperm-cervical mucus interactions and subsequent fertility. Fert. Steril., 38 : 439 (1982).
12) Naz RK, Menge AC. Antisperm antibodies: origin, regμlation, and sperm reactivity in human infertility. Fertil. Steril., 61 (6) : 1001-1013 (1994).
13) Paμl S, Baukloh V and Mettler L. Enzyme-linked immunosorbent assays for sperm-antibody detection and antigenic analysis. J. Immunolog. Methods, 56 : 193 (1983).
14) Rose NR, Hjort T, Rühme P, Harper MJK and Vyazov O (eds.). Techniques for detection of iso- and autoantibodies to human spermatozoa. Clin. Exp. Immunol., 23 : 175 (1976).
15) Rühme P, Renckes CNM, Bezemer PD and van Amstel N. Prognosis of fertility in women with unexplained infertility and sperm agglutinins in the serum. Fertil. Steril., 42 : 561 (1984).
16) Scheit KH, von der Kammer H, Wuttke W, Neumann G and Hinney B. Ein Enzymimmuno-Test für den Nachweis von Spermatozoen-Antikörpern. Lab. med., 10 : 61 (1986).
17) Shμlman JF, Shμlman S. Methylprednisolone treatment of immunologic infertility in the male. Fertil. Steril., 38 : 591 (1982).
This translation is for reference only, please refer to the original for details.