Wet ELISA data table record

• Period and code, not your name: ____________________
• Date: ____________________
• Tasks included: [ ] T1 [ ] T2 [ ] T3 [ ] T4 [ ] T5 [ ] T6
• Route: [ ] paper handed to Mr. Mendoza [ ] one PDF to Wk5 Data table: Wet ELISA lab

R1. Your one record

Complete one Wet ELISA data table record with six tasks, T1 to T6: your two control predictions, a six-well data table, the step order with one wash sentence, a control check, a check of a flagged strip, and one limit sentence. Keep the six tasks together; they are one submission, on paper or as one PDF.

Your route line tells you where your well colors come from. On Route W you run a classroom kit with your group and read your own wells. On Route P you read a supplied illustrative record called Strip 1. The six tasks, the table and the limit are the same on both routes. Nothing on Route P claims you ran a kit, and nothing on either route calls a sample well positive or negative.

Thursday's paper dilution plan and Friday's four labeled model wells were separate records. Today's six wells carry no concentration label and no unit. You will record color words, compare each well with the negative control, and check the two controls. Tuesday's debate weighed the harm of false results. Today you see what a run has to show before anyone can trust a color at all.

R2. The binding chain

An antigen is a substance an antibody can bind. An antibody has a binding site that contacts a particular part of the antigen. Binding is selective, but an antibody can sometimes bind another target; that is cross-reaction, and it is one reason a specific test is still not an error-free test. Janeway, Immunologists' Toolbox, introductory Immunization paragraphs and A-8.

In a color-based ELISA the layers stack in a set order. Antigen, or a sample that may hold it, goes into the well and attaches to the surface. A primary antibody is added and binds antigen if antigen is there. An enzyme-linked antibody is added and binds the antibody layer below it. Last, a substrate is added; the enzyme turns it into a colored product, so color appears where the whole chain is present. Color is the signal, the detectable response of the test. ELISA background, Specimen Requirements and Procedure; Assay Guidance Manual, Sandwich Immunoassay (ELISA).

Kits differ. Some attach an antibody to the well first and add the sample second; some name the steps differently. On Route W, your kit's printed procedure is the order you follow and record. On Route P, the supplied step order in R3 is the one you record.

R3. Order and washing

Each reagent can only bind the layer added before it. Add a reagent out of order and it has nothing to hold onto; it washes away, or it stays behind as loose material that has nothing to do with binding. Either way the final color stops meaning what it should.

Between the main steps the wells are washed: liquid is added and emptied to carry away everything that did not bind. That is what makes the color specific. Skip a wash, or shorten it, and enzyme-linked antibody that never bound anything can stay in the well and make color where there is no antigen. ELISA background, Specimen Requirements and Procedure (between each of the four primary steps, a wash step removes unbound material); Assay Guidance Manual, Basic Steps for Developing and Running an Immunoassay.

The classroom model order, used for T3 on Route P and for comparison on Route W:

Incubation means the waiting time that lets a layer bind before the next step. On Route W, use the times printed in your kit's procedure and record when each one started. This reading does not give you a time; the kit does.

R4. Two controls decide whether to read the rest

A control is a well with an expected result, run the same way as every other well. The positive control holds material that should complete the chain, so it should develop color. The negative control holds no antigen, so it should stay nearly clear. Both are recommended for tests that give a yes-or-no style result, and both are treated exactly like the samples. ELISA background, Quality Control and Lab Safety; Assay Guidance Manual, Control Samples.

Read the two controls before you read anything else. If the positive control developed color and the negative control stayed nearly clear, the run passed its own check and you can read the sample wells. If the negative control developed color, the run made color without antigen; contamination, a skipped wash, a reused tip or a timing slip can all do that. The sample wells on that strip cannot be read, because the same problem could be adding color everywhere. The run is flagged, and a careful group repeats it or asks the teacher to review it. Do not subtract the negative control's color and keep going.

A clean control pair lets you read the other wells. It does not make any sample well positive or negative, and it does not guarantee that every well behaved.

