Section: 802, (Thursday)
Group: 4
Names: John Gibson
Instructor: S. Royan, PhD
Date: Dec 06, 2024
University Of Massachusetts,
Lowell, Fall 2024
Immunology Lab Report 7:
Severe Acute Respiratory Syndrom Antibody Diagnosis And Blood Typing
Objective
The experiment aimed to diagnose two patients, Patients A and B, for SARS-Cov viral infection serum antibody presentation of the Severe Acute Respiratory Syndrome (SARS) using indirect enzyme-linked immunosorbent assay (indirect ELISA). Also, this experiment aimed to give blood ABO group typing and Rhesus (Rh) group typing of other separate four simulated patients, Patients 1 through 4, with synthetic blood samples.
Material And Methods (4)
ELISA Antibody Tests
A flat-bottom microtiter array strip with 12 wells was utilized for indirect ELISA assay. Each well was loaded with 50 μL of purified, non-infectious SARS-Cov antigen particles (produced from human cell culture infected with SARS-Cov) to coat the wells at room temperature for 5 minutes. After coating the antigen, the antigen fluid was emptied and discarded, and the wells were immediately washed with 0.01 M phosphate buffered solution (PBS), about 0.1 mL, repeated 2 times, and emptied.
The microtiter strip was then loaded with positive control antibody serum (from a known SARS-Cov patient who produced antibodies against the virus) in wells 1-3, negative control human serum without anti-SARS-Cov antibodies in wells 4-6, patient A’s serum in wells 7-9, and patient B’s serum in wells 10-12. Each 50 μL volume. The sera were incubated at room temperature for 5 minutes, allowing the primary antibody to bind to the antigen.
After primary antibody binding, the wells were emptied, and the wells were washed with PBS, the same as before, repeated 2 times, and emptied. Then, the secondary antibody (mouse anti-human immunoglobulin) conjugated with horse radish peroxidase (HRP) was added to all wells, 50 μL each, and given 5 minutes of incubation time.
After secondary antibody binding, the wells were emptied again, and the wells were washed with PBS, repeated 3 times, and emptied. Then, all wells were loaded with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate with hydrogen peroxide H2O2, 50 μL each well, for oxidation reaction with HRP enzyme. The microtiter strip was photographed at 5 minutes of reaction time.
Blood ABO group and Rh group typing
A blood typing card with A, B, and Rh marking shallow concaves was loaded with one drop of anti-A, anti-B, and anti-D synthetic antibody serum for their respective shallow concave. 1 drop of Patient 1’s blood was given to each shallow concave. Then, a plastic toothpick stirred and mixed the blood with antibodies, and a photograph of the blood typing card was taken. The process was repeated for Patients 2 to 4, with 70% alcohol washing the blood typing card and drying with a paper towel between uses.
Data
Figure 1 shows the ELISA antibody test result.
Figure 1. ELISA Antibody Test Result
Caption: + is positive control; - is negative control; A is patient A serum; B is patient B serum.
Figures 2 to 5 show pictures of blood typing cards of Patients 1 to 4, respectively.
Table 1 shows the ABO group and Rh group blood typing results.
Table 1. Blood ABO Group and Rh Group Typing Result
Caption: Anti-A, serum with antibody against A antigen; Anti-B, serum with antibody against B antigen; Rh, serum with antibody against D antigen.
Results
As shown in Figure 1, patient A had a qualitatively positive antibody test result (blue), while patient B had a negative test result (no color change, colorless).
As shown in Table 1, patient 1 had an anti-A antibody clumping and an anti-Rh antibody clumping, hence denoted as A+; patient 2 had an anti-B antibody clumping and an anti-Rh antibody clumping, hence denoted as B+; patient 3 had an anti-A antibody clumping and an anti-B antibody clumping, hence denoted as AB-; patient 4 had only anti-Rh antibody clumping, hence denoted as O+.
Discussion
An ELISA antibody test was performed in the first part of this experiment to diagnose the infection status of patients A and B with the SARS-Cov virus. The antibodies against the SARS-Cov virus could be detected in the two patients 21 days after SARS disease symptom onset with immune response delay (4). This experiment set out to detect both patient A's and B’s serum antibodies.
The SARS-Cov virus originated in China and caused SARS disease in 2003. The symptoms of SARS included fever, coughing, and flu-like symptoms. As the disease progressed 2 to 7 days, the patient could experience difficulty breathing (4). The mortality rate was 8% (4). The symptoms started about 10 days after the virus infection (4). The prevention measures included wearing masks, frequent hand washing, avoiding going into crowds, and disinfecting living spaces (1). Treatment of the disease was often the same as general treatment for pneumonia (4). There were no effective vaccines as of 2014 (2).
In this experiment, the indirect ELISA procedure was performed. The microtiter wells were coated with purified, non-infectious SARS-Cov virus particles as the first step. The primary antibodies were positive control serum in wells 1 to 3 from a known SARS patient who produced antibodies against the virus and from patients A or B in wells 7 to 12. The secondary antibody was mouse anti-human antibodies conjugated with HRP. Patient A’s serum contained anti-SARS-Cov antibodies, hence the secondary antibody binding to the primary antibody. Subsequently, the HRP catalyzed TMB oxidation reaction, resulting in blue color in Patient A’s wells 7 to 9, as shown in Figure 1.
