Domain: Healthcare — Basic Immunology
Target concept: Immunoglobulin - IgG Subclass Testing

Video generation prompt: Spot IgG1, IgG2, IgG3, IgG4 standards and the test serum sequentially from left to right onto a nitrocellulose membrane; after blocking, incubate with a pooled set of four fluorescently labeled anti-human IgG subclass-specific secondary antibodies (anti-IgG1, anti-IgG2, anti-IgG3, anti-IgG4) at 4°C for 1 hour; the image shows the nitrocellulose membrane observed under a fluorescence imaging system after washing.

Evaluation guide:
(i) Target concept: Antigen-antibody binding specificity enables subclass-resolved detection of human IgG on solid-phase nitrocellulose via fluorescent secondary antibodies.
Minimum mechanism: Fluorescent signal must arise only where primary IgG subclasses (or test serum IgG) are immobilized and bound by complementary subclass-specific secondary antibodies; blocking must suppress background; membrane must remain structurally intact and stationary during imaging.
(ii)Storyline: [Frame 1–2] Membrane laid flat, five unlabeled spots deposited left-to-right → [Frame 3–4] Blocking solution applied uniformly → [Frame 5–6] Pooled subclass-specific fluorescent secondary antibodies added and incubated (implied by static 4°C context) → [Frame 7–10] Post-wash fluorescence image revealed: five sharp, spatially segregated bands emit light in order IgG1–IgG4–test serum; viewpoint remains fixed frontal; no hand/tool intrusion; membrane stays rigid and flat; lighting is darkfield-compatible; fluorescence is band-localized and non-diffuse.

Manual:
Experimental Procedure
1. Spot purified IgG1, IgG2, IgG3, IgG4 standards and test serum sequentially left-to-right onto nitrocellulose membrane.
2. Block membrane to prevent nonspecific binding.
3. Incubate with a pooled set of four fluorescently labeled anti-human IgG subclass-specific secondary antibodies (anti-IgG1, anti-IgG2, anti-IgG3, anti-IgG4) at 4°C for 1 hour.
4. Wash thoroughly and image under a fluorescence imaging system.
Observation of Experimental Phenomena
Five discrete fluorescent bands aligned left-to-right: IgG1, IgG2, IgG3, IgG4, test serum; membrane background non-fluorescent; band intensities differ across the four standards, reflecting differences in binding efficiency among the four subclass-specific secondary antibodies rather than a single fixed ranking.
Summary of Experimental Principles
IgG subclass testing uses a pool of secondary antibodies, each specific to one IgG subclass, to detect differential expression of IgG1–IgG4 in serum; nitrocellulose enables immobilization and selective antigen-antibody binding; fluorescence imaging provides high sensitivity and multiplex capability across the full membrane.

Prompt Grounding rubric:
level_1: No nitrocellulose membrane is visible; none of the five expected spots (IgG1, IgG2, IgG3, IgG4, test serum) appear; there is no left-to-right sequential arrangement; the membrane is not shown as a flat, imaged strip at all.
level_2: A nitrocellulose membrane is visible, but the spot count is wrong (fewer or more than five), the left-to-right order does not match IgG1, IgG2, IgG3, IgG4, test serum, or individual spots cannot be told apart from one another; the shot is a tight close-up on a single spot rather than the whole strip.
level_3: The nitrocellulose membrane is shown with exactly five distinct, spatially separated spots in the correct left-to-right order (IgG1, IgG2, IgG3, IgG4, test serum); the membrane surface has a uniform matte look consistent with having been blocked; the camera frames the membrane widely enough that all five spot positions are visible together.
level_4: All of 3-score, plus the full membrane width is shown with neither the leftmost (IgG1) nor rightmost (test serum) spot cropped at the frame edge; spot spacing is even and resembles a standard multiplex dot-blot strip; no extraneous on-screen labels, arrows, or annotations are overlaid on the image.
level_5: All of 4-score, plus the camera holds a fixed frontal framing of the entire membrane from start to finish with no hand, tool, or overlay ever entering the frame; the five spots keep the same order and spacing in every frame; the membrane itself never appears cropped, tilted, or partially out of frame at any point in the clip.

Scientific and Causal Correctness rubric:
level_1: Fluorescent signal is not confined to the five spot locations (e.g., a diffuse glow covers large areas of the membrane or the inter-spot buffer zones); or none of the five spots (including test serum) show any signal; or spot brightness carries no relationship to spot identity, so the test serum spot cannot be told apart from a blank control.
level_2: Signal stays confined to the five spot locations with no membrane-wide glow, but all five spots (including test serum) show visually indistinguishable intensity; there is no sign that the different subclass-specific secondary antibodies are binding differently, so subclass-specific detection is not demonstrated.
level_3: Signal appears at all five correctly-positioned spots; the test serum spot shows a clearly detectable signal, confirming IgG is present in the test sample; at least one control spot is visibly brighter or dimmer than another, showing that some differential subclass detection is occurring.
level_4: All of 3-score, plus signal stays confined to the five spot locations with no visible glow on the surrounding membrane; each of the four purified-standard spots (IgG1, IgG2, IgG3, IgG4) shows a brightness level visibly distinguishable from every other one (no two identical), consistent with each subclass-specific secondary antibody having a somewhat different binding efficiency; no spot's signal merges or smears into a neighboring spot.
level_5: All of 4-score, plus the five spots stay spatially discrete with no visible bleed-through or crosstalk between neighbors and no visible fading of signal over the course of the clip; the test serum spot's brightness is clearly detectable and falls within the range spanned by the four standard spots rather than appearing brighter than all four or dimmer than all four, consistent with test serum containing a natural mixture of these subclasses.

Spatiotemporal Consistency rubric:
level_1: The membrane appears to jump to a different position, stretch, or change size between one frame and the next; individual spots flicker on and off inconsistently; the overall scene brightness suddenly shifts partway through the clip; a fluorescent spot appears or disappears with no gradual transition; the camera's viewpoint suddenly rotates or zooms rather than holding steady.
level_2: The membrane stays on screen, but lighting is uneven from one side to the other in a way that shifts over time; spot positions visibly shift sideways between frames; fluorescent signal flickers or strobes rather than staying steady; something (e.g., a hand or tool) partially blocks the membrane and then disappears without ever fully leaving the frame.
level_3: The membrane stays stationary within a stable, unchanging frame; all five spots keep the same spacing and left-to-right order from start to finish of the clip; once fluorescent signal appears it stays continuously visible on the spots without pulsing, fading, or visibly spreading outward over time; overall scene brightness stays even; no object briefly enters and blocks part of the membrane.
level_4: All of 3-score, plus the membrane remains fully visible and motionless for the entire clip; all five spots keep identical spacing and alignment across every frame; fluorescent signal becomes visible on all five spots at essentially the same moment (consistent with a single wash/imaging step) rather than appearing on some spots before others; no visible perspective change, warping, or tearing of the membrane edges.
level_5: All of 4-score, plus the membrane occupies the same screen region in every frame and the spot positions show no visible drift or repositioning across frames; fluorescent signal reaches its full visible brightness at the moment it first appears and stays visually constant afterward; no visible camera shake, no visible refocusing, and no visible change in depth of field at any point in the clip.