Two students performed an ELISA to determine the amount of anti-Spike antibody in serum of a Covid-19 patient. They used the same ELISA plates, the same reagents for coating, blocking and detection, and the same ELISA reader. Both generated independent standard curves of absorbance vs concentration using the same Spike protein. Student 'A' correctly reported a concentration of 100 μg/ml, but student 'B' reported 450 μg/ml. Which one of the following could most likely explain the wrong result of student 'B'?
1
The ELISA plate was not washed properly between coating with antigen and blocking.
2
The ELISA plate was not washed properly after addition of sample.
3
The slope of the standard curve generated by student B was lower than optimal.
4
The negative control showed very little absorbance.