To quantitatively determine both patients’ ELISA antibody tests, a spectrophotometer such as the Spectra Max would be needed to measure light absorbance at 450 nm at 5 minutes of reaction time and with 0.18 M sulfuric acid added, stopping the reaction (3). Stopping the reaction by 0.18 M sulfuric acid would turn the blue TMB reaction product yellow. Quantitative measurements were not performed in this experiment.
In conclusion, in the ELISA experiment, patient A was diagnosed with SARS-Cov virus infection, as shown in Figure 1. Patient B’s serum did not have qualitatively detectable antibodies. However, Patient B might or might not have had an infection if the blood had been drawn during the 21-day immune response time when antibody production against the virus was insufficient.
The A, B, and H antigens are red blood cell surface oligosaccharides produced by the H gene’s fucosyltransferase activities, producing the basic H antigen with fucose added to the terminal galactose, which is linked to N-acetylglucosamine, which, is linked to another galactose, which, in turn, is linked to red blood cell surface lipids (3). The A and B antigens are H antigens amended, to the terminal galactose, with an additional N-acetylgalacosamine and galactose, respectively. The Rhesus-positive (+) antigen D is a protein on the red blood cell surface (3).
The ABO and Rh blood group systems discovery by Landsteiner and Weiner is vital in modern medicine blood transfusions, along with 36 other blood group systems (3). Whichever surface antigens the red blood cells lack, the person’s serum contains the antibodies against the antigens. A person with A antigens but no B antigens on red blood cells has anti-B antibodies in the serum and is denoted as having an A blood type. A person with B antigens but no A antigens on red blood cells has anti-A antibodies in the serum and is denoted as having a B blood type. A person with neither A nor B antigens on red blood cells has both anti-A and anti-B antibodies in the serum and is denoted as having an O blood type. A person with D antigens on red blood cells has no anti-D antibodies in the serum and is denoted as having an Rh+ blood type. During blood transfusion, when donated blood contains antigens on red blood cells that have corresponding antibodies in the recipient’s serum, the antibodies cross-link red blood cells causing misshapen, crossed-linked red blood cell clumps, which is a life-threatening condition (3). Hence, ABO and Rh blood typings are performed during blood donation and blood transfusion to ensure safety.
A hemolytic disease occurs when a Rh- blood type mother is pregnant for the second time with a second Rh+ fetus. During the first pregnancy with the first Rh+ fetus, no hemolytic disease occurs because the mother’s immune system has not been exposed to D antigens, and there is no anti-D antibody production. During the delivery of the first Rh+ fetus, the birth trauma exposes the fetus’ D antigen to the mother or any leaking of the fetal blood into the maternal bloodstream, and the mother’s immune system starts producing anti-D antibodies, including immunoglobulin G (IgG), which is permeable through the placenta. Subsequently, during the second pregnancy of a second Rh+ fetus, the antibodies diffuse into the fetus’s bloodstream, attaching to red blood cells with D antigen, causing severe anemia (hemolytic disease) of the fetus or death (3).
RhoGAM antibodies are often administered to a Rh- mother pregnant with a Rh+ fetus. The RhoGAM antibodies bind to D antigens that leak into the maternal bloodstream and mask the antigens so that the mother’s immune system does not activate to produce anti-D antibodies.
In this experiment, Patients 1 and 2 sought to check heredity and relations, and blood typings were performed to give exclusions of relations if applicable. Patients 3 and 4 were subjected to blood typing tests in the hospital for undisclosed reasons. As shown in Table 1, patient 1 had an anti-A antibody reaction and an anti-Rh antibody reaction, hence denoted as A+; patient 2 had an anti-B antibody reaction and an anti-Rh antibody reaction, hence denoted as B+; patient 3 had an anti-A antibody reaction and an anti-B antibody reaction, hence denoted as AB-; patient 4 had only anti-Rh antibody reaction, hence denoted as O+. In conclusion, Patients 1 and 2’s blood relation was not excluded from the blood typing test. An A+ person could be a sibling of a B+ person providing that the parents have A and B alleles in genotypes such as IAi and IBi, which could produce children of IAi, IBi, IAIB, and ii, four phenotypes. The four phenotypes represent A, B, AB, and O blood types. Patients 1 and 2 had Rh+ blood type, so their blood relation was not precluded either because they could inherit the D allele from either shared parent.
In conclusion, Patient 3 had an Rh- blood type, which is rare because missing the D allele in human populations is rare. Patient 4 had an O+ blood type, which made him/her a blood donor to people of A+, B+, and AB+ blood types because the O blood type means no A or B antigens on red blood cells.
References
Lau, J. T., Tsui, H., Lau, M., & Yang, X. (2004). SARS transmission, risk factors, and prevention in Hong Kong. Emerging infectious diseases, 10(4), 587.
Roper, R. L., & Rehm, K. E. (2009). SARS vaccines: where are we?. Expert review of vaccines, 8(7), 887-898.
Royan, S.V. & A. Alton. (2024b). Immunology Lab 9 Recitation Slides 2024. [Unpublished Immunology Laboratory Slides]. Department of Biological Sciences, University of Massachusetts, Lowell.
Royan, S.V. (2024b). Immunology Lab 9 Protocol Handout 2024. [Unpublished Immunology Laboratory Slides]. Department of Biological Sciences, University of Massachusetts, Lowell.
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