R5. Read color, then stop

Use four words for every well: Nearly clear, Light, Medium, Dark. They are an observation scale, in order, not amounts. Write the hue (for example, blue) once on the hue line above your table, because kits differ. Read each well on its own against a white background, then write whether it shows more color than the negative control, about the same, or less. Light is still more color than Nearly clear.

Today's wells have no concentration label. The darker-means-more relationship you used on Friday was that model's stated relationship, not a rule for every format, and some formats run the other way. Assay Guidance Manual, Sandwich Immunoassay and Competitive Binding Assay. You are not estimating an amount, and there is no curve to read one from.

"More color than the negative control" is a comparison you wrote down. It is not a positive result. Calling a sample well positive or negative needs the test's decision rule, the stated cutoff for what counts, and that rule is not supplied today. A classroom kit is also not a test of a person: in a clinic a screening ELISA is not a diagnosis by itself, and a confirmatory test follows it. ELISA background, Clinical Significance. Your T6 sentence names one thing the record cannot establish. Naming it is enough; do not invent the missing rule.

R6. Your route

Only your route's section is on your page. If you were absent, or the class ran Route W and you did not, use R6-P.

#### R6-P. Route P: reading a supplied record

Strip 1 is a supplied illustrative record: six wells, a hue, and a color word for each well. It is what a careful group might have written down; it is not a plate you looked at and not a measurement. You copy its colors into T2, compare each with NC, list the supplied step order in T3, and check its controls in T4 exactly as a bench student would. Strip 2, the flagged strip in T5, is supplied to everyone on both routes.

Nothing on this route claims you ran a kit, opened a reagent, or observed a plate. Write "Supplied Strip 1" on your record's route line. If a well word seems odd, record it as supplied and use your T6 limit; do not change a supplied word.

Workflow for the six-well record. This is a teaching diagram, not a kit procedure, a reagent list or a picture of a plate. On Route W your kit's printed procedure supplies the actual steps and times; on Route P the supplied step order in R3 is what you record.

BUILD THE CHAIN, ONE LAYER AT A TIME
Step 1  antigen or sample goes into the well
        wash: unbound material leaves
Step 2  primary antibody binds antigen, if antigen is there
        wash
Step 3  enzyme-linked antibody binds the layer below it
        wash
Step 4  substrate: color is meant to appear where the chain is complete, unless the run has an error
        (stop step only if the kit prints one)
                       |
                       v
READ EACH WELL ON ITS OWN
Nearly clear / Light / Medium / Dark
then compare with NC: more color / about the same / less
                       |
                       v
CHECK THE TWO CONTROLS FIRST
PC developed color?      NC stayed nearly clear?
Both yes: read the sample wells.
NC colored: the strip is flagged; repeat or review, do not subtract.
                       |
                       v
STATE THE LIMIT
More color than NC is a comparison you wrote down.
Positive or negative needs a decision rule, which is not supplied.
A classroom kit is not a test of a person.

Full text equivalent. The chain is built one layer at a time, and between layers the wells are washed so that anything that did not bind is carried away. Antigen or sample attaches to the well. Primary antibody binds antigen if it is there. Enzyme-linked antibody binds the antibody layer below it. Substrate is added last, and the enzyme is meant to make color where the full chain is present, unless the run has an error; some kits print a stop step after that. Read every well on its own, using the four color words, then compare each well with the negative control. Before reading any sample well, check the two controls: the positive control should have developed color and the negative control should have stayed nearly clear. If both did, the sample wells can be read. If the negative control developed color, the strip is flagged, and the group repeats the run or asks for review rather than subtracting the color. Whatever the colors show, the record stops at a comparison. A positive or negative call for a sample well needs the test's decision rule, which this record does not supply, and a classroom kit does not test a person.

T1. Predict the two controls. Before any color is read, write what the positive control well should look like at the end of the run and what the negative control well should look like. Then write one sentence saying why a run needs both.

*Space: 3 writing lines. Help: Expected is not observed. Do not fill this from the data table.*

Positive control expected:

________________________________________________________________________________

Negative control expected:

________________________________________________________________________________

Why a run needs both:

________________________________________________________________________________

T2. Six-well data table

Fill one row for each of the six wells: the color you record using the four color words, and whether the well shows more color than the negative control, about the same, or less. Write the hue once on the hue line. Do not assign a concentration or a positive or negative call to any well.

