This isn't a question with a cut-and-dry answer. I would say you could make a good argument in favor of several characters—Crooks springs first to mind, but one could also argue that Candy is a very lonely character, given his advanced age, which sets him apart from the rest of the workers. You could also argue that Curley's wife, despite the fact that she flirts with other men (particularly Slim), is the loneliest character, as the sole woman and someone who is ignored by her husband, the person who is supposed to care about her most. The most important thing, when you're asked to write an ACE-IT paragraph like this, is to choose your argument and stick with it, giving the best evidence you can for it.
As an example, let's say we're going to argue that Crooks is the loneliest character. That's your assertion.
Next, we need to give some context and citation. That means we need to explain who Crooks is and use some quotations from the book to demonstrate that he's lonely. Crooks is described as "the Negro stable buck," the only black worker in the story. On Saturday night, Lennie finds him sitting alone in his room. Crooks tells Lennie that he "ain't wanted" in the bunkhouse with the other men, and explains why: "I can't play because I'm black. They say I stink. Well, I tell you, you all of you stink to me."
Explication and interpretation is the part where you explain how the quotations you've chosen indicate that Crooks is lonely—or rather, how your citations support your assertion. In this case, we can argue that Crooks is forced to keep to himself; he can't have any friends among his supposed peers, because they have unfairly rejected him based on the color of his skin. In turn, Crooks has dealt with this by deciding he doesn't want to be around them anyway—they all "stink" to him. Furthermore, because he's a "cripple," this isn't a situation he can easily find his way out of. He's stuck in it; he's a "permanent" resident in a place where he isn't wanted. Because he sees no escape route, his loneliness is intensified.
In your conclusion, then, you draw together the ideas you have already put forward and summarize what you are saying. You may wish to acknowledge early in your paragraph that Crooks is not the only candidate for the position of "loneliest" character. If so, you could reiterate that here, but also reiterate the key reasons why he is lonelier than the others—for example, although Curley's wife is the only woman, she is white and therefore able to fraternize, even if she can't spend time with the person she really cares about. Crooks, on the other hand, is racially isolated and has nobody to turn to. His loneliness, moreover, is something he seemingly cannot escape from, and its ongoing status means that he becomes more and more lonely as time passes.