How to get setting from separate file?

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Simple answer: Don’t add a separate PHP file for AJAX requests. Declare the functions that will be used in your plugin and load them via admin-ajax.php.

add_action('wp_ajax_custom_action_name','custom_action_handler');
// to make AJAX action available for non-logged in users:
add_action('wp_ajax_nopriv_custom_action_name','custom_action_handler');

function custom_action_handler() {
    global $wpdb;

    // optionally keep the actual functions separately
    // include(dirname(__FILE__).'/ajax-handler.php');
    // custom_ajax_handler_process(); // handle request

    // or just handle request here

    exit;
}

$endpoint = admin_url('admin-ajax.php').'?action=custom_action_name';

WordPress will automatically take the action and match it to the wp_ajax_ (and/or wp_ajax_nopriv_) hook suffix to load the hooked function (custom_action_handler).

Complex answer: you can have a separate PHP file to handle the AJAX requests, but you need to be able to load the WordPress functions and classes you need (eg. for $wpdb) within it first. You can do this by:

  1. define('SHORTINIT',true);
  2. Require wp-blog-header.php from WordPress root path.
  3. Include any needed WordPress includes manually.
  4. Process the AJAX request.

Step 3 is the hard part as depending on what you are doing you may need a number of inter-related functions for the file to even load without a fatal error crashing your code and thus request response. Though if you are just accessing the database functions/class then these may be minimal. Overall this is a doable approach, but may mean much more work for only a slight increase in AJAX response performance time, so probably the simple answer is better.