*Space: Six printed rows with blank Color and Compared-with-NC cells at least 3 cm wide and an optional Note column; one route line and one hue line above the table, and on Route W two more lines above the table for the kit name and the incubation start times. Help: Read each well on its own before comparing. Light is still more color than Nearly clear. The four words are an observation scale, not amounts.*

• Route line: Route P: supplied Strip 1
• Record source: Supplied Strip 1 (illustrative)
• Hue supplied: blue

Blank fields on every row.

• Hue line: ____________________
• Color (Nearly clear / Light / Medium / Dark): one per row, left blank for you
• Compared with NC (More color / About the same / Less color): one per row, left blank for you
• Note (optional): one per row, left blank for you

Supplied Strip 1 (illustrative). Hue: blue. Supplied illustrative observation words, not measurements; no concentration, no decision rule, no positive or negative call for any sample well; nothing claims a student ran a kit

Copy these supplied colors into your T2 rows; the Compared-with-NC column is yours to fill.

T3. Order and wash. List the main steps of the run in the order they happened, with a wash between each (four in the model; use your kit's count if it prints more). Then write one sentence: what does the wash remove, and what would a skipped wash do to the color?

*Space: 4 short lines plus 2 writing lines. Help: Your kit may name a step differently. Keep the order it printed. The wash sentence is about what the color would mean, not about being neat.*

*Template: The wash removes ______, so a skipped wash would ______.*

Steps in order:

________________________________________________________________________________

Wash sentence:

________________________________________________________________________________

T4. Control check. Answer two questions from your table: Did the positive control develop color? Did the negative control stay nearly clear? Then write one sentence saying whether the other four wells can be read from this run, and why.

*Space: 2 short lines plus 2 writing lines. Help: A clean pair lets you read the other wells. It does not make them positive or negative.*

*Template: The positive control ______ and the negative control ______, so the other wells ______ because ______.*

PC developed color: yes or no:

________________________________________________________________________________

NC stayed nearly clear: yes or no:

________________________________________________________________________________

Can the sample wells be read, and why:

________________________________________________________________________________

Supplied Strip 2 (for T5), printed for both routes

Supplied Strip 2 (illustrative, flagged). Hue: blue. Supplied illustrative observation words; the negative control developed color, so the strip is flagged; no positive or negative call for any sample well

T5. Flagged strip, Strip 2. Strip 2 is a supplied illustrative record for every student. Its negative control is Medium. In two sentences, say what that means for reading the sample wells S1 to S4 on that strip, and what should happen next with the run.

*Space: 4 writing lines. Help: Do not subtract the negative control's color and keep going. Say what the run shows and what a careful group does with it.*

*Template: Because Strip 2's negative control is Medium, ______. Next, ______.*

What the Medium negative control means for the sample wells:

________________________________________________________________________________

________________________________________________________________________________

What happens next:

________________________________________________________________________________

________________________________________________________________________________

T6. One limit. Write one sentence naming something this record cannot establish about a sample well, or one kind of information that would be needed to establish it. Do not invent that information.

*Space: 2 writing lines. Help: More color than the negative control is a comparison you wrote down. A positive or negative call needs the test's decision rule, and a classroom kit is not a test of a person.*

*Template: This record cannot establish ______ because it does not supply ______.*

Limit sentence:

________________________________________________________________________________

• [ ] I predict what both controls should show before I read any color.
• [ ] I record each of six wells with the four color words and compare each with the negative control.
• [ ] I list the steps in the order they ran and explain what the wash removes.
• [ ] I read the two controls first and say whether the other wells can be read.
• [ ] I explain what a colored negative control does to a strip.
• [ ] I name one thing my record cannot establish, without inventing the missing rule.

Hand the paper record to Mr. Mendoza before leaving, or submit one PDF to Wk5 Data table: Wet ELISA lab by the due time on the assignment. Use your period and code. Do not submit